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Posts Tagged ‘Oxidative phosphorylation’


Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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New Insights on the Warburg Effect [2.2]

Larry H. Bernstein, MD, FCAP, Curator, Writer

https://pharmaceuticalintelligence.com/8/05/15/lhbern/New_Insights_on_the_Warburg_Effect_%5B2.2%5D

 

New Insights on the Warburg Effect [2.2]

Defective Mitochondria Transform Normal Cells into Tumors

GEN News Jul 9, 2015

Ninety-one years ago Otto Warburg demonstrated that cancer cells have impaired respiration, which became known as the Warburg Effect. The interest in this and related work was superceded in the last quarter of the twentieth century by work on the genetic code. Now there is renewed interest.

An international research team reports that a specific defect in mitochondria plays a key role in the transition from normal cells to cancerous ones. The scientists disrupted a key component of mitochondria of otherwise normal cells and the cells took on characteristics of malignant cells.

Their study (“Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming”) is published Oncogene and was led by members of the lab of Narayan G. Avadhani, Ph.D., the Harriet Ellison Woodward Professor of Biochemistry in the department of biomedical sciences in the school of veterinary medicine at the University of Pennsylvania. Satish Srinivasan, Ph.D., a research investigator in Dr. Avadhani’s lab, was the lead author.

This is consistent with the 1924 observation by Warburg that cancerous cells consumed glucose at a higher rate than normal cells (Meyerhof ratio) and had defects in their grana, the organelles that are now known as mitochondria. He postulated that these defects led to problems in the process by which the cell produces energy. But the process called oxidative phosphorylation was not yet known. Further work in his laboratory was carried out by Hans Krebs and by Albert Szent Gyorgyi elucidating the tricarboxylic acid cycle.  The discovery of the importance of cytochrome c and adenosine triphosphate in oxidative phosphorylation was made in the post World War II period by Fritz Lippman, with an important contribution by Nathan Kaplan. All of the name scientists, except Kaplan, received Nobel Prizes. The last piece of the puzzle became the demonstation of a sequence of hydrogen transfers on the electron transport chain. The researchers above have now shown that mitochondrial defects indeed contributed to the cells becoming cancerous.

“The first part of the Warburg hypothesis has held up solidly in that most proliferating tumors show high dependence on glucose as an energy source and they release large amounts of lactic acid,” said Dr. Avadhani. “But the second part, about the defective mitochondrial function causing cells to be tumorigenic, has been highly contentious.”

To see whether the second part of Warburg’s postulation was correct, the researchers took cell lines from the skeleton, kidney, breast, and esophagus and used RNA molecules to silence the expression of select components of mitochondrial cytochrome oxidase C, or CcO, a critical enzyme involved in oxidative phosphorylation. CcO uses oxygen to make water and set up a transmembrane potential that is used to synthesize ATP, the molecule used for energy by the body’s cells.

The biologists observed that disrupting only a single protein subunit of cytochrome oxidase C led to major changes in the mitochondria and in the cells themselves. “These cells showed all the characteristics of cancer cells,” noted Dr. Avadhani.

The normal cells that converted to cancerous cells displayed changes in their metabolism, becoming more reliant on glucose by utilization of the glycolytic pathway. They reduced their synthesis of ATP.  Oxidative phosphorylation was reduced in concert with the ATP reduction. The large switch to glycolysis as primary energy source is a less efficient means of making ATP that is common in cancer cells.

The cells lost contact inhibition and gained an increased ability to invade distant tissues, both hallmarks of cancer cells. When they were grown in a 3D medium, which closely mimics the natural environment in which tumors grow in the body, the cells with disrupted mitochondria formed large, long-lived colonies, akin to tumors.

The researchers also silenced cytochrome oxidase C subunits in breast and esophageal cancer cell lines. They found that the cells became even more invasive, according to Dr. Srinivasan. The team then looked at actual tumors from human patients and found that the most oxygen-starved regions, which are common in tumors, contained defective versions of CcO.

“That result alone couldn’t tell us whether that was the cause or effect of tumors, but our cell system clearly says that mitochondrial dysfunction is a driving force in tumorigenesis,” explained Dr. Avadhani.

The researchers observed that disrupting CcO triggered the mitochondria to activate a stress signal to the nucleus, akin to an SOS alerting the cell that something was wrong. Dr. Avadhani and his colleagues had previously seen a similar pathway activated in cells with depleted mitochondrial DNA, which is also linked to cancer.

Building on these findings, Dr. Avadhani and members of his lab will examine whether inhibiting components of this mitochondrial stress signaling pathway might be a strategy for preventing cancer progression.

“We are targeting the signaling pathway, developing a lot of small molecules and antibodies,” said Dr. Avadhani. “Hopefully if you block the signaling the cells will not go into the so called oncogenic mode and instead would simply die.”

In addition, they noted that looking for defects in CcO could be a biomarker for cancer screening.

 

Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism

Rafael Moreno-Sánchez, Alvaro Marín-Hernández, Emma Saavedra, Juan P. Pardo, Stephen J. Ralph, Sara Rodríguez-Enríquez
Intl J Biochem Cell Biol 7 Feb 2014; 50:10-23
http://dx.doi.org/10.1016/j.biocel.2014.01.025

The supply of ATP in mammalian and human cells is provided by glycolysis and oxidative phosphorylation (OxPhos). There are no other pathways or processes able to synthesize ATP at sufficient rates to meet the energy demands of cells. Acetate thiokinase or acetyl-CoA synthetase, a ubiquitous enzyme catalyzing the synthesis of ATP and acetate from acetyl-CoA, PPi and AMP, might represent an exception under hypoxia in cancer cells, although the flux through this branch is negligible (≤10%) when compared to the glycolytic flux (Yoshii et al., 2009).

Glycolysis in human cells can be defined as the metabolic process that transforms 1 mol of glucose (or other hexoses) into 2 moles of lactate plus 2 moles of ATP. These stoichiometric values represent a maximum and due to the several reactions branching off glycolysis, they will be usually lower under physiological conditions, closer to 1.3–1.9 for the lactate/glucose ratio (Travis et al., 1971; Jablonska and Bishop, 1975; Suter and Weidemann, 1975; Hanson and Parsons, 1976; Wu and Davis, 1981; Pick-Kober and Schneider, 1984; Sun et al., 2012). OxPhos is the metabolic process that oxidizes several substrates through the Krebs cycle to produce reducing equivalents (NADH, FADH2), which feed the respiratory chain to generate an H+.

Applying basic biochemical principles, this review analyzes data that contrasts with the Warburg hypothesis that glycolysis is the exclusive ATP provider in cancer cells. Although disregarded for many years, there is increasing experimental evidence demonstrating that oxidative phosphorylation (OxPhos) makes a significant contribution to ATP supply in many cancer cell types and under a variety of conditions.

Substrates oxidized by normal mitochondria such as amino acids and fatty acids are also avidly consumed by cancer cells. In this regard, the proposal that cancer cells metabolize glutamine for anabolic purposes without the need for a functional respiratory chain and OxPhos is analyzed considering thermodynamic and kinetic aspects for the reductive carboxylation of 2-oxoglutarate catalyzed by isocitrate dehydrogenase.

In addition, metabolic control analysis (MCA) studies applied to energy metabolism of cancer cells are reevaluated. Regardless of the experimental/environmental conditions and the rate of lactate production, the flux-control of cancer glycolysis is robust in the sense that it involves the same steps:

  • glucose transport,
  • hexokinase,
  • hexosephosphate isomerase, and
  • glycogen degradation,

all at the beginning of the pathway; these steps together with phosphofructokinase 1 also control glycolysis in normal cells.

The respiratory chain complexes exert significantly higher flux-control on OxPhos in cancer cells than in normal cells. Thus, determination of the contribution of each pathway to ATP supply and/or the flux-control distribution of both pathways in cancer cells is necessary in order to identify differences from normal cells which may lead to the design of rational alternative therapies that selectively target cancer energy metabolism.

Fig. 1. Labeling patterns of 13C-glutamate or 13C-glutamine mitochondrial metabolism in cancer cells.

Fig. 2. Survey in PubMed of papers published in the field of tumor mitochondrial metabolism from 1951 to September 2013.

 

Emerging concepts in bioenergetics and cancer research: Metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy

Emilie Obre, Rodrigue Rossignol
Intl J Biochem Cell Biol 2015; 59:167-181
http://dx.doi.org/10.1016/j.biocel.2014.12.008

The field of energy metabolism dramatically progressed in the last decade, owing to a large number of cancer studies, as well as fundamental investigations on related transcriptional networks and cellular interactions with the microenvironment. The concept of metabolic flexibility was clarified in studies showing the ability of cancer cells to remodel the biochemical pathways of energy transduction and linked anabolism in response to glucose, glutamine or oxygen deprivation.

A clearer understanding of the large scale bioenergetic impact of C-MYC, MYCN, KRAS and P53 was obtained, along with its modification during the course of tumor development. The metabolic dialog between different types of cancer cells, but also with the stroma, also complexified the understanding of bioenergetics and raised the concepts of metabolic symbiosis and reverse Warburg effect.

Signaling studies revealed the role of respiratory chain derived reactive oxygen species for metabolic remodeling and metastasis development. The discovery of oxidative tumors in human and mice models related to chemoresistance also changed the prevalent view of dysfunctional mitochondria in cancer cells. Likewise, the influence of energy metabolism-derived oncometabolites emerged as a new means of tumor genetic regulation. The knowledge obtained on the multi-site regulation of energy metabolism in tumors was translated to cancer preclinical studies, supported by genetic proof of concept studies targeting LDHA, HK2, PGAM1, or ACLY.

Here, we review those different facets of metabolic remodeling in cancer, from its diversity in physiology and pathology, to the search of the genetic determinants, the microenvironmental regulators and pharmacological modulators.

 

Pyruvate kinase M2: A key enzyme of the tumor metabolome and its medical relevance

Mazurek, S.
Biomedical Research 2012; 23(SPEC. ISSUE): Pages 133-142

Tumor cells are characterized by an over expression of the glycolytic pyruvate kinase isoenzyme
type M2 (abbreviations: M2-PK or PKM2). In tumor metabolism the quaternary structure of M2-PK (tetramer/dimer ratio) determines whether glucose is used for glycolytic energy regeneration (highly active tetrameric form, Warburg effect) or synthesis of cell building blocks (nearly inactive dimeric form) which are both prerequisites for cells with a high proliferation rate. In tumor cells the nearly inactive dimeric form of M2- PK is predominant due to direct interactions with different oncoproteins. Besides its key functions in tumor metabolism recent studies revealed that M2-PK may also react as protein kinase as well as co activator of transcription factors. Of medical relevance is the quantification of the dimeric form of M2-PK with either an ELISA or point of care rapid test in plasma and stool that is used for follow-up studies during therapy (plasma M2-PK) and colorectal cancer (CRC) screening (fecal M2-PK; mean sensitivity for CRC in 12 independent studies with altogether 704 samples: 80% ± 7%). An intervention in the regulation mechanisms of the expression, activity and tetramer: dimer ratio of M2-PK has significant consequences for the proliferation rate and tumorigenic capacity of the tumor cells, making this enzyme an intensively

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Oxidation and Synthesis of Fatty Acids

Author and Curator: Larry H. Bernstein, MD, FCAP 

 

Lipid Metabolism

http://www.elmhurst.edu/~chm/vchembook/622overview.html

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidation to produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.

metabolism of fats

metabolism of fats

http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

fattyacidspiral

fattyacidspiral

http://www.elmhurst.edu/~chm/vchembook/images/620fattyacidspiral.gif

 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.

 

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2
 NET 12 ATP
 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP
GRAND TOTAL  130 ATP

Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.

This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.

  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.

.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.

My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.

The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1964/lynen-bio.html

http://onlinelibrary.wiley.com/store/10.1002/anie.201106003/asset/image_m/mcontent.gif?v=1&s=1e6dc789dfa585fe48947e92cc5dfdcabd8e2677

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

http://science.howstuffworks.com/dictionary/famous-scientists/biologists/feodor-lynen-info.htm

Fyodor Lynen

Fyodor Lynen

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Summary, Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP 

 

This portion of a series of chapters on metabolism, proteomics and metabolomics dealt mainly with carbohydrate metabolism. Amino acids and lipids are presented more fully in the chapters that follow. There are features on the

  • functioning of enzymes and proteins,
  • on sequential changes in a chain reaction, and
  • on conformational changes that we shall also cover.

These are critical to developing a more complete understanding of life processes.

I needed to lay out the scope of metabolic reactions and pathways, and their complementary changes. These may not appear to be adaptive, if the circumstances and the duration is not clear. The metabolic pathways map in total
is in interaction with environmental conditions – light, heat, external nutrients and minerals, and toxins – all of which give direction and strength to these reactions. A developing goal is to discover how views introduced by molecular biology and genomics don’t clarify functional cellular dynamics that are not related to the classical view.  The work is vast.

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from carbon dioxide and water by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are consumed by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts.

Carbohydrates are used for short-term fuel, and even though they are simpler to metabolize than fats, they don’t produce as equivalent energy yield measured by ATP.  In animals, the concentration of glucose in the blood is linked to the pancreatic endocrine hormone, insulin. . In most organisms, excess carbohydrates are regularly catabolized to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates.

Glucose is metabolized obtaining ATP and pyruvate by way of first splitting a six-carbon into two three carbon chains, which are converted to lactic acid from pyruvate in the lactic dehydrogenase reaction. The reverse conversion is by a separate unidirectional reaction back to pyruvate after moving through pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that convert pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the citric acid cycle. This multi-enzyme complex is related structurally and functionally to the oxoglutarate dehydrogenase and branched-chain oxo-acid dehydrogenase multi-enzyme complexes. In eukaryotic cells the reaction occurs inside the mitochondria, after transport of the substrate, pyruvate, from the cytosol. The transport of pyruvate into the mitochondria is via a transport protein and is active, consuming energy. On entry to the mitochondria pyruvate decarboxylation occurs, producing acetyl CoA. This irreversible reaction traps the acetyl CoA within the mitochondria. Pyruvate dehydrogenase deficiency from mutations in any of the enzymes or cofactors results in lactic acidosis.

PDH-rxns The acetyl group is transferred to coenzyme A

PDH-rxns The acetyl group is transferred to coenzyme A

http://guweb2.gonzaga.edu/faculty/cronk/biochem/images/PDH-rxns.gif

Typically, a breakdown of one molecule of glucose by aerobic respiration (i.e. involving both glycolysis and Kreb’s cycle) is about 33-35 ATP. This is categorized as:

Glycogenolysis – the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues.

Glycogenolysis in liver provides circulating glucose short term.

Glycogenolysis in muscle is obligatory for muscle contraction.

Pyruvate from glycolysis enters the Krebs cycle, also known as the citric acid cycle, in aerobic organisms.

Anaerobic breakdown by glycolysis – yielding 8-10 ATP

Aerobic respiration by Kreb’s cycle – yielding 25 ATP

The pentose phosphate pathway (shunt) converts hexoses into pentoses and regenerates NADPH. NADPH is an essential antioxidant in cells which prevents oxidative damage and acts as precursor for production of many biomolecules.

Glycogenesis – the conversion of excess glucose into glycogen as a cellular storage mechanism; achieving low osmotic pressure.

Gluconeogenesis – de novo synthesis of glucose molecules from simple organic compounds. An example in humans is the conversion of a few amino acids in cellular protein to glucose.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

The hormone insulin is the primary glucose regulatory signal in animals. It mainly promotes glucose uptake by the cells, and it causes the liver to store excess glucose as glycogen. Its absence

  1. turns off glucose uptake,
  2. reverses electrolyte adjustments,
  3. begins glycogen breakdown and glucose release into the circulation by some cells,
  4. begins lipid release from lipid storage cells, etc.

The level of circulatory glucose (known informally as “blood sugar”) is the most important signal to the insulin-producing cells.

  • insulin is made by beta cells in the pancreas,
  • fat is stored n adipose tissue cells, and
  • glycogen is both stored and released as needed by liver cells.
  • no glucose is released to the blood from internal glycogen stores from muscle cells.

The hormone glucagon, on the other hand, opposes that of insulin, forcing the conversion of glycogen in liver cells to glucose, and then release into the blood. Growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon.  These hormones are referred to as stress hormones because they are released under the influence of catabolic proinflammatory (stress) cytokines – interleukin-1 (IL1) and tumor necrosis factor α (TNFα).

Net Yield of GlycolysisThe preparatory phase consumes 2 ATP

The pay-off phase produces 4 ATP.

The gross yield of glycolysis is therefore

4 ATP – 2 ATP = 2 ATP

The pay-off phase also produces 2 molecules of NADH + H+ which can be further converted to a total of 5 molecules of ATP* by the electron transport chain (ETC) during oxidative phosphorylation.

Thus the net yield during glycolysis is 7 molecules of ATP*
This is calculated assuming one NADH molecule gives 2.5 molecules of ATP during oxidative phosphorylation.

Cellular respiration involves 3 stages for the breakdown of glucose – glycolysis, Kreb’s cycle and the electron transport system. Kreb’s cycle produces about 60-70% of ATP for release of energy in the body. It directly or indirectly connects with all the other individual pathways in the body.

The Kreb’s Cycle occurs in two stages:

  1. Conversion of Pyruvate to Acetyl CoA
  2. Acetyl CoA Enters the Kreb’s Cycle

Each pyruvate in the presence of pyruvate dehydrogenase (PDH) complex in the mitochondria gets converted to acetyl CoA which in turn enters the Kreb’s cycle. This reaction is called as oxidative  decarboxylation as the carboxyl group is removed from the pyruvate molecule in the form of CO2 thus yielding 2-carbon acetyl group which along with the coenzyme A forms acetyl CoA.

The PDH requires the sequential action of five co-factors or co-enzymes for the combined action of dehydrogenation and decarboxylation to take place. These five are TPP (thiamine phosphate), FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide), coenzyme A (denoted as CoA-SH at times to depict role of -SH group) and lipoamide.

Acetyl CoA condenses with oxaloacetate (4C) to form a citrate (6C) by transferring its acetyl group in the presence of enzyme citrate synthase. The CoA liberated in this reaction is ready to participate in the oxidative decarboxylation of another molecule of pyruvate by PDH complex.

Isocitrate undergoes oxidative decarboxylation by the enzyme isocitrate dehydrogenase to form oxalosuccinate (intermediate- not shown) which in turn forms α-ketoglutarate (also known as oxoglutarate) which is a five carbon compound. CO2 and NADH are released in this step. α-ketoglutarate (5C) undergoes oxidative decarboxylation once again to form succinyl CoA (4C) catalysed by the enzyme α-ketoglutarate dehydrogenase complex.

Succinyl CoA is then converted to succinate by succinate thiokinase or succinyl coA synthetase in a reversible manner. This reaction involves an intermediate step in which the enzyme gets phosphorylated and then the phosphoryl group which has a high group transfer potential is transferred to GDP to form GTP.

Succinate then gets oxidised reversibly to fumarate by succinate dehydrogenase. The enzyme contains iron-sulfur clusters and covalently bound FAD which when undergoes electron exchange in the mitochondria causes the production of FADH2.

Fumarate is then by the enzyme fumarase converted to malate by hydration(addition of H2O) in a reversible manner.

Malate is then reversibly converted to oxaloacetate by malate dehydrogenase which is NAD linked and thus produces NADH.

The oxaloacetate produced is now ready to be utilized in the next cycle by the citrate synthase reaction and thus the equilibrium of the cycle shifts to the right.

The NADH formed in the cytosol can yield variable amounts of ATP depending on the shuttle system utilized to transport them into the mitochondrial matrix. This NADH, formed in the cytosol, is impermeable to the mitochondrial inner-membrane where oxidative phosphorylation takes place. Thus to carry this NADH to the mitochondrial matrix there are special shuttle systems in the body. The most active shuttle is the malate-aspartate shuttle via which 2.5 molecules of ATP are generated for 1 NADH molecule. This shuttle is mainly used by the heart, liver and kidneys. The brain and skeletal muscles use the other shuttle known as glycerol 3-phosphate shuttle which synthesizes 1.5 molecules of ATP for 1 NADH.

Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH. The importance of this pathway can easily be underestimated.  The main source for energy in respiration was considered to be tied to the high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+. The pentose phosphate shunt is essential for the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

The pyridine nucleotide transhydrogenase reaction concerns the energy-dependent reduction of TPN by DPNH. In 1959, Klingenberg and Slenczka made the important observation that incubation of isolated liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate acceptor resulted in a rapid and almost complete reduction of  the intramitochondrial TPN. These and related findings led Klingenberg and co-workers (1-3) to postulate the occurrence of a ATP-controlled transhydrogenase reaction catalyzing the reduction of TPN by DPNH.  (The role of transhydrogenase in the energy-linked reduction of TPN.  Fritz Hommes, Ronald W. Estabrook, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6. http://dx.doi.org:/10.1016/0006-291X(63)90017-2/).

Further studies observed the coupling of TPN-specific dehydrogenases with the transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine nucleotide (TPN). The studies showed the direct interaction between TPNHz and DPN, in the presence of transhydrogenase to yield products having the properties of TPN and DPNHZ. The reaction involves a transfer of electrons (or hydrogen) rather than a phosphate. (Pyridine Nucleotide Transhydrogenase  II. Direct Evidence for and Mechanism of the Transhydrogenase Reaction* by  Nathan 0. Kaplan, Sidney P. Colowick, And Elizabeth F. Neufeld. (From The Mccollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland) J. Biol. Chem. 1952, 195:107-119.) http://www.JBC.org/Content/195/1/107.Citation
Notation: TPN, NADP; DPN, NAD+; reduced pyridine nucleotides: TPNH (NADPH2), DPNH (NADH).

Note: In this discussion there is a detailed presentation of the activity of lactic acid conversion in the mitochondria by way of PDH. In a later section there is mention of the bidirectional reaction of lactate dehydrogenase.  However, the forward reaction is dominant (pyruvate to lactate) and is described. This is not related to the kinetics of the LD reaction with respect to the defining characteristic – Km.

Biochemical Education Jan 1977; 5(1):15. Kinetics of Lactate Dehydrogenase: A Textbook Problem.
K.L. MANCHESTER. Department of Biochemistry, University of Witwatersrand, Johannesburg South Africa.

One presupposes that determined Km values are meaningful under intracellular conditions. In relation to teaching it is a simple experiment for students to determine for themselves the Km towards pyruvate of LDH in a post-mitochondrial supernatant of rat heart and thigh muscle. The difference in Km may be a factor of 3 or 4-fold.It is pertinent then to ask what is the range of suhstrate concentrations over which a difference in Km may be expected to lead to significant differences in activity and how these concentrations compare with pyruvate concentrations in the cell. The evidence of Vesell and co-workers that inhibition by pyruvate is more readily seen at low than at high enzyme concentration is important in emphasizing that under intracellular conditions enzyme concentrations may be relatively large in relation to the substrate available. This will be particularly so in relation to [NADH] which in the cytoplasm is likely to be in the ~M range.

A final point concerns the kinetic parameters for LDH quoted by Bergmeyer for lactate estimations a pH of 9 is recommended and the Km towards lactate at that pH is likely to be appreciably different from the quoted values at pH 7 — Though still at pH 9 showing a substantially lower value for lactate with the heart preparationhttp://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2877%2990013-9/pdf

Several investigators have established that epidermis converts most of the glucose it uses to lactic acid even in the presence of oxygen. This is in contrast to most tissues where lactic acid production is used for energy production only when oxygen is not available. This large amount of lactic acid being continually produced within the epidermal cell must be excreted by the cell and then carried away by the blood stream to other tissues where the lactate can be utilized. The LDH reaction with pyruvate and NADH is reversible although at physiological pH the equilibrium position for the reaction lies very far to the right, i.e., in favor of lactate production. The speed of this reaction depends not only on the amount of enzyme present but also on the concentrations of the substances involved on both sides of the equation. The net direction in which the reaction will proceed depends solely on the relative concentrations of the substances on each side of the equation.
In vivo there is net conversion of pyruvate (formed from glucose) to lactate. Measurements of the speed of lactate production by sheets of epidermis floating on a medium containing glucose indicate a rate of lactate production of approximately 0.7 rn/sm/
mm/mg of fresh epidermis.Slice incubation experiments are presumably much closer to the actual in vivo conditions than
the homogenate experiments. The discrepancy between the
two indicates that in vivo conditions are far from optimal for the conversion of pyruvate to lactate. Only 1/100th of the maximal activity of the enzyme present is being achieved. The concentrations of the various substances involved are not
optimal in vivo since pyruvate and NADH concentrations are
lower than lactate and NAD concentrations and this might explain the in vivo inhibition of LDH activity. (Lactate Production And Lactate Dehydrogenase In The Human Epidermis*. KM. Halprin, A Ohkawara. J Invest Dermat 1966; 47(3): 222-6.)
http://www.nature.com/jid/journal/v47/n3/pdf/jid1966133a.pdf

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Introduction to Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP

 

Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence.  It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.

There are essentially two major sources of fuel, mainly, carbohydrate and fat.  A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.

Carbohydrates are a major fuel utilized by way of either of two pathways.  They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle.  Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.

Carbohydrate metabolism has either of two routes of utilization.  This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle.  In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate.  In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital.  Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld,  who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.

This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.

The subchapters are as follows:

1.1      Carbohydrate Metabolism

1.2      Studies of Respiration Lead to Acetyl CoA

1.3      Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

1.4      The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy

Complex I or NADH-Q oxidoreductase

Complex I or NADH-Q oxidoreductase

Fatty acid oxidation and ETC

Fatty acid oxidation and ETC

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The multi-step transfer of phosphate bond and hydrogen exchange energy

Curator: Larry H. Bernstein, MD, FCAP, Leaders in Pharmaceutical Intelligence

In this subtext of the series we expand on a tie between respiration and glycolysis, and the functioning of the mitochondrion to discover the key role played by oxidative phosphorylation, “acetyl coenzyme A, and electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue, and the criticality of the multi-step sequence of reactions in energy transfer.

This portion considerably provides a response to the TWO points made by Jose EDS Rosallis:

  1. Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. This poiny needs further clarification, but he makes important observations here.
  • Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it had changed from active form of the previous day to a non-active form.

The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity.

  • This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity.

This led to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead

  • it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency,
  • stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and
  • increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat these assays with

  • PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case.

I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer)
(Nathan O. Kaplan discovery) indicating this as his idea. The reason was my “chief” (SP) more than once,  said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things. ” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.

[This reminds me of when I was studying the emergence of lactic dehysrogenase isoenzyme patterns in the developing eye lens of cattle, I raised reservations about Elliott Vessells challenge to Nathan Kaplan, but that not being my primary problem, my brilliant mentor (H.M.), a very young full professor of anatomy said – leave that to NOK.}

Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is

  • glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved.

This dialogue explains why during the Schroedinger’s “What is life?” reading with him he asked me if from biochemist in exile, to biochemist I expressed all of my thoughts to him. Since I had considered that Schrödinger did not confront Darlington & Haldane for being in exile. This may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggests the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of
that year(1971).

  1. show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/
  1. Signaling transduction tutorial.
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-transduction-tutorial/
  1. Carbohydrate metabolism
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Oxidation-Reduction Reactions

Rachel Casiday, Carolyn Herman, and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/cytochromes.html

 

OX-Phos steps

OX-Phos steps

http://s1.hubimg.com/u/6583902_f496.jpg

 

Key Concepts:

  • ATP as Free-Energy Currency in the Body
  • Coupled Reactions
    • Standard Free-Energy Change for Coupled Reactions
    • ATP Dephosphorylation Coupled to Nonspontaneous Reactions
    • Coupled Reactions to Generate ATP
  • Structure and Function of the Mitochondria
  • Oxidation-Reduction Reactions in the Electron-Transport Chain
    • Electron-Carrier Proteins (NOTE: This section includes a separate link and an animation.)
    • Relationship Between Reduction Potentials and Free Energy
  • Proton Gradient as Means of Coupling Oxidative and Phosphorylation Components of Oxidative Phosphorylation
  • ATP Synthetase Uses Energy From Proton Gradient to Generate ATP

Every day, we build bones, move muscles, eat food, think, and perform many other activities with our bodies. All of these activities are based upon chemical reactions. However, most of these reactions are not spontaneous (i.e., they are accompanied by a positive change in free energy, DG>0) and do not occur without some other source of free energy. Hence, the body needs some sort of “free-energy currency,” (Figure 1) a molecule that can store and release free energy when it is needed to power a given biochemical reaction.

The four questions:

  1. How does the body “spend” free-energy currency to make a nonspontaneous reaction spontaneous? The answer, which is based on thermodynamics, is to use coupled reactions.
  2. How is food used to produce the reducing agents (NADH and FADH2) that can regenerate the free-energy currency? The answer, from biology, is found in glycolysis and the citric-acid cycle.
  3. How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)? Once again, coupled reactions are key.
  4. What mechanism does the body use to couple the reducing agent reactions and the generation of ATP? ATP is synthesized primarily by a two-step process consisting of an electron-transport chain and a proton gradient.  This process is based on electrochemistry and equilibrium, as well as thermodynamics.

The free-energy change (DG) for the net reaction is given by the sum of the free-energy changes for the individual reactions.  The phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.This step actually consists of two reactions:

(1) the phosphorylation of glycerol, and

(2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

      Glycerol + HPO42- –>  (Glycerol-3-Phosphate)2- + H2O DGo= +9.2 kJ
(nonspontaneous)
(2)
+      ATP4- + H2O –>       ADP3- + HPO42- + H+ DGo30.5 kJ
(spontaneous)
(3)
     Glycerol + ATP4- –> (Glycerol-3-Phosphate)2- +ADP3- + H+ DGo21.3 kJ
(spontaneous)
(4)
   

ATP is the most important “free-energy-currency” molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous; i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body’s use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

structure of ATP

structure of ATP

This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

flowchart of food energy

flowchart of food energy

This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy, so that it is available to the body in a readily accessible form.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart1.jpg

Glycolysis   Glucose + 2 HPO42- + 2 ADP3- + 2 NAD+ –>
2 Pyruvate + 2 ATP4- + 2 NADH + 2 H+ + 2 H2O
(5)
Intermediate Step   2(Pyruvate + Coenzyme A + NAD+ –>
Acetyl CoA + CO2 + NADH)
(6)
Citric-Acid Cycle 2(Acetyl CoA + 3 NAD++ FAD + GDP3-
+ HPO42- + 2H2O –> 2 CO2 + 3 NADH + FADH2
+ GTP4- + 2H+ + Coenzyme A)
(7)

The structures of the important molecules in Equations 5-7 are shown in Table 1, below.

How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.

pyruvate

pyruvate

  Pyruvate

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/pyruvate.jpg

acetylCoA

acetylCoA

Acetyl CoA

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

NADH

NADH

NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

 

FADH2

FADH2

FADH2

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/FADH2.jpg

two-dimensional representations of several important molecules in Equations 5-7.

As seen in Equations 5-7 in the blue box, glycolysis and the citric-acid cycle produce a net total of only four ATP or GTP molecules (GTP is an energy-currency molecule similar to ATP) per glucose molecule. This yield isfar below the amount needed by the body for normal functioning, and in fact is far below the actual ATP yield for glucose in aerobic organisms (organisms that use molecular oxygen). For each glucose molecule the body processes, the body actually gains approximately 30 ATP molecules! (See Figure 4, below.)  So, how does the body generate ATP?

The process that accounts for the high ATP yield is known as oxidative phosphorylation. A quick examination of Equations 5-7 shows that glycolysis and the citric-acid cycle generate other products besides ATP and GTP, namely NADH and FADH2 (blue). These products are molecules that are oxidized (i.e., give up electrons) spontaneously. The body uses these reducing agents (NADH and FADH2) in an oxidation-reduction reaction .  As you will see later in this tutorial, it is the free energy from these redox reactions that is used to drive the production of ATP.

flowchart - making of ATP

flowchart – making of ATP

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart2.jpg

This flowchart shows the major steps involved in breaking down glucose from the diet and converting its chemical energy to the chemical energy in the phosphate bonds of ATP, in aerobic (oxygen-using) organisms. Note: In this flowchart, red denotes a source of carbon atoms (originally from glucose),green denotes energy-currency molecules, and blue denotes the reducing agents that can be oxidized spontaneously.

In the discussion above, we see that glucose by itself generates only a tiny amount of ATP. However, during the breakdown of glucose, a large amount of NADH and FADHis produced; it is these reducing agents that dramatically increase the amount of ATP produced. How does this work?

How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)?

As discussed in an earlier section about coupling reactions, ATP is used as free-energy currency by coupling its (spontaneous) dephosphorylation (Equation 3) with a (nonspontaneous) biochemical reaction to give a net release of free energy (i.e., a net spontaneous reaction). Coupled reactions are also used to generate ATP by phosphorylating ADP. The nonspontaneous reaction of joining ADP to inorganic phosphate to make ATP (Equation 8, below, and Figure 2, above) is coupled to the oxidation reaction of NADH or FADH(Equation 9, below). (Recall, NADH and FADH2 are produced in glycolysis and the citric-acid cycle as described in the blue box). For simplicity, we shall henceforth discuss only the oxidation of NADH; FADH2 follows a very similar oxidation pathway.

The oxidation reaction for NADH has a larger, but negative, DG than the positive DG required for the formation of ATP from ADP and phosphate. This set of coupled reactions is so important that it has been given a special name: oxidative phosphorylation. This name emphasizes the fact that an oxidation (of NADH) reaction (Equation 9 and Figure 5, below) is being coupled to a phosphorylation (of ADP) reaction (Equation 8, below, and Figure 2, above). In addition, we must consider the reduction reaction (gaining of electrons) that accompanies the oxidation of NADH. (Oxidation reactions are always accompanied by reduction reactions, because an electron given up by one group must be accepted by another group.) In this case, molecular oxygen (O2) is the electron acceptor, and the oxygen is reduced to water (Equation 10, below) .

The individual reactions of interest for oxidative phosphorylation are:

Phosphorylation

ADP3- + HPO42- + H+ –>
ATP4- + H2O

DGo= +30.5 kJ
(nonspontaneous)
(8)
oxidation

NADH –> NAD+ + H+ +  2e

DGo158.2 Kj
(spontaneous)
(9)
reduction

1/2 O2 + 2H+ + 2e –> H2O

DGo61.9 kJ
(spontaneous)

                                                                       (10)                                    

The net reaction is obtained by summing the coupled reactions, as shown in Equation 11, below.

ADP3- + HPO42- + NADH + 1/2 O2 + 2H+ –>
ATP4- + NAD+ + 2 H2O
DGo= -189.6 kJ
(spontaneous)
(11)

The molecular changes that occur upon oxidation of NADH are shown:

NAD+_NADH

NAD+_NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/NAD+_NADH.jpg

This is a two-dimensional (ChemDraw) representation showing the change that occurs when NADH is oxidized to NAD+. “R” represents the part of the structure that is shown in black in the drawing of NADH in Table 1, and does not change during the oxidation half-reaction. The molecular changes that occur upon oxidation are shown in red.

In this tutorial, we have seen that nonspontaneous reactions in the body occur by coupling them with a very spontaneous reaction (usually the ATP reaction shown in Equation 3). We have just seen that ATP is produced by coupling the phosphorylation reaction with NADH oxidation (a very spontaneous reaction). But we have not yet answered the question: by what mechanism are these reactions coupled?

Coupling Reactions in Biological Systems

Every day your body carries out many nonspontaneous reactions. As discussed earlier, if a nonspontaneous reaction is coupled to a spontaneous reaction, as long as the sum of the free energies for the two reactions is negative, the coupled reactions will occur spontaneously. How is this coupling achieved in the body? Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme. Consider again the phosphorylation of glycerol (Equations 2-4). Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver. The product of glycerol phosporylation, glycerol-3-phosphate (Equation 2), is used in the synthesis of phospholipids.

Glycerol kinase is a large protein comprised of about 500 amino acids. X-ray crystallography of the protein shows us that there is a deep groove or cleft in the protein where glycerol and ATP attach (see Figure 6, below). Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol. Instead of two separate reactions where ATP loses a phosphate (Equation 3) and glycerol picks up a phosphate (Equation 2), the enzyme allows the phosphate to move directly from ATP to glycerol (Equation 4).

The coupling in oxidative phosphorylation uses a more complicated (and amazing!) mechanism, but the end result is the same: the reactions are linked together, the net free energy for the linked reactions is negative, and, therefore, the linked reactions are spontaneous.

glyckin

glyckin

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/glyckin.jpg

This is a schematic representation of ATP and glycerol bound (attached) to glycerol kinase. The enzyme glycerol kinase is a dimer (consists of two identical subuits). There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate (spontaneous) and glycerol gaining a phosphate (nonspontaneous) are linked together as one spontaneous process

Questions on ATP: The Body’s Free-Energy Currency (How Free-Energy Currency Works)

  • Biological systems involve many molecules containing phosphate groups, such as ATP. Although ATP is the most commonly used free-energy currency, any of these phosphorylated molecules could, in theory, be used as free-energy currency. The standard free-energy change (DGo) for the dephosphorylation (removal of a phosphate group) of several biological compounds is given below:
Acetyl phosphate DGo = -47.3 kJ/mol
Adenosine triphosphate (ATP) DGo = -30.5 kJ/mol
Glucose-6-phosphate DGo = -13.8 kJ/mol
Phosphoenolpyruvate (PEP) DGo = -61.9 kJ/mol
Phosphocreatine DGo = -43.1 kJ/mol

Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency (i.e., the amount of nonspontaneous reactions enabled per phosphate removed from a molecule of free-energy currency) from the most efficient to the least efficient.

  • What, if any, changes are there in the shape of the ring as NADH is oxidized to NAD+(see Figure 5)? (Hint: Consider which atoms lie in the same plane in each structure.)

Mechanism of Coupling the Oxidative-Phosphorylation Reactions

In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles (specialized cellular components) known as mitochondria. A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions.

Synthesis of ATP (Equation 8) is coupled with the oxidation of NADH (Equation 9) and the reduction of O2 (Equation 10). There are three key steps in this process:

  1. Electrons are transferred from NADH, through a series of electron carriers, to O2. The electron carriers are proteins embedded in the inner mitochondrial membrane. (More detail about the structure of the mitochondria is presented in the next section.) (See Figure 7a.)
  2. Transfer of electrons by these carriers generates a proton (H+) gradient across the inner mitochondrial membrane. (See Figure 7b.)
  3. When Hspontaneously diffuses back across the inner mitochondrial membrane, ATP is synthesized. The large positive free energy of ATP synthesis is overcome by the even larger negative free energy associated with proton flow down the concentration gradient. (See Figure 7c.)

These steps are outlined below.

  1. Electron Transport (Oxidation-Reduction Reactions) Through a Series of Proteins in the Inner Membrane of the Mitochondria
e_transfer

e_transfer

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/e_transfer.jpg

Generation of H+(Proton) Concentration Gradient Across the Inner Mitochondrial Membrane During the Electron-Transport Process (via a Proton Pump)

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/gradient.jpg

Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+Back to the Matrix Inside the Inner Mitochondrial Membrane

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP_produced.jpg

The three major steps in oxidative phosphorylation are

(a) oxidation-reduction reactions involving electron transfers between specialized proteins embedded in the inner mitochondrial membrane; 

(b) the generation of a proton (H+) gradient across the inner mitochondrial membrane (which occurs simultaneously with step (a)); and 

(c) the synthesis of ATP using energy from the spontaneous diffusion of electrons down the proton gradient generated in step (b).

Note: Steps (a) and (b) show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron (green) from another protein in the electron-transport chain, an Fe(III) ion in the center of a heme group (purple) embedded in the protein is reduced to Fe(II). The coordinates for the protein were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

Cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP. Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs.

Structure and Function of the Mitochondria

mitochondria

mitochondria

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/mitochondria.jpg

This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below. An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure.

The mitochondrial membranes are crucial for this organelle’s role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane ispermeable to most small molecules and ions, because it contains large protein channels called porins. The inner membrane is impermeable to most ions and polar molecules. The inner membrane is the site of oxidative phosphorylation. Although the membrane is mostly impermeable, it contains special H+ (proton) channels and pumps that enable the coupling of the redox reaction involving NADH and O2 (Equations 9-10) to the phosphorylation reaction of ADP (Equation 8), as described below (“Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation”). (Recall the discussion of protein channels in the “Maintaining the Body’s Chemistry: Dialysis in the Kidneys” Tutorial .)

As shown in Figure 8, inside the inner membrane is a space known as the matrix; the space between the two membranes is known as the intermembrane space. The matrix side of the inner membrane has a negative electrical charge relative to the intermembrane space due to an H+ gradient set up by the redox reaction (Equations 9 and 10). This charge difference is used to provide free energy (G) for the phosphorylation reaction (Equation 8).

Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation

Phosphorylation of ADP (Equation 8) is coupled to the oxidation-reduction reaction of NADH and O2 (Equations 9 and 10). Electrons are not transferred directly from NADH to O2, but rather pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. Why? This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion. As we shall see, it is this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis.

Two major types of mitochondrial proteins (see Figure 9, below) are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.

  1. The electron carriers (NADH-Q reductase, ubiquinone (Q), cytochrome reductase, cytochrome c, and cytochrome oxidase shown in shades of purple in Figure 9 below) transport electrons in a stepwise fashion from NADH to O2.  Three of these carriers (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) are also proton pumps, and simultaneously pump H+ ions (protons) from the matrix to the intermembrane space. (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.) The protons that are pumped across the membrane complete the redox reaction (Equations 9 and 10). The creation of a proton gradient across the membrane is one way of storing free energy.
  2. ATP synthetase (shown in red in Figure 9 below) allows H+ ions to diffuse back into the matrix and uses the free energy released to synthesize ATP from ADP and HPO42-. The ATP synthetase is essential for the phosphorylation to occur (Equation 8). (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.)

The electron carriers can be divided into three protein complexes (NADH-Q reductase (1), cytochrome reductase (3), and cytochrome oxidase (5)) that pump protons from the matrix to the intermembrane space, and two mobile carriers (ubiquinone (2) and cytochrome c (4)) that transfer electrons between the three proton-pumping complexes. (Gold numbers refer to the labels on each protein in Figure 9, below.) Because electrons move from one carrier to another until they are finally transferred to O2, the electron carriers (shown in Figure 9,below) are said to form an electron-transport chain.

Figure  below, is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on “View the Movie.”

Proteins of inner space

Proteins of inner space

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/Proteins.jpg

This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation. Click the blue button below to download QuickTime 4.0 to view the movie.

NADH-Q reductase (1), cytochrome reductase (3) , and cytochrome oxidase (5) are electron carriers as well as proton pumps, using the energy gained from each electron-transfer step to move protons (H+) against a concentration gradient, from the matrix to the intermembrane space.Ubiquinone (Q) (2) and cytochrome c (Cyt C) (4) are mobile electron carriers. (Ubiquinone is not actually a protein.) All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials. Together, these electron carriers form a “chain” to transport electrons from NADH to O2. The path of the electrons is shown with the green dotted line.

ATP synthetase (red) has two components: a proton channel (allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix), and a catalytic component to catalyze the formation of ATP.

For a more complete description of each step in oxidative phosphorylation (indicated by the gold numbers), click here.

view movie

view movie

http://www.apple.com/quicktime/

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/movie.jpg

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/QuickTime.jpg

Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that, although NADH donates two electrons and O2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time. Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial.

In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron tranfers from one electron carrier to the next spontaneous?

What causes electrons to be transferred down the electron-transport chain?

As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in a iron-sulfur (Fe-S) group or in a heme group. (Recall the heme group from the Chem 151 tutorial “Hemoglobin and the Heme Group: Metal Complexes in the Blood“.) The iron in these groups is alternately oxidized and reduced between Fe(II) (reduced) or Fe(III) (oxidized) states.

Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential (e) of a reduction half reaction is, the greater the tendency is for the species to accept an electron. Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change.

Reduction Potentials and Relationship to Free Energy

An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential (e). By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction (i.e., the opposite reaction). The electrical potential for an oxidation-reduction reaction is calculated by

erxn = eoxidation + ereduction (12)

For example, for the overall reaction of the oxidation of NADH paired with the reduction of O2, the potential can be calculated as shown below.

Reduction Potentials ereduction
NAD+ + 2H+ + 2e –> NADH + H+ -0.32 V
(1/2) O2 + 2H+ + 2e –> H2O +0.82 V

The overall reaction is

NADH + H–> NAD+ + 2H+ + 2e eoxidation = 0.32 V
(1/2) O2 + 2H+ + 2e –> H2O ereduction = 0.82 V
net: NADH + (1/2)O2 + H+ –>
H2O + NAD+
erxn = 1.14 V

The electrical potential (erxn) is related to the free energy (DG) by the following equation:

DG= -nFerxn (13)

where n is the number of electrons transferred (in moles, from the balanced equation), and F is the Faraday constant (96,485 Coulombs/mole). (Using this equation, DG is given in Joules; one Joule = 1 Volt x 1 Coulomb.)

Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy (DG =-220 kJ); i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16, below.

reduced cytochrome c–> oxidized cytochrome c+ e eoxidation = – .220 V (14)
oxidized cytochrome c + e –> reduced cytochrome c ereduction = .250 V (15)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = 0.030 V (16) Spontaneous

We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

reduced cyt c–> oxidized cyt c+ e eoxidation = – .220 V (17)
oxidized cyt b + e –> reduced cyt b ereduction = – 0.34 V (18)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = – 0.56 V (19) NOT Spontaneous

Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in DG (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

Complex Name Half Reaction Reduction Potential
Cytochrome reductase

(also known as cytochrome b-c1 complex)

(3 in Figure 9)

Cytochrome b (Fe(III) center)
+ e –>
Cytochrome b (Fe(II) center)
-0.34 V
(at pH 7, T=30oC)
Cytochrome c1 (Fe(III) center)
+ e– –>
Cytochrome c1 (Fe(II) center)
+0.220 V
(at pH 7, T=30oC)
Cytochrome c

(4 in Figure 9)

Cytochrome c (Fe(III) center)
+ e– –>
Cytochrome c (Fe(II) center)
+0.250 V
(at pH 7, T=30oC)
Cytochrome oxidase

(5 in Figure 9)

Cytochrome oxidase
( Fe(III) center) + e– –>
Cytochrome oxidase
(Fe(II) center)
+0.285 V
(at pH 7.4, T=25oC)
Table 2

To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

Hence, the electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H+. As we shall see below, this huge concentration gradient leads to the production of ATP.

Questions on Electron Carriers: Steps in the Electron-Transport Chain; Reduction Potentials and Relationship to Free Energy

  • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
  • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H+ ions) in the intermembrane space is increased.  Could cells move protons (H+ ions) from the matrix to the intermembrane space without transporting electrons?  Why or why not?

 ATP Synthetase: Production of ATP

We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily-available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

ATP synthetase is a protein consisting of two important segments: a transmembrane proton channel, and a catalytic component located inside the matrix. The proton-channel segment allows H+ ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Thus, since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.

Questions on ATP Synthetase: Production of ATP

  • A scientist has created a phospholipid-bilayer membrane containing ATP-synthetase proteins. Instead of a proton gradient, this scientist has created a large Cs+ gradient (many Cs+ ions on the side of the membrane without the catalytic unit, and few Cs+ ions on the side of the membrane with the catalytic unit). Would you expect the ATP-synthetase proteins in this membrane to be able to generate ATP, given an abundant supply of ADP and phosphate? Briefly, explain your answer. (HINT: Draw on your knowledge of the structure of protein channels to predict what effect replacing H+ ions with Cs+ ions would have.)
  • Certain toxins allow H+ ions to move freely across the inner mitochondrial membrane (i.e., without needing to pass through the channel in ATP synthetase). What effect do you expect these toxins to have on the production of ATP? Briefly, explain your answer.

Summary

In this tutorial, we have learned that the ability of the body to perform daily activities is dependent on thermodynamic, equilibrium, and electrochemical concepts.   These activities, which are typically based on nonspontaneous chemical reactions, are performed by using free-energy currency. The common free-energy currency is ATP, which is a molecule that easily dephosphorylates (loses a phosphate group) and releases a large amount of free energy. In the body, the nonspontaneous reactions are coupled to this very spontaneous dephosphorylation reaction, thereby making the overall reaction spontaneous (DG < 0). As the coupled reactions occur (i.e., as the body performs daily activities), ATP is consumed and the body regenerates ATP by using energy from the food we eat (Figure 3). As seen in Figure 4, the breakdown of glucose (glycolysis) obtained from the food we eat cannot by itself generate the large amount of ATP that is needed for metabolic energy by the body. However, glycolysis and the subsequent step, the citric-acid cycle, produce two easily oxidized molecules: NADH and FADH2. These redox molecules are used in an oxidative-phosphorylation process to produce the majority of the ATP that the body uses. This oxidative-phosphorylation process consists of two steps: the oxidation of NADH (or FADH2) and the phosphorylation reaction which regenerates ATP. Oxidative phosphorylation occurs in the mitochondria, and the two reactions (oxidation of NADH or FADHand phosphorylation to generate ATP) are coupled by a proton gradient across the inner membrane of the mitochondria (Figure 9). As seen in Figures 7 and 9, the oxidation of NADH occurs by electron transport through a series of protein complexes located in the inner membrane of the mitochondria. This electron transport is very spontaneous and creates the proton gradient that is necessary to then drive the phosphorylation reaction that generates the ATP. Hence, oxidative-phosphorylation demonstrates that free energy can be easily transferred by proton gradients. Oxidative-phosphorylation is the primary means of generating free-energy currency for aerobic organisms, and as such is one of the most important subjects in the study of bioenergetics (the study of energy and its chemical changes in the biological world).

Additional Link:

  • This fun description of oxidative phosphorylation by Dr. E.J.Oakeley contains step-by-step animated illustrations of the redox reactions involved, as well as a quiz to test your understanding of the material.

References:

Alberts, B. et al. In Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.: New York, 1994, pp. 653-684.

Becker, W.M. and Deamer, D.W. In The World of the Cell, 2nd ed., The Benjamin/Cummings Publishing Co., Inc.: Redwood City, CA, 1991, pp. 291-307.

Fasman, G.D. In Handbook of Biochemistry and Molecular Biology, 3rd ed., CRC Press, Inc.: Cleveland, OH, 1976, Vol. I (Physical and Chemical Data), pp. 132-137.

Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.

Moa, C., Ozer, Z., Zhou, M. and Uckun, F. X-Ray Structure of Glycerol Kinase Complexed with an ATP Analog Implies a Novel Mechanism for the ATP-Dependent Gylcerol Phosphorylation by Glycerol Kinase.Biochemical and Biophysical Reaearch Communications. 1999, 259, 640-644.

Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.

Stryer, L. In Biochemistry, 4th. ed., W.H. Freeman and Co.: New York, 1995, pp. 490, 509, 513, 529-557.

Zubay, G. Biochemistry, 3rd. ed., Wm. C. Brown Publishers: Dubuque, IA, 1983, p. 42.

Acknowledgements:

The authors thank Dewey Holten (Washington University in St. Louis) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.

Copyright 1999, Washington University, All Rights Reserved.

 

 

 

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Studies of Respiration Lead to Acetyl CoA

Curator: Larry H. Bernstein, MD, FCAP

In this series of discussions it has become clear that the studies of carbohydrate metabolism were highlighted by Meyerhof’s work on the glycolytic pathway, and the further elucidation of a tie between Warburg’s studies of impaired respiration for malignant aerobic cells relying on glycolysis, comparanle to Pasteur’s observations 60 years earlier by for yeast.   The mitochondrion was unknown at the time, and it took many years to discover the key role played by oxidative phosphorylation and Fritz Lipmann’s discovery of “acetyl coenzyme A, and the later explanation of electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue.  I shall here embark on a journey to trace these important connected developments.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Phosphorylation

In some reactions, the purpose of phosphorylation is to “activate” or “volatize” a molecule, increasing its energy so it is able to participate in a subsequent reaction with a negative free-energy change. All kinases require a divalent metal ion such as Mg2+ or Mn2+ to be present, which stabilizes the high-energy bonds of the donor molecule (usually ATP or ATP derivative) and allows phosphorylation to occur.This is a major focus of this discussion.

In other reactions, phosphorylation of a protein substrate can inhibit its activity (as when AKT phosphorylates the enzyme GSK-3). When src is phosphorylated on a particular tyrosine, it folds on itself, and thus masks its own kinase domain, and is thus turned “off”. In still other reactions, phosphorylation of a protein causes it to be bound to other proteins which have “recognition domains” for a phosphorylated tyrosine, serine, or threonine motif. In the late 1990s it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. This is all that needs to be said at this time about proteins.

 

Oxidative Phosphorylation

ATP is the molecule that supplies energy to metabolism. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.

During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within the cell’s intermembrane wall mitochondria, whereas, in prokaryotes, these proteins are located in the cells’ intermembrane space.

O t to  W a r b u r g
Nobel Lecture, December 10, 1931

The oxygen-transferring ferment of respiration

The effects of iron are very great, and it follows that oxidation and reduction of the ferment iron must occur extremely rapidly. In fact, almost every molecule of oxygen that comes into contact with an atom of ferment iron reacts with it.  Complex-bound bivalent iron in compounds reacts, in vitro as well as in the cell, with molecular oxygen. tt is not yet possible to reduce in vitro trivalent iron with the cell fuel: it is always necessary to add a substance of unknown composition, a ferment, that activates the combustible material for the attack of the iron. It must, therefore, be concluded that activation of the combustible substance in the breathing cell precedes the attack of the ferment iron; this corresponds with “hydrogen activation” as postulated in the theory of Wieland and Thunberg. According to the results of a joint research with W. Christian, this is a cleavage comparable with those known as fermentation.

It is possible that the interplay of splitting ferment and oxygen-transferring ferment does not fully explain the mechanism of cellular respiration; that the iron that reacts with the molecular oxygen does not directly oxidize the activated combustible substances, but that it exerts its effects indirectly through still other iron compounds – the three non-auto-oxidizable cell hemes of MacMunn, which occur in living cells according to the spectroscopic observations of MacMunn and Keilin, and which are reduced in the cell under exclusion of oxygen. It is still not possible to answer the question whether the MacMunn hemes form part of the normal respiratory cycle, i.e., whether respiration is not a simple iron catalysis but a four-fold one. The available spectroscopic observations are also consistent with the view that the MacMunn hemes in the cell are only reduced when the concentration of activated combustible substance is physiologically above normal. This will suffice to indicate that oxygen transfer by the iron of the oxygen transferring ferment is not the whole story of respiration. Respiration requires not only oxygen-transferring ferment and combustible substance, but oxygen-transferring ferment and the living cell.

Inhibition of cellular respiration by prussic acid was discovered some 50 years ago by Claude Bernard, and has interested both chemists and biologists ever since. It takes place as the result of a reaction between the prussic acid and the oxygen-transferring ferment iron, that is, with the ferment iron in trivalent form. [In the prussic acid reaction] the oxidizing OH-group of the trivalent ferment-iron is replaced by the non-oxidizing CN-group, thus bringing transfer of oxygen to a standstill. Prussic acid inhibits reduction of the ferment iron. Inhibition of respiration by carbon monoxide was discovered only a few years ago. [Given] the initial reaction in respiration, then, in the presence of carbon monoxide, the competing reaction will also occur and, varying with the pressures of the carbon monoxide and of the oxygen, more or less of the ferment iron will be removed from the catalytic process on account of fixation of carbon monoxide to the ferment iron. Unlike prussic acid, therefore, carbon monoxide affects the bivalent iron of the ferment. Carbon monoxide inhibits oxidation of the ferment iron.

Thus inhibition of respiration by carbon monoxide, unlike that by prussic acid, depends upon the partial pressure of oxygen. The toxic action of prussic acid in the human subject is based on its inhibitory action on cellular respiration. The toxic effect of carbon monoxide on man has nothing to do with inhibition of cellular respiration by carbon monoxide but is based on the reaction of carbon monoxide with blood iron. For, the effect of carbon monoxide on blood iron occurs at pressures of carbon monoxide far from the level at which cellular respiration would be inhibited.

If carbon monoxide is added to the oxygen in which living cells breathe, respiration ceases, as has already been mentioned, but if exposure to ultraviolet or visible light is administered, respiration recurs. By alternate illumination and darkness it is possible to cause respiration and cessation of respiration in living, breathing cells in mixtures of carbon monoxide and oxygen. In the dark, the iron of the oxygen-transferring ferment becomes bound to carbon monoxide, whereas in the light the carbon monoxide is split off from the iron which is, thus, liberated for oxygen transfer. This fact was discovered in 1926 in collaboration with Fritz Kubowitz. Photochemical dissociation of iron carbonyl compounds was discovered in 1891 by Mond and Langer, by exposing iron pentacarbonyl. This reaction is specific for carbonyl compounds of iron, most of which appear to dissociate in the presence of light, e.g., carbon-monoxide hemoglobin (John Haldane, 1897) carbon-monoxide hemochromogen (Anson and Mirsky, 1925), carbon-monoxide pyridine hemochromogen (H. A. Krebs, 1928), and carbon-monoxide ferrocysteine (W. Cremer, 1929).

When the photochemical dissociation of iron carbonyl compounds is measured quantitatively (we followed hereby Emil Warburg’s photochemical experiments), by using monochromatic light and comparing the amount of light energy absorbed with the amount of carbon monoxide set free, it is found that Einstein’s law of photochemical equivalence is very exactly fulfilled. The number of FeCO-groups set free is equal to the number of light quanta absorbed, and this is independent of the wavelength employed.

Photochemical dissociation of iron carbonyl compounds can be used to determine the absorption spectrum of a catalytic oxygen-transferring iron compound. One combines the catalyst in the dark with carbon monoxide, and so abolishes the oxygen-transferring power of the iron. If then this is exposed to monochromatic light of various wavelengths and of measured quantum intensity, and the effect of light W measured the increase in the rate of catalysis – it is found that the effects of the light are proportional to the quanta absorbed. The arrangement becomes very simple if the catalyst is present, as is usually the case, in infinitesimally low concentration in the exposed system. Then the thickness of the layers related to the amount of absorption of light can be considered to be infinitely thin, the number of quanta absorbed is proportional to the number of quanta supplied by irradiation.

In collaboration with Erwin Negelein, this principle was employed to measure the relative absorption spectrum of the oxygen-transferring respiratory ferment. The respiration of living cells was inhibited by carbon monoxide which was mixed with the oxygen. We then irradiated with monochromatic light of various wavelengths and of measured quantum intensity, and [measured] the increase of respiration together with the relative absorption spectrum. Only practically colorless cells are suitable for this type of experiment, [which requires] a layer infinitely thin with regard to light absorption.

Imagine living cells whose respiration is inhibited by carbon monoxide. If these are irradiated, respiration does not increase suddenly from the dark to the light-value, but there is a definite, although very short, interval until the combination of carbon monoxide with the ferment is broken down by the light. Even without calculation, it is obvious that the rate of increase in the effect of light must be related to the depth of colour of the ferment. If the ferment absorbs strongly, the -monoxide compound will be rapidly broken down, and vice versa.

The time of increase of the action of light can be measured. The time taken for a given intensity of light to cause dissociation of approximately half the carbon-monoxide compound of the ferment can be measured and, from this time, and from the effective intensity of light, the absolute absorption coefficient of the ferment for every wavelength can be calculated. The absorption capacity of the ferment, measured in accordance with this principle, was found to be of the same order as the power of light absorption of our strongest pigments. If one imagines a ferment solution of molar concentration, a layer of 2 x 10-6 cm thickness would weaken the blue mercury line 436 µµ up by half. The fact that the ferment in spite of this cannot be seen in the cells is due to its low concentration.

Monochromators and color filters were used to isolate the lines from these sources of light. If the absorption coefficient is entered as a function of the wavelength, the absorption spectrum of the carbon-monoxide compound of the ferment is obtained. The principal absorption-band or y-band lies in the blue.
This is the spectrum of a heme compound, according to the position of the bands, the intensity state of the bands, and the absolute magnitude of the absorption coefficients.

It appeared essential to have a control to ascertain whether heme as an oxidation catalyst of carbon monoxide and prussic acid really behaves like the ferment. If cysteine is dissolved in water containing pyridine, and a trace of heme is added, and this is shaken with air, the cysteine is catalytically oxidized by the oxygen-transferring power of the heme. According to Krebs, the catalysis is inhibited by carbon monoxide in the dark, but the inhibition ceases when the mixture is illuminated. Prussic acid too acts on this model on cellular respiration, inasmuch as it combines with the trivalent heme and inhibits its reduction. Just as in life, inhibition by carbon monoxide is dependent on the oxygen pressure, while inhibition by prussic acid is independent of the oxygen pressure.

In conjunction with Negelein, this model was also used to test the ferment experiments quantitatively. Heme catalysis in the model was inhibited by carbon monoxide in the dark. Then monochromatic light of known quantum intensity was used to irradiate it, and the absorption spectrum of the catalyst calculated from the effect of the light which was known from direct measurements on the pure substance. The calculation gave the absorption spectrum of the heme that had been added as a catalyst, and so the method was verified as a technique for the determination of the ferment spectrum, both the calculation and the measurement method.

The positions of the principal band and a-band of the ferment are:

Principal band            α-band

433 µµ                    590 µµ

These will be referred to as the “ferment bands” because the ferment was the first for which they were determined. Hemes are the complex iron compounds of the porphyrins, in which two valencies of the iron are bound to nitrogen. The porphyrins, of which Hans Fischer determined the chemical structure, are tetrapyrrole compounds in which the four pyrrole nuclei are held together by four interposed methane groups in the cr-position. Green, red, and mixed shades of hemes are known. If magnesium is replaced by iron in chlorophyll, green hemes are obtained. Their color is due to a strong band in the red which is already recognized in chlorophyll. The ferment does not absorb in the red and cannot, therefore, be a green heme. Red hemes are the usual hemes in blood pigment and in its related substances, such as mesoheme and deuteroheme. Coproheme is also a red heme which is an iron compound of the coproporphyrin that H. Fischer recognized in the body. Other red hemes are 20 µµ further from the red than the ferment bands. It follows that the ferment is not a red heme.

The pheoporphyrins are closely related to blood pigment but, as H. Fischer showed, pheoporphyrin a is simply mesoporphyrin in which the one propionic acid has been oxidized so that ring closure with the porphyrin nucleus is made possible. Pheoporphyrin a is a reduction product of chlorophyll a or an oxidation product of blood pigment, and connects together, in an amazingly simple manner, the principal pigments of the organic world the blood pigment and the leaf pigment.

Chlorophyll b has, in general, bands of longer wavelength than chlorophyll a, and for this reason,

  1. Christian and I applied Fischer’s reduction method to it. In this way we obtained pheoheme b, which, when linked with protein, corresponds with the ferment in respect to the position of the principal band. The principal band of the carbon-monoxide compound of pheohemoglobin b is 435 µµ.
  2. However, while the principal band of pheohemoglobin corresponds with the ferment bands within the permitted limits, the α-band shifts so far beyond them because it lies too near the red. It is, nevertheless, interesting that
  3. when ‘chlorophyll b is reduced, one obtains a pheoporphyrin of which the heme of all the pheohemes that have been demonstrated up to the present time is the most like the ferment.

 

Still nearer the ferment in its spectrum, is a heme occurring in Nature. This is

  • spirographis heme, which has been isolated from chlorocruorin, the blood pigment of the bristle-worm Spirographis,

in collaboration with Negelein and Haas, the bands of spirographis heme, coupled to globin, are :

  • carbon-monoxyhemoglobin of spirographis:  principle band, 434 µµ; α-band, 594 µµ.

Spirographis heme differs from the red hemes by the surplus or ketone oxygen-atom, and is classified as pheoheme. Like Fischer’s pheohemes, spirographis heme is intermediate between chlorophyll and blood pigment in respect of

  • the degree of oxidation of the side-chains.

The two hemes with a spectrum most like that of the ferment – pheoheme b and spirographis heme – possess a remarkable property. If they are dissolved in dilute sodium-hydroxide solution, in the form of ferrous compounds,

  • the absorption bands slowly wander towards the blue, near the bands of blood heme. In this way,
  • mixed-color hemes have been converted into red hemes.

On acidification, the change reverts: the <<blood bands>> disappear and

  • the ferment bands appear.

This experiment shows that

  1. oxidation of the side-chains does not suffice to give rise to the ferment bands, but
  2. some process of the type of anhydride formation must also occur.

The unique intermediate status of the ferment-like hemes demonstrated by these simple experiments suggests

  1. the suspicion that blood pigment and leaf pigments have both arisen from the ferment –
  2. blood pigments by reduction, and leaf pigment by oxidation.
  • For evidently, the ferment existed earlier than hemoglobin and chlorophyll.

The investigations on the oxygen-transferring ferment have been supported from the start by the Notgemeinschaft der deutschen Wissenschaft and the Rockefeller Foundation, without whose help they could not have been carried out. I have to thank both organizations here.

A L B E R T S Z E N T- GY Ö R G Y I      Nobel Lecture, December 11, 1937

Oxidation, energy transfer, and vitamins

A living cell requires energy not only for all its functions, but also

  • for the maintenance of its structure.
  • The source of this energy is the sun’s radiation.

Energy from the sun’s rays is trapped by green plants, and

  • converted into a bound form, invested in a chemical reaction.

When sunlight falls on green-plants, they liberate oxygen from carbon dioxide, and

  1. store up carbon, bound to the elements of water, as carbohydrate.

The radiant energy is now locked up in this carbohydrate molecule. This molecule is our food. When energy is required,

  • the carbohydrate is again combined with oxygen to form carbon dioxide, oxidized, and energy released.

Investigations during the last few decades have brought hydrogen instead of carbon, and instead of CO2 water, the mother of all life, into the foreground. It is becoming increasingly probable that

  1. radiant energy is used primarily to break water down into its elements,
  2. while CO2, serves only to fix the elusive hydrogen thus released.

While this concept of energy fixation was still being developed, the importance of hydrogen in the reversal of this process, whereby energy is liberated by oxidation, had already been confirmed by H. Wieland’s experiments.

Our body really only knowns one fuel, hydrogen. The foodstuff, carbohydrate, is essentially a packet of hydrogen, a hydrogen supplier, a hydrogen donor, and the main event during its combustion is

  • the splitting off of hydrogen.

So the combustion of hydrogen is

  • the real energy-supplying reaction;

To the elucidation of reaction (6), which seems so simple, I have devoted all my energy for the last fifteen years.

When I first ventured into this territory, the foundations had already been laid by the two pioneers H. Wieland and
O. Warburg, and Wieland’s teaching had been applied by Th. Thunberg to the realm of animal physiology.Wieland and Thunberg showed, with regard to foodstuffs, how

  1. the first step in oxidation is the “activation” of hydrogen, whereby
  2. the bonds linking it to the food molecule are loosened, and
  3. hydrogen prepared for splitting off.

But at the same time oxygen is also, as Warburg showed,

  • activated for the reaction by an enzyme.
  • the hydrogen-activating enzymes are called dehydrases or dehydrogenases.

Warburg called his oxygen-activating catalyst, “respiratory enzyme”.These concepts of Wieland and Warburg were apparently contradictory, and

  1. my first task was to show that the two processes are complementary to one another, and that
  2. in muscle cells activated oxygen oxidizes activated hydrogen.

This picture was enriched by the English worker D. Keilin, who showed that

  • activated oxygen does not oxidize activated hydrogen directly, but
  • that a dye, cytochrome, is interposed between them.

In keeping with this function, the “respiratory enzyme” is now also called “cytochrome oxidase”.

About ten years ago, when I tried to construct this system of respiration artificially and added together the respiratory enzyme with cytochrome and some foodstuff together with its dehydrogenase, I could justifiably expect that this system would use up oxygen and oxidize the food. But the system remained inactive. I found that

  • the dehydrogenation of certain donors is linked to the presence of a co-enzyme.

Analysis of this co-enzyme showed it to be a nucleotide, identical with v. Euler’s co-zymase, which H. v. Euler and R. Nilsson had already shown to accelerate the process of dehydration. As a result of Warburg’s investigations,this co-dehydrogenase has recently come very much into the foreground. Warburg showed that

  • it contains a pyridine base, and that it accepts hydrogen directly
    [pyridine nucleotide, triphosphopyridine nucleotide, TPN]

from food when the latter is dehydrogenated. It is therefore, the primary H-acceptor.

While working on the isolation of the co-enzyme with Banga, I found a remarkable dye, which showed clearly by its reversible oxidation that it, too, played a part in the respiration. We called this new dye cytoflav. Later Warburg showed that

  • this substance exercised its function in combination with a protein.

He called this protein complex of the dye, “yellow enzyme”. R. Kuhn, to whom we owe the structural analysis of the dye, called the dye lactoflavin and, with Györgyi and Wagner-Jauregg, showed it to be identical with vitamin B,.But the respiratory system stayed inactive even

  • after the addition of both these new components, codehydrogenase and yellow enzyme.

The C4-dicarboxylic acids and their activators which Thunberg discovered are

  • interposed between cytochrome and the activation of hydrogen as intermediate hydrogen-carriers.

In the case of carbohydrate, hydrogen from the food is first taken up by oxaloacetic acid, which

  • is reacted with the cytoplasmic malic dehydrogenase (and pyridine nucleotide –
    reduced DPN[H])
    , and thereby activated.

By taking up two hydrogen atoms, oxaloacetic acid is changed into malic acid.

  • OAA + NADH – (MDH) – malate + NAD+ + H+

This malic acid now passes on the H-atoms, and thus reverts to oxaloacetic acid,

  • which can again take up new H-atoms.

Malate + NAD+ + H+ — MDH – OAA + NADH

The H-atoms released by malic acid are taken up by fumaric acid, which is similarly

  • activated by the so-called succinic dehydrogenase.

The uptake of two H-atoms

  • converts the fumarate to succinate, to succinic acid.

The two H-atoms of succinic acid are then

  • oxidized away by the cytochrome.

Finally the cytochrome is oxidized by the respiratory enzyme, and

  • the respiratory enzyme by oxygen.

The function of the C4-dicarboxylic acids is not to be pictured as consisting of a certain amount of C4-dicarboxylic acid in the cell which is alternately oxidized and reduced. Fig. 2 corresponds more to the real situation. The protoplasmic surface, which is represented by the semi-circle, has single molecules of oxaloacetate and fumarate attached to it as prosthetic groups. These fused, activated dicarboxylic molecules then temporarily bind the hydrogen from the food. The co-dehydrogenases and the yellow enzymes also take part in this system. I have attempted to add them in at the right place.

This diagram, which will probably still undergo many more modifications, states that the “foodstuff” – H-donor – starts by

  1. passing its hydrogen, which has been activated by dehydrase, to the co-dehydrogenase.
  2. The coenzyme passes it to the oxaloacetic acid*.
  3. The malic acid then passes it on again to a co-enzyme,
  4. which passes the hydrogen to the yellow enzyme.
  5. The yellow enzyme passes the hydrogen to the fumarate.
  6. The succinate so produced is then oxidized by cytochrome,
  7. the cytochrome by respiratory enzyme,
  8. the respiratory enzyme by oxygen.

So the reaction 2H + O – H2O, which seems such a simple one,

  • breaks down into a long series of separate reactions.

With each new step, with each transfer between substances,

  • the hydrogen loses some of its energy,
  • finally combining with oxygen in its lowest-energy compound.

So each hydrogen atom is gradually oxidized in a long series of reactions, and

  • its energy released in stages.

This oxidation of hydrogen in stages seems to be one of the basic principles of biological oxidation. The reason for it is probably mainly that

  • the cell would not be able to harness and transfer to other processes
  • the large amount of energy which would be released by direct oxidation.

The cell needs small change if it is to be able to

  • pay for its functions without losing too much in the process.

So it oxidizes the H-atom by stages, converting the large banknote into small change.

About half of all plants – contain a polyphenol, generally a pyrocatechol derivative, together with an enzyme, polyphenoloxidase, which oxidizes polyphenol with the help of oxygen. The current interpretation of the mode of action of this oxidase was a confused one. I succeeded in showing that the situation was simply this, that

the oxidase oxidizes the polyphenol to quinone with oxygen.

  • In the intact plant the quinone is reduced back again
  • with hydrogen made available from the foodstuff.

Phenol therefore acts as a hydrogen-carrier between oxygen and the H-donor, and we are here again faced with a probably still imperfectly understood system for

  • the stepwise combustion of hydrogen.

——————————————————————————————————————————–

Vitamin C

If benzidine is added to a peroxide in the presence of peroxidase, a deep-blue color appears immediately, which is caused by the oxidation of the benzidine. This reaction does not occur without peroxidase. I simply used some juice which had been squeezed from these plants instead of a purified peroxidase, and added benzidine and peroxide, and the blue pigment appeared, after a small delay of about a second. Analysis of this delay showed that it was due to the presence of a powerful reducing substance, which reduced the oxidized benzidine again, until it had itself been used up. Thanks to the invitation from F. G. Hopkins and the help of the Rockefeller Foundation, I was able ten years ago to transfer my workshop to Cambridge, where for the first time I was able to pay more serious attention to chemistry. Soon I succeeded in isolating the substance in question from adrenals and various plants, and in showing that it corresponded to the formula C6H8O6 and was related to the carbohydrates. This last circumstance induced me to apply to Prof. W. N. Haworth, who immediately recognized the chemical interest of the substance and asked me for a larger quantity to permit analysis of its structure.

The Mayo Foundation and Prof. Kendall came to my help on a large scale, and made it possible for me to work, regardless of expense, on the material from large American slaughter-houses. The result of a year’s

work-was 25 g of a crystalline substance, which was given the name “hexuronic acid”. I shared this amount of the substance with Prof. Haworth. He undertook to investigate the exact structural formula of the substance. I used the other half of my preparation to gain a deeper understanding of the substance’s function. The substance could not replace the adrenals, but caused the disappearance of pigmentation in patients with Addison’s disease.

In 1930 I settled down in my own country at the University of Szeged. I also received a first-rate young American collaborator, J. L. Svirbely, who had experience in vitamin research, but besides this experience brought only the conviction that my hexuronic acid was not identical with vitamin C. In the autumn of 1931 our first experiments were completed, and showed unmistakably that hexuronic acid was power- fully anti-scorbutic, and that the anti-scorbutic acitvity of plant juices corresponded to their hexuronic acid content. We did not publish our results till the following year after repeating our experiments. At this time Tillmans was already directing attention to the connection between the reducing strength and the vitamin activity of plant juices. At the same time King and Waugh also reported crystals obtained from lemon juice, which were active anti-scorbutically and resembled our hexuronic acid.

My town, Szeged, is the centre of the Hungarian paprika industry. Since this fruit travels badly, I had not had the chance of trying it earlier. The sight of this healthy fruit inspired me one evening with a last hope, and that same night investigation revealed that this fruit represented an unbelievably rich source of hexuronic acid, which, with Haworth, I re-baptized ascorbic acid. I also had the privilege of providing my two prize-winning colleagues P. Karrer and W. N. Haworth with abundant material, and making its structural analysis possible for them. I myself produced with Varga the mono-acetone derivative of ascorbic acid, which forms magnificent crystals; from which, after repeated dissolving and recrystallization, ascorbic acid can be separated again with undiminished activity. This was the first proof that ascorbic acid was identical with vitamin C.
————————————————————————————————————————————-

Returning to the processes of oxidation, I now tried to analyse further the system of respiration in plants, in which ascorbic acid and peroxidase played an important part. I had already found in Rochester that the peroxidase plants contain an enzyme which reversibly oxidizes ascorbic acid with two valencies in the presence of oxygen. Further analysis showed that here again a system of respiration was in question, in which hydrogen was oxidized by stages. I would like, in the interests of brevity, to summarize the end result of these experiments, which I carried out with St. Huszák. Ascorbic acid oxidase oxidizes the acid with oxygen to reversible dehydroascorbic acid, whereby the oxygen unites with the two labile H-atoms from the acid to form hydrogen peroxide. This peroxide reacts with peroxidase and oxidizes a second molecule of ascorbic acid. Both these molecules of dehydro-ascorbic acid again take up hydrogen from the foodstuff, possibly by means of SH-groups. But peroxidase does not oxidize ascorbic acid directly. Another substance is interposed between the two, which belongs to the large group of yellow, water-soluble phenol-benzol-r-pyran plant dyes (flavone, flavonol, flavanone). Here the peroxidase oxidizes the phenol group to the quinone, which then oxidizes the ascorbic acid directly, taking up both its H-atoms.

At the time that I had just detected the rich vitamin content of the paprika, I was asked by a colleague of mine for pure vitamin C. This colleague himself suffered from a serious haemorrhagic diathesis. Since I still did not have enough of this crystalline substance at my disposal then, I sent him paprikas. My colleague was cured. But later we tried in vain to obtain the same therapeutic effect with pure vitamin C. Guided by my earlier studies into the peroxidase system, I investigated with my friend St. Rusznyák and his collaborators Armentano and Bentsáth the effect of the other link in the chain, the flavones. Certain members of this group of substances, the flavanone hesperidin (Fig. 5) and the formerly unknown eriodictyolglycoside, a mixture of which we had isolated from lemons and named citrin, now had the same therapeutic effect as paprika itself.

H U G O T H E O R E L L          Nobel Lecture, December 12, 1955

The nature and mode of action of oxidation enzymes

 

Practically all chemical reactions in living nature are started and directed in their course by enzymes. This being the case, Man has of course since time immemorial seen examples of what we now call enzymatic reactions, e.g. fermentation and decay. It would thus be possible to trace the history of enzymes back to the ancient Greeks, or still further for that matter. But it would be rather pointless, since to observe a phenomenon is not the same thing as to explain it. It is more correct to say that our knowledge of enzymes is essentially a product of twentieth-century research.

Jöns Jacob Berzelius, wrote in his yearbook in 1835: “…The catalytic force appears actually to consist thought herein that through their mere presence, and not through their affinity, bodies are able to arouse affinities which at this temperature are slumbering…”  Enzymes are the catalyzers of the biological world, and Berzelius’ description of catalytic force is surprisingly far-sighted…  if one could once understand the mechanism it would doubtless prove that the forces of ordinary chemistry would suffice to explain also these as yet mysterious reactions.

The year 1926 was a memorable one. The German chemist Richard Willstitter gave a lecture then in Deutsche Chemische Gesellschaft, in which he summarized the experiences gained in his attempts over many years to produce pure enzymes. Willstätter drew the conclusion that the enzymes did not belong to any known class of chemical substances, and that the effects of the enzymes derived from a new natural force, thus taking the view that 90 years earlier Berzelius thought to be improbable. That same year, through an irony of fate, the American researcher J. B. Sumner published a work in which he claimed to have crystallized in pure form an enzyme, urease. In the ensuing years J. H. Northrop and his collaborators crystallized out a further three enzyme preparations, pepsin, trypsin, and chymotrypsin, like urease, hydrolytic enzymes that split linkages by introducing water. If these discoveries had been undisputed from the outset it would probably not have been 20 years before Sumner, together with Northrop and Stanley, received a Nobel Prize.

When in 1933 I went on a Rockefeller fellowship to Otto Warburg’s institute in Berlin, Warburg and Christian had in the previous year produced a yellow-coloured preparation of an oxidation enzyme from yeast. The yellow colour was of particular interest: it faded away on reduction and returned on oxidation with e.g. oxygen, so that it was evident that the yellow pigment had to do with the actual enzymatic process of oxido-reduction. It was possible to free the yellow pigment from the high-molecular carrier substance, whose nature was still unknown, for example by treatment with acid methyl alcohol, whereupon the enzyme effect disappeared. Through simultaneous works by Warburg in Berlin, Kuhn in Heidelberg and Karrer in Zurich the constitution of the yellow pigment (lactoflavin, later riboflavin or vitamin B,) was determined. It was here for the first time possible to localize the enzymatic effect to a definite atomic constellation: hydrogen freed from the substrate (hexose monophosphate) is, with the aid of a special enzyme system (TPN-Zwischenferment) whose nature was elucidated somewhat later, placed on the nitrogen atoms of the flavin (1) and (10), giving rise to the colourless leucoflavin. This is reoxidized by oxygen, hydrogen peroxide being formed, and may afterwards be reduced again, and so forth. This cyclic process then continues until the entire amount of substrate has been deprived of two hydrogen atoms and been transformed into phosphogluconic acid; and a corresponding amount of hydrogen peroxide has been formed. At the end of the process the yellow enzyme is still there in unchanged form, and has thus apparently, as Berzelius expressed himself, aroused a chemical affinity through its mere presence.

The polysaccharides, which constituted 80-90% of the entire weight, were completely removed, together with some inactive colourless proteins. After fractionated precipitations with ammonium sulphate I produced a crystalline preparation which on ultracentrifuging and electrophoresis appeared homogeneous. The enzyme was a protein with the molecular weight 75,000 and strongly yellow-colored by the flavin part. The result of the Flavin analysis was 1 mol flavin per 1 mol protein. With dialysis against diluted hydrochloric acid at low temperature the yellow pigment was separated from the protein, which then became colorless. In the enzyme test the flavin part and the protein separately were inactive, but if the flavin part and the protein were mixed at approximately neutral reaction the enzyme effect returned, and the original effect came back when one mixed them in the molecular proportions 1 : 1. That in this connection a combination between the pigment and the protein came about was obvious, moreover, for other reasons: the green-yellow colour of the flavin part changed to pure yellow,and its strong. yellow fluorescence disappeared with linking to the protein.

In my electrophoretic experiments lactoflavin behaved as a neutral body, while the pigment part separated from the yellow enzyme moved rapidly towards the anode and was thus an acid. An analysis for phosphorus showed 1 P per mol flavin, and when after a time (1934) I succeeded in isolating the natural pigment component this proved to be a lactoflavin phosphoric acid ester, thus a kind of nucleotide, and it was obvious that the phosphoric acid served to link the pigment part to the protein. I will now show some simple experiments with the yellow enzyme, its colored part, which we now generally refer to as FMN (flavin mononucleotide), and the colorless enzyme protein.

  • The ferment-solution is pure yellow, the FMN-solution green-yellow,owing to the 1st that the light-absorption band in the blue of the free FMN is displaced somewhat in the long-wave direction on being linked with the protein component. A reducing agent (Na2S2O4) is now added to the one cuvette, it is indifferent which. The colour disappears in consequence of the formation of leucoflavin. Oxygen-gas is bubbled through the solution: the colour comes back as soon as the excess of reducing agent has been consumed. The experiment demonstrates the reaction cycle of the yellow enzyme: reduction through hydrogen from the substrate side, reoxidation with oxygen-gas.
  • A flask containing FMN-solution so diluted that its yellow color is not descernible to the eye is placed on a lamp giving long-wave ultraviolet light. The solution gives a strong, yellow fluorescence which disappears on reduction and returns on bubbling with oxygen-gas.
  • Two flasks are placed on the fluorescence lamp. The one contains a diluted solution of the free protein in phosphate buffer (pH 7), the other phosphate buffer alone. An equal amount of FMN-solution is dripped into each flask. In the flask with protein the fluorescence is at once extinguished,

but in the flask with buffer-solution alone it remains. The experiment demonstrates the resynthesis of  yellow enzyme, and since the fluorescence is extinguished by the protein, one may draw the conclusion that some group in the protein is in this connection linked to the imino-group NH(3) of the flavin, which according to Kuhn must be free for the fluorescence to appear.

The significance of these investigations on the yellow enzyme may be summarized

as follows.

  1. The reversible splitting of the yellow enzyme to apo-enzyme + coenzyme in the simple molecular relation 1 : 1 proved that we had here to do with a pure enzyme; the experiments would have been incomprehensible if the enzyme itself had been only an impurity.
  2. This enzyme was thus demonstrably a protein. In the sequel all the enzymes which have been isolated have proved to be proteins.
  3. The first coenzyme, FMN, was isolated and found to be a vitamin phosphoric acid ester. This has since proved to be something occurring widely in nature: the vitamins nicotinic acid amide, thiamine and pyridoxine form in an analogous way nucleotide-like coenzymes, which like the nucleic acids

themselves combine reversibly with proteins.

During the past 20 years a large number of flavoproteins with various enzyme effects have been produced. Instead of FMN many of them contain a dinucleotide, FAD, which consists of FMN + adenylic acid.

We constructed a very sensitive apparatus to record changes in the intensity of the fluorescence, and were thus able to follow the rapidity with which the fluorescence diminishes when FMN and protein are combined, or increases when they are split. Under suitable conditions the speed of combination is very high. Thanks to the great sensitivity of the fluorescent method my Norwegian collaborator Agnar Nygaard and I were able to make accurate determinations of the speed-constant simply by working in extremely diluted solutions, where the speed of combination is low because an FMN molecule so seldom happens to collide with a protein-molecule. We then varied the degree of acidity, ionic milieu and temperature, and we treated the protein with a large number of different reagents which affect in a known way different groups in proteins. In this way we succeeded with a rather high degree of certainty in ascertaining that phosphoric acid in FMN is linked to primary amino-groups in the protein, and the imino-group (3) in FMN to the phenolic hydroxyl group in a tyrosine residue, whereby the fluorescence is extinguished.

We still do not quite understand how through its linkage to the coenzyme the enzyme-protein “activates” the latter to a rapid absorption and giving off of hydrogen. But something we do know. The so-called oxido-reduction potential of the enzyme is in any case of great importance, and it is determined by a simple relation to the dissociation constants for the oxidized and for the reduced coenzyme-enzyme complex. The dissociation constants are in their turn functions of the velocity constants for the combination between coenzyme and enzyme and for the reverse process, and these velocity constants we have been able to determine both in the yellow ferment and in a number of enzyme systems. Without going into any details I may mention that the linkage of coenzyme to enzyme was found to have surprisingly big effects upon the potential of the former.

Alcohol dehydrogenase

 

Alcohol dehydrogenases occur in both the animal and the vegetable kingdoms, e.g. in liver, in yeast, and in peas. They are colourless proteins which together with DPN may either oxidize alcohol to aldehyde, as occurs chiefly in the liver, or conversely reduce aldehyde to alcohol, as occurs in yeast.

The yeast enzyme was crystallized by Negelein & Wulf (1936) in Warburg’s institute, the liver enzyme (from horse liver) by Bonnichsen & Wassén at our institute in Stockholm in 1948. These two enzymes have come to play a certain general rôle in biochemistry on account of the fact that it has been possible to investigate their kinetics more accurately than is the case with other enzyme systems. The liver enzyme especially, we have on repeated occasions studied with particular thoroughness, since especially favourable experimental conditions here presented themselves. For all reactions with DPN-system it is possible to follow the reaction DPN+ + 2H =+ DPNH + H+ spectrophotometrically, since DPNH has an absorption-band in the more long-wave ultraviolet region, at 340 rnp, and thousands of such experiments have been performed all over the world. A couple of years ago, moreover, we began to apply our fluorescence method, which is based on the fact that DPNH but not DPN fluoresces, even if considerably more weakly than the flavins. Asregards the liver enzyme there is a further effect, which proved extremely useful for certain spectrophotometrical determinations of reaction speeds; together with Bonnichsen I found in 1950 that the 340 rnp band of the reduced coenzyme was displaced, on combination with liver alcohol dehydrogenase, to 325 rnp, and together with Britton Chance we were thus able with the help of his extremely refined rapid spectrophotometric methods to determine the velocity constant for this very rapid reaction. This reaction belongs to the 3 bost problem involving the enzyme, the coenzyme, and the substrate, and both the coenzyme and the substrate occur in both oxidized and reduced forms.

It is a curious whim of nature that the same coenzyme which in the yeast makes alcohol by attaching hydrogen to aldehyde also occurs in the liver to remove from alcohol the same hydrogen, so that the alcohol becomes aldehyde again, which is then oxidized further
————————————————————————————————————————————–
Heme proteins

In 1936 we had obtained cytochrome approximately 80% pure, and in 1939 close to 100%.It is a beautiful red, iron-porphyrin-containing protein which functions as a link in the chain of the cell-respiration enzymes, the iron atom now taking up and now giving off an electron, and the iron thus alternating valency between the 3-valent ferri and the 2-valent ferro stages. It is a very pleasant substance to work with, not merely because it is lovely to look at, but also because it is uncommonly stable and durable. From 100 kg horse heart one can produce 3-4 grams of pure cytochrome c. The molecule weighs about 12,000 and contains one mol iron porphyrin per-mol.

Exp. 4. Two cuvettes each contain a solution of ferricytochrome c. The colour is blood-red. To the one are added some grains of sodium hydrosulphite: the color is changed to violet-red (ferrocytochrome). Oxygen is now bubbled through the ferrocytochrome-solution: no visible change occurs. The ferrocyto-chrome can thus not be oxidized by oxygen. A small amount of cytochrome oxidase is now added: the ferricytochrome color returns.

From this experiment we can draw the conclusion that reduced cytochrome c cannot react with molecular oxygen. In a chain of oxidation enzymes it will thus not be able to be next to the oxygen. The incapacity of cytochrome to react with oxygen was a striking fact that required an explanation. Another peculiarity was the extremely firm linkage between the red heme pigment and the protein part; in contradistinction to the majority of other heme protides, the pigment cannot be split off by the addition of acetone acidified with hydrochloric acid. Further, there was a displacement of the light-absorption bands which indicated that the two unsaturated vinyl groups occurring in ordinary protohemin were saturated in the hematin of

the cytochrome. In 1938 we succeeded in showing that the porphyrin part of the cytochrome was linked to the protein by means of two sulphur bridges from cysteine residues in the protein of the porphyrin in such a way that the vinyl groups were saturated and were converted to α-thioether groups. The firmness of the linkage and the displacement of the spectral bands were herewith explained. This was the first time that it had been possible to show the nature of chemical linkages between a “prosthetic” group (in this case iron porphyrin) and the protein part in an enzyme.

The light-absorption bands of the cytochrome showed that it is a so-called hemochromogen, which means that two as a rule nitrogen-containing groups are linked to the iron, in addition to the four pyrrol-nitrogen atoms in the porphyrin. From magnetic measurements that I made at Linus Pauling’s institute in Pasadena and from amino-acid analyses, titration curves and spectrophotometry together with Å. Åkeson it emerged (1941) that the nitrogen-containing, hemochromogen-forming groups in cytochrome c were histidine residues, or to be more specific, their imidazole groups.   Recently we have got a bit farther. Tuppy & Bodo in Vienna began last year with Sanger’s method to elucidate the amino-acid sequence in the hemin-containing peptide fragment that one obtains with the proteolytic breaking down of cytochrome c, and succeeded in determining the sequence of the amino acids nearest the heme. The experiments were continued and supplemented by Tuppy, Paléus & Ehrenberg at our institute in Stockholm with the following result:

The peptide chain 1-12 (“Val”) = the amino acid valine, “Glu” = glutamine,”Lys” = lysine, and so forth) is by means of two cysteine-S-bridges and a linkage histidine-Fe linked to the heme. When in 1954 Linus Pauling delivered his Nobel Lecture in Stockholm he showed a new kind of models for the study of the steric configuration of peptide chains, which as we know may form helices or “pleated sheets” of various kinds. It struck me then that it would be extremely interesting to study the question as to which of these possibilities might be compatible with the sulphur bridges to the hemin part and with the linkage of nitrogen containing groups to the iron. Pauling was kind enough to make me a present of his peptide-model pieces, which I shall show presently. This is thus the second time they figure in a Lecture.

Anders Ehrenberg and I now made a hemin model on the same scale as the peptide pieces and constructed models of hemin peptides with every conceivable variant of hydrogen bonding. It proved that many variants could be definitely excluded on steric grounds, and others were improbable for other reasons. Of the original, at least 20 alternatives, finally only one remained – a left-twisting a-helix with the cysteine residue no. 4 linked to the porphyrin side-chain in 4-position, and cysteine no. 7 to the side-chain in 2-position. The imidazole residue fitted exactly to linkage with the iron atom. The peptide spiral becomes parallel with the plane of the heme disc.

Through calculations on the basis of the known partial specific volume of the cytochrome we now consider it extremely probable that the heme plate in cytochrome c is surrounded by peptide spirals on all sides in such a way that the heme iron is entirely screened off from contact with oxygen; here is the explanation of our experiment in which we were unable to oxidize reduced cytochrome c with oxygen-gas. The oxygen simply cannot get at the iron atom. There is, on the other hand, a possibility for electrons to pass in and out in the iron atom via the imidazole groups.  It strikes us as interesting that even at this stage the special mode of reacting of the cytochrome is beginning to be understood from what we know of its chemical constitution.

F r i t z  L i p m a n n           Nobel Lecture, December 11, 1953

Development of the acetylation problem: a personal account

 

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation. For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1. The decision to explore this particular reaction started me on a rather continuous journey into partly virgin territory to meet with some unexpected discoveries, but also to encounter quite a few nagging disappointments

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, to my surprise, as shown in Fig. 1, pyruvate oxidation was very slow, but the addition of a little phosphate caused a  remarkable increase in rate. The next figure, Fig. 2, shows the phosphate effect more drastically, using a preparation from which all phosphate was removed by washing with acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate. In spite of such a phosphate dependence, the phosphate balance measured by the ordinary Fiske-Subbarow procedure did not at first indicate any phosphorylative step. Nevertheless, the suspicion remained that phosphate in some manner was entering into the reaction and that a phosphorylated intermediary was formed. As a first approximation, a coupling of this pyruvate

oxidation with adenylic acid phosphorylation was attempted. And, indeed, addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate (Table 11). In parallel with the then just developing fermentation now concluded that the missing link in the reaction chain was acetyl phosphate. In partial confirmation it was shown that a crude preparation of acetyl phosphate, synthesized by the old method of Kämmerer and Carius2

would transfer phosphate to adenylic acid (Table 2). However, it still took quite some time from then on to identify acetyl phosphate definitely as the initial product of the pyruvic oxidation in this system3,4.

At the time when these observations were made, about a dozen years ago, there was, to say the least, a tendency to believe that phosphorylation was rather specifically coupled with the glycolytic reaction. Here, however, we had found a coupling of phosphorylation with a respiratory system. This observation immediately suggested a rather sweeping biochemical significance, of transformations of electron transfer potential, respiratory or fermentative, to phosphate bond energy and therefrom to a wide range of biosynthetic reactions7.

There was a further unusual feature in this pyruvate oxidation system in that the product emerging from the process not only carried an energy-rich phosphoryl radical such as already known, but the acetyl phosphate was even more impressive through its energy-rich acetyl. It rather naturally became a contender for the role of “active” acetate, for the widespread existence of which the isotope experience had already furnished extensive evidence. I became, therefore, quite attracted by the possibility that acetyl phosphate could serve two rather different purposes, either to transfer its phosphoryl group into the phosphate pool, or to supply its active acetyl for biosynthesis of carbon structures. Thus acetyl phosphate should be able to serve as acetyl donor as well as phosphoryl donor, transferring, as shown in Fig. 3, on either side of the oxygen center, such as indicated by Bentley’s early experiments on cleavage7a of acetyl phosphate in H2 18O.

These two novel aspects of the energy problem, namely

(1) the emergence of an energy-rich phosphate bond from a purely respiratory reaction; and

(2) the presumed derivation of a metabolic building-block through this same there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first

focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated. reaction, prompted me to propose

  • not only the generalization of the phosphate bond as a versatile energy distributing system,
  • but also to aim there towards a general concept of transfer of activated groupings by carrier as the fundamental reaction in biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a metabolic intermediary first focussed attention to possible mechanisms for the metabolic elaboration of group activation, it soon turned out that the relationship between acetyl phosphate and acetyl transfer was much more complicated than anticipated.

It appeared that as an energy source the particle bound oxidative phosphorylation of the kind observed first by Herman Kalckar14 could be replaced by ATP, as had first been observed with the acetylation of choline in brain preparations by Nachmansohn and his group15,16. Using ATP and acetate as precursors, it was possible to set up a homogeneous particle-free acetylation system obtained by extraction of acetone pigeon liver. In this extract acetyl phosphate was unable to replace the ATP acetate as acetyl precursor.

In spite of this disappointment with acetyl phosphate, our decision to turn to a study of acetylation started then to be rewarding in another way. During these studies we became aware of the participation of a heat-stable factor which disappeared from our enzyme extracts on aging or dialysis. This cofactor was present in boiled extracts of all organs, as well as in microorganisms and yeast. It could not be replaced by any other known cofactor. Therefore, it was suspected that we were dealing with a new coenzyme. From then on, for a number of years, the isolation and identification of this coenzyme became the prominent task of our laboratory. The problem now increased in volume and I had the very good fortune that a group of exceedingly able people were attracted to the laboratory; first Constance Tuttle, then Nathan O. Kaplan and shortly afterwards, G. David Novelli, and then others.

Early data on the replacement of this heat-stable factor by boiled extracts are shown in the next table (Table 3). The pigeon liver acetylation system proved to be a very convenient assay system for the new coenzyme17 since on aging for 4 hours at room temperature, the cofactor was completely autolyzed.

Fortunately, on the other hand, the enzyme responsible for the decomposition of this factor was quite unstable and faded out during the aging, while the acetylation apoenzymes were unaffected.

The next figure, Fig. 4, shows coenzyme A (CoA) assay curves obtained with acetone pigeon liver extract. Finding pig liver a good source for the coenzyme, we set out to collect a reasonably large quantity of a highly purified preparation and then to concentrate on the chemistry with this material. In this analysis we paid particular attention to the possibility of finding in this obviously novel cofactor one of the vitamins.

The subsequence finding of a B-vitamin in the preparation gave us further confidence that we were dealing here with a key substance. We still felt, however, slightly dissatisfied with the proof for pantothenic acid. Therefore, to liberate the chemically rather unstable pantothenic acid from CoA, we made use of observations on enzymatic cleavage of the coenzyme. Two enzyme preparations, intestinal phosphatase and an enzyme in pigeon liver extract, had caused independent inactivation. It then was found that through combined action of these two enzymes, pantothenic acid was liberated18,19.

The two independent enzymatic cleavages indicated early that in CoA existed two independent sites of attachment to the pantothenic acid molecule. One of these obviously was a phosphate link, linking presumably to one of a hydroxyl group in pantothenic acid. The other moiety attached to pantothenic acid, which, cleaved off by liver enzyme, remained unidentified for a long time. In addition to pantothenic acid, our sample of 40 per cent purity had been found to contain about 2 per cent sulfur by elementary analysis and identified by cyanide-nitroprusside test as a potential SH grouping 20,21. Furthermore, the coenzyme preparation contained large amounts of adenylic acid21.

Units Coenzyme

Fig. 4. Concentration-activity curves for coenzyme A preparations of different purity. The arrow indicates the point of 1 unit on the curve. (o) crude coenzyme, 0.25 unit per mg; (x) purified coenzyme, 130 units per mg.

In the subsequent elaboration of the structure, the indications by enzyme analysis for the two sites of attachment to pantothenic acid have been most helpful. The phosphate link was soon identified as a pyrophosphate bridge22; 5-adenylic acid was identified by Novelli23 as enzymatic split product and by Baddiley 24, through chemical cleavage. At the same time, Novelli made observations which indicated the presence of a third phosphate in addition to the pyrophosphate bridge. These indications were confirmed by analysis of a nearly pure preparation which was obtained by Gregoryas from Streptomyces fradiae in collaboration with the research group at the Upjohn Company26.

It was at this period that we started to pay more and more attention to the sulfur in the coenzyme. As shown in Table 5, our purest preparation contained 4.13 per cent sulfur corresponding to one mole per mole of pantothenate. We also found26 that dephosphorylation of CoA yielded a compound containing pantothenic acid and the sulfur carrying moiety, which we suspected as bound through the carboxyl. Through the work of Snell and his group27, the sulfur-containing moiety proved to be attached to pantothenic acid through a link broken by our liver enzyme. It was identified as thioethanolamine by Snell and his group, linked peptidically to pantothenic acid.

Through analysis and synthesis, Baddiley now identified the point of attachment of the phosphate bridge to pantothenic acid in 4-position24 and Novelli et al.28 completed the structure analysis by enzymatic synthesis of “dephospho-CoA” from pantetheine-4’-phosphates and ATP. Furthermore, the attachment of the third phosphate was identified by Kaplan29 to attach in s-position on the ribose of the 5-adenylic acid (while in triphosphopyridine nucleotide it happens to be in 2-position). Therefore, the structure was now

established, as shown in Fig. 5.

Fig. 5. Structure of coenzyme A

 

The metabolic function of CoA


Parallel with this slow but steady elaboration of the structure, all the time we explored intensively metabolic mechanisms in the acetylation field. By use of the enzymatic assay, as shown in Tables 6, 7, 8, and 9, CoA was found present in all living cells, animals, plants and microorganisms17. Furthermore,

the finding that all cellular pantothenic acid could be accounted for by CoA17 made it clear that CoA represented the only functional form of this vitamin. The finding of the vitamin furnished great impetus; nevertheless, a temptation to connect the pantothenic acid with the acetyl transfer function has

blinded us for a long time to other possibilities.

The first attempts to further explore the function of CoA were made with pantothenic acid-deficient cells and tissues. A deficiency of pyruvate oxidation in pantothenic acid-deficient Proteus morganii, an early isolated observation by Dorfman30 and Hills31, now fitted rather well into the picture. We soon became quite interested in this effect, taking it as an indication for participation of CoA in citric acid synthesis. A parallel between CoA levels and pyruvate oxidation in Proteus morganii was demonstrated32. Using panto thenic aciddeficient yeast, Novelli et al.33 demonstrated a CoA-dependence of acetate oxidation (Fig. 5a) and Olson and Kaplan34 found with duck liver a striking parallel between CoA content and pyruvic utilization, which is shown in Fig. 6.

But more important information was being gathered on -the enzymatic level. The first example of a generality of function was obtained by comparing the activation of apoenzymes for choline- and sulfonamide-acetylation respectively, using our highly purified preparations9 of CoA. As shown in Fig. 7, similar activation curves obtained for the two respective enzymes. Through these experiments, the heat-stable factor for choline acetylation that had been found by Nachmansohn and Berman35 and by Feldberg and Mann36 was identified with CoA. The next most significant step toward a generalization of CoA function for acetyl transfer was made by demonstrating its functioning in the enzymatic synthesis of acetoacetate. The CoA effect in acetoacetate synthesis was studied by Morris Soodak37, who obtained for this reaction a reactivation curve quite similar to those for enzymatic acetylation, as shown in Fig. 8.

Soon afterwards Stern and Ochoa38 showed a CoA-dependent citrate synthesis with a pigeon liver fraction similar to the one used by Soodak for acetoacetate synthesis. In our laboratory, Novelli et al. confirmed and extended this observation with extracts of Escherichia coli39.

In the course of this work, which more and more clearly defined the acetyl transfer function of CoA, Novelli once more tried acetyl phosphate. To our surprise and satisfaction, it then appeared, as shown in Table 9, that in Escherichia coli extracts in contrast to the animal tissue, acetyl phosphate was more than twice as active as acetyl donor for citrate synthesis than ATP acetate 39. Acetyl phosphate, therefore, functioned as a patent microbial acetyl donor. Acetyl transfer from acetyl phosphate, like that from ATP-acetate, was CoA-dependent, as shown in Table 9. Furthermore, a small amount of “microbial conversion factor”, as we called it first, primed acetyl phosphate for activity with pigeon liver acetylation systems40, as shown in Table 10.

Eventually the microbial conversion factor was identified by Stadtman et al.40 with the transacetylase first encountered by Stadtman and Barker in extracts of Clostridium kluyveri41 and likewise, although not clearly defined as such, in extracts of Escherichia coli and Clostridium butylicum by Lipmann and Tuttle42. The definition of such a function was based on the work of Doudoroff et al.43 on transglucosidation with sucrose phosphorylase. Their imaginative use of isotope exchange for closer definition of enzyme mechanisms has been most influential. Like glucose-I-phosphate with sucrose phosphorylase, acetyl phosphate with these various microbial preparations equilibrates its phosphate rapidly with the inorganic phosphate of the solution. As in Doudoroff et al. experiments, first a covalent substrate enzyme derivative had been proposed 43. However, then Stadtman et al.40, with the new experience of CoA dependent acetyl transfer, could implicate CoA in this equilibration between acetyl- and inorganic phosphate and thus could define the transacetylase as an enzyme equilibrating acetyl between phosphate and CoA:

In the course of these various observations, it became quite clear that there existed in cellular metabolism an acetyl distribution system centering around CoA as the acetyl carrier which was rather similar to the ATP-centered phosphoryl distribution system. The general pattern of group transfer became recognizable, with donor and acceptor enzymes being connected through the CoA —- acetyl CoA shuttle. A clearer definition of the donor-acceptor enzyme scheme was obtained through acetone fractionation of our standard system for acetylation of sulfonamide into two separate enzyme fractions, which were inactive separately but showed the acetylation effect when combined. A fraction, A-40, separating out with 40 per cent acetone, was shown by Chou44 to contain the donor enzyme responsible for the ATP-CoA-acetate reaction, while with more acetone precipitated, the acceptor function, A-60, the acetoarylamine kinase as we propose to call this type of enzyme. The need for a combination of the two for overall acetyl transfer is shown in Fig. 9. This showed that a separate system was responsible for acetyl CoA formation through interaction of ATP, CoA and acetate (cf. below) and that the overall acetylation was a two-step reaction:

These observations crystallized into the definition of a metabolic acetyl transfer territory as pictured in Fig. 10. This picture had developed from the growing understanding of enzymatic interplay involving metabolic generation of acyl CoA and transfer of the active acyl to various acceptor systems. A most important, then still missing link in the picture was supplied through the brilliant work of Feodor Lynen45 who chemically identified acetyl CoA as the thioester of CoA. Therewith the thioester link was introduced as a new energy-rich bond and this discovery added a very novel facet to our understanding of the mechanisms of metabolic energy transformation.

Enzyme Localization In The  Anaerobic Mitochondria Of Ascaris L Umbricoides

 

Robert S. Rew And Howard J. Saz

From the Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556

 

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides   var. suum function anaerobically in electron transport-associated  phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria.  Succinate and pyruvate dehydrogenases as well as NADH  oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c  reductases  were  determined to be distributed  as  in mammalian mitochondria.  In contrast  with  the  mammalian systems, fumarase and NAD-linked “malic” enzyme were isolated primarily from the intermembrane  space fraction of the worm mitochondria. These enzymes required for the anaerobic  energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space.  The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.                                Downloaded from jcb.rupress.org

                                                                                                                                                      

                          

                               

                               

                               

                               

David Keilin’s Respiratory Chain Concept and its Chemiosmotic Consequences

Peter Mitchell              Nobel Lecture, 8 December, 1978

Glynn Research Institute, Bodmin, Cornwall, U. K.
“for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory”

Peter D. Mitchell (1920-1992) received the Nobel Prize in 1978 for developing the Chemiosmotic Theory to explain ATP synthesis resulting from membrane-associated electron transport [Ubiquinone and the Proton Pump].

Mitchell is the last of the gentleman scientists. He first proposed the chemiosmotic principle in a 1961 Nature article while he was at the University of Edinburgh. Shortly after that, ill health forced him to move to Cornwall where he renovated an old manor house and converted it into a research laboratory. From then on, he and his research colleague, Jennifer Moyle, continued to work on the chemiosmotic theory while being funded by his private research foundation. [Peter Mitchell: Wikipedia]

The Chemiosmotic Theory was controversial in 1978 and it still has not been fully integrated into some biochemistry textbooks in spite of the fact that it is now proven. The main reason for the resistance is that it overthrows much of traditional biochemistry and introduces a new way of thinking. It is a good example of a “paradigm shift” in biology.

Because he was such a private, and eccentric, scientist there are very few photos of Peter Mitchell or his research laboratory at Glynn House . The best description of him is in his biography Wandering in the Gardens of the Mind: Peter Mitchell and the Making of Glynn by John Prebble, and Bruce Weber. A Nature review by E.C. Slater [Metabolic Gardening] gives some of the flavor and mentions some of the controversy.

Wandering_in_the_Gardens_of_the_Mind_Peter_Mitchell

Wandering_in_the_Gardens_of_the_Mind_Peter_Mitchell

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Many scientists believe that the Chemiosmotic Theory was the second greatest contribution to biology in the 2oth century (after the discovery of the structure of DNA). Mitchell had to overcome many critics including Hans Krebs. The case is strong.

In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was created in the mitochondria was assumed to be by substrate-level phosphorylation. Mitchell’s chemiosmotic hypothesis was the basis for understanding the actual process of oxidative phosphorylation. At the time, the biochemical mechanism of ATP synthesis by oxidative phosphorylation was unknown.

Mitchell realised that the movement of ions across an electrochemical potential difference could provide the energy needed to produce ATP. His hypothesis was derived from information that was well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of positive to negative charges). Their movement is also affected by thermodynamic forces, the tendency of substances to diffuse from regions of higher concentration. He went on to show that ATP synthesis was coupled to this electrochemical gradient.[11]

His hypothesis was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP.

Growth, development and metabolism are some of the central phenomena in the study of biological organisms. The role of energy is fundamental to such biological processes. The ability to harness energy from a variety of metabolic pathways is a property of all living organisms. Life is dependent on energy transformations; living organisms survive because of exchange of energy within and without.

In a living organism, chemical bonds are broken and made as part of the exchange and transformation of energy. Energy is available for work (such as mechanical work) or for other processes (such as chemical synthesis and anabolic processes in growth), when weak bonds are broken and stronger bonds are made. The production of stronger bonds allows release of usable energy.

One of the major triumphs of bioenergetics is Peter D. Mitchell‘s chemiosmotic theory of how protons in aqueous solution function in the production of ATP in cell organelles such as mitochondria.[5] This work earned Mitchell the 1978 Nobel Prize for Chemistry. Other cellular sources of ATP such as glycolysis were understood first, but such processes for direct coupling of enzyme activity to ATP production are not the major source of useful chemical energy in most cells. Chemiosmotic coupling is the major energy producing process in most cells, being utilized in chloroplasts and several single celled organisms in addition to mitochondria.

Cotransport

In August 1960, Robert K. Crane presented for the first time his discovery of the sodium-glucose cotransport as the mechanism for intestinal glucose absorption.[2] Crane’s discovery of cotransport was the first ever proposal of flux coupling in biology and was the most important event concerning carbohydrate absorption in the 20th century.[3][4]

The free energy (ΔG) gained or lost in a reaction can be calculated: ΔG = ΔH – TΔS
where G = Gibbs free energy, H = enthalpy, T = temperature, and S = entropy.

How inositol pyrophosphates control cellular phosphate homeostasis?

Adolfo Saiardi*

Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, Department of Cell and Developmental Biology,

University College London, Gower Street, London WC1E 6BT, United Kingdom

Advances in Biological Regulation 52 (2012) 351–359

Phosphorus in his phosphate PO43_ configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control.

Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production.

Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate.

Two distinct classes of proteins the inositol hexakisphosphates kinases (IP6Ks) and the diphosphoinositol pentakisphosphate kinases (PP-IP5Ks or IP7Ks) are capable of synthesizing inositol pyrophosphates.

IP6Ks utilize ATP as a phosphate donor to phosphorylate IP6 to IP7, generation the isomer 5PP-IP5 (Fig. 1A), and inositol pentakisphosphate I(1,3,4,5,6)P5 to PP-IP4 (Saiardi et al., 1999, 2000; Losito et al., 2009). Furthermore, at least in vitro, IP6Ks generate more complex molecules containing two or more pyrophosphate moieties, or even three-phosphate species (Draskovic et al., 2008; Saiardi et al., 2001). Three IP6K isoforms referred to as IP6K1, 2, 3 exist in mammal; however, there is a single IP6K in the yeast Saccharomyces cerevisiae called Kcs1.

The PP-IP5Ks enzymes, synthesize inositol pyrophosphate from IP6, but not from IP5, (Losito et al., 2009) generating the isomer 1PP-IP5. Kinetic studies performed in vitro suggested that IP7, the 5PP-IP5 isomer generated by IP6Ks, is the primary substrate of this new enzyme, and this finding was confirmed in vivo by analysing PP-IP5K null yeast (vip1D) that accumulate the un-metabolized substrate IP7 (Azevedo et al., 2009; Onnebo and Saiardi, 2009). Thus PP-IP5K is responsible for IP8,

isomer 1,5PP2-IP4 synthesis (Fig. 1A). Two PP-IP5K isoforms referred to as PP-IP5Ka and b exist in mammal while a single PP-IP5K called Vip1 is present in S. cerevisiae.

Inositol pyrophosphates are hydrolysed by the diphosphoinositol-polyphosphate phosphohydrolases (DIPPs) (Safrany et al., 1998). Four mammalian enzymes DIPP1,2,3,4 have been identified, while only one DIPP protein exists in S. cerevisiae called Ddp1. These phosphatases are promiscuous enzymes able to hydrolyse inositol pyrophosphate as well as nucleotide analogues, such as diadenosine hexaphosphate (Ap6A) (Caffrey et al., 2000; Fisher et al., 2002). More recently, it has been shown that DIPPs also degrade polyP (Lonetti et al., 2011). Inositol pyrophosphates control the most disparate biological processes, from telomere length to vesicular trafficking. It is conceivable that all these function can be focused on the fact that inositol pyrophosphates are controlling cellular energy metabolism and consequently, ATP production. We have recently, demonstrated that inositol pyrophosphates control glycolysis and mitochondrial oxidative phosphorylation by both inhibiting the glycolytic flux and increasing mitochondrial activity (Szijgyarto et al., 2011).

Another important molecule to briefly introduce is polyP (Fig. 1B). The interested reader is encouraged to read the following comprehensive reviews (Kornberg et al., 1999; Rao et al., 2009). The polyP polymer likely represents a phosphate buffer that is synthesized and degraded in function of the phosphate needs of the cells. Furthermore, it also functions as a chelator of metal ions, thereby regulating cellular cation homeostasis. However, polyP also possesses more classical signalling roles.

In bacteria for example, it influences pathogenicity (Brown and Kornberg, 2008) and in mammalian cells it has been proposed to regulate fibrinolysis and platelet aggregation (Caen and Wu, 2010). In prokaryotes, polyP synthesis is carried out by a family of conserved polyP kinases (PPKs), whereas degradation is mediated by several polyP phosphatases (Rao et al., 2009). In higher eukaryotes polyP synthesis remains poorly characterized.

In humans alteration of phosphate metabolism is implicated in several pathological states. Higher serum phosphate leads to vascular calcification and cardiovascular complications. Although only very small amount of phosphate circulates in the serum, its concentration is tightly regulated and it is independent from dietary phosphorus intake (de Boer et al., 2009). Therefore, it is not surprising that intense research efforts are aimed to elucidate phosphate uptake and metabolism. IP6K2 was initially cloned while searching for a novel mammalian intestinal phosphate transporter that the group of Murer identified as PiUS (Phosphate inorganic Uptake Stimulator) (Norbis et al., 1997). Once transfected into Xenopus oocytes, PiUS stimulated the cellular uptake of radioactive phosphate.

Subsequently, two groups discovered that PiUS was capable of converting IP6 to IP7 and rename it to IP6K2 (Saiardi et al., 1999; Schell et al., 1999). The ability of inositol pyrophosphate to control the uptake of phosphate is an evolutionary conserved feature; in fact, kcs1D yeast with undetectable level of IP7 exhibits a reduced uptake of phosphate from the culture medium (Saiardi et al., 2004).

In mammals, regulation of phosphate homeostasis is not restricted to IP6K2, all three mammalian IP6Ks are likely to play a role. A genome-wide study aimed at identifying genetic variations associated with changes of serum phosphorus concentration identified IP6K3 (Kestenbaum et al., 2010). This human genetic study identified two independent single nucleotide polymorphisms (SNP) at locus 6p21.31, which are localised within the first intron of the IP6K3 gene. Interestingly, this study that analysed more than 16,000 humans identified SNP variant in only seven genes. Three of which, the sodium phosphate cotransporter type IIa, the calcium sensing receptor and the fibroblast growth factor 23, are well known regulators of phosphate homeostasis. These evidences support a role for IP6K3 in controlling serum phosphate levels in humans (Kestenbaum et al., 2010).

 

The hypothesis

 

Although, inositol pyrophosphate may have acquired unique organism-specific functions, the conserved ability of this class of molecules to regulate phosphate metabolism suggests an evolutionary ancient role. In this last paragraph, I will formulate few hypotheses that I hope will stimulate further research aimed at elucidating the biological link between phosphate, inositol pyrophosphates and polyP.

Inositol pyrophosphates regulate the entry of phosphate into the cells (Norbis et al., 1997), suggesting that they could affect phosphate uptake either directly (by stimulating a transporter, for example) or a indirectly by helping ‘fixing’ free phosphates in organic molecules. The cytosolic concentration of free phosphate [Pi] cannot fluctuate widely. Therefore, cellular entry of phosphates and its utilization may well be coupled. For example, the synthesis of polyP may be linked to phosphate entry in the cell. Inositol pyrophosphate control of energy metabolism (Szijgyarto et al., 2011) affects not only ATP levels but it can also alter the entire cellular balance of adenine nucleotides. Given that phosphate transfer reactions mainly use ATP as a vehicle for the phosphate groups, inositol pyrophosphate could affect phosphate metabolism by regulating the adenylate cellular pool. Moreover, it is tempting to speculate the existence of a feedback mechanism that coordinates the metabolic balance between ATP, phosphate and inositol pyrophosphates.

Inositol pyrophosphates could either contribute to the regulation of polyP synthesis, play a role in polyP degradation, or both. The yeast polyP polymerase has been identified with the subunit four (Vtc4) of the vacuolar membrane transporter chaperone (VTC) complex (Hothorn et al., 2009). Interestingly, pyrophosphates (Pi–Pi) dramatically accelerate the polyP polymerase reaction. It would therefore be interesting to determine whether the pyrophosphate moiety of IP7 can stimulate polyP vacuolar synthesis in a similar fashion. Similarly, it would be interesting to analyse the effect of inositol pyrophosphates on controlling the activity of the actin-like DdIPK2 enzyme. It should be noted however, that the existence even in yeast or Dictyostelium of other enzymes able to synthesize different polyP pools cannot be excluded. Thus, we will be able to validate and fully appreciate the role played by inositol pyrophosphates on polyP synthesis only after the identification of higher eukaryotes polyp synthesizing peptide/s.

The most abundant form of organic phosphate on earth is IP6, or phytic acid, a molecule that is highly abundant in plant seeds from which was originally characterised. In plant seeds, IP6 represents a phosphate storage molecule that it is hydrolysed during germination, releasing phosphates and cations. It will be an astonishing twist of event if inositol pyrophosphates were controlling the levels of their own precursor IP6 (Raboy, 2003), although due to the evolutionary conserved ability of inositol pyrophosphate to control phosphate homeostasis we should not be entirely surprised.

Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link.

Auesukaree C, Tochio H, Shirakawa M, Kaneko Y, Harashima S. Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae. J Biol Chem 2005;280:25127–33.

Azevedo C, Burton A, Ruiz-Mateos E, Marsh M, Saiardi A. Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc Natl Acad Sci U S A 2009;106:21161–6.

Bennett M, Onnebo SM, Azevedo C, Saiardi A. Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 2006;63:552–64.

Boer VM, Crutchfield CA, Bradley PH, Botstein D, Rabinowitz JD. Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations. Mol Biol Cell 2010;21:198–211.

Brown MR, Kornberg A. The long and short of it – polyphosphate, PPK and bacterial survival. Trends Biochem Sci 2008;33:284–90.

Burton A, Hu X, Saiardi A. Are inositol pyrophosphates signalling molecules? J Cell Physiol 2009;220:8–15.

Caen J, Wu Q. Hageman factor, platelets and polyphosphates: early history and recent connection. J Thromb Haemost 2010;8:1670–4.

Caffrey JJ, Safrany ST, Yang X, Shears SB. Discovery of molecular and catalytic diversity among human diphosphoinositol polyphosphate phosphohydrolases. An expanding Nudt family. J Biol Chem 2000;275:12730–6.

A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

Nathan J. Lanning,1 Brendan D. Looyenga,1,2 Audra L. Kauffman,1 Natalie M. Niemi,1 Jessica Sudderth,3

Ralph J. DeBerardinis,3 and Jeffrey P. MacKeigan1,*

Cell Reports   http://dx.doi.org/10.1016/j.celrep.2014.03.065

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels.

We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity.

Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Comments to be further addressed by JES Roselino

I will add some observations or at least one single observation.
Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity. This lead to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency, stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and increases glycogen phosphorylation as long as AMP levels are high).
I did everything I was able to do by the end of 1970 in order to repeat this assays with PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case. I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, have said it to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also have a faulty ability for recollection she also uses to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during “What is life” reading with him he asked me if from biochemist in exile, to biochemist I talked everything to him. Since I have considered that Schrödinger did not have confronted Darlington & Haldane for being in exile. Also, may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggest the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year(1971).

Another aspect I think you must call attention, in my opinion, is the following, show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

Yours,

JES Roselino

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Larry H. Benstein, MD, FCAP, Gurator and writer

https://pharmaceuticalintelligence.com/7/8/2014/Update on mitochondrial function, respiration, and associated disorders

This is a condensed account of very recent published work on respiration and disturbed mitochondrail function.  We know that their is an equilibrium between respiration and autophagy in eukaryotic cells.  The Krebs Cycle produces 32 ATPs in oxidative phosphorylation, which is far more efficient than glycolysis.  There is also a different contribution of mitochondrial metabolism, in the balance, between tissues that are synthetic and those that are catabolic.  This is a subject long understood, essential for cellular energetics, and not adequately explored.

 

Gain-of-Function Mutant p53 Promotes Cell Growth and Cancer Cell Metabolism via Inhibition of AMPK Activation.

Zhou G1Wang J2Zhao M2Xie TX2Tanaka N2, et al.
Mol Cell. 
2014 Jun 19;54(6):960-974.   doi: 10.1016/j.molcel.2014.04.024. 

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined.

We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells.

Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth.

Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation.

Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function. PMID:24857548

Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin.

Adekola KUAydemir SDMa SZhou ZRosen STShanmugam M.
Leuk Lymphoma. 2014 May 14:1-23.

Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity.

Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal.

Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation.

CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

KEYWORDS:  Basic Biology; Chemotherapeutic approaches; Lymphoid Leukemia; Signal transduction             PMID: 24828872

Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance.

Lee ZW1Teo XYTay EYTan CHHagen TMoore PKDeng LW.
Br J Pharmacol. 2014 May 15.    doi: 10.1111/bph.12773

Many disparate studies have reported the ambiguous role of hydrogen sulfide (H2 S) in cell survival. The present study investigated the effect of H2 S on viability of cancer and non-cancer cells.

Cancer and non-cancer cells were exposed to H2 S (using sodium hydrosulfide, NaHS and GYY4137) and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis process by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi) was determined by ratiometric pHi measurement using BCECF staining.

Continuous, but not single, exposure to H2 S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H2 S-releasing donor, GYY4137, significantly increased glycolysis leading to overproduction of lactate. H2 S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells.

Low and continuous exposure to H2 S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy.

KEYWORDS:  cancer cell death; cancer glucose metabolism; hydrogen sulfide; pH homeostasis          PMID: 24827113


Agonism of the 5-Hydroxytryptamine 1F Receptor Promotes Mitochondrial Biogenesis and Recovery from Acute Kidney Injury

Garrett SMWhitaker RMBeeson CC, and Schnellmann RG

Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (S.M.G., R.M.W., C.C.B., R.G.S.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina (R.G.S.)
Address correspondence to: Dr. Rick G. Schnellmann, Department of Drug Discovery and Biomedical Sciences, MUSC, Charleston, SC 29425.
E-mail: schnell@musc.edu

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB.

The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT1F receptor agonists,

  1. LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and
  2. LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM)

increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and

  • validation studies confirmed both agonists increased mitochondrial proteins  in vitro.
    [e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)]

Small interfering RNA knockdown of the 5-HT1F receptor

  • blocked agonist-induced MB.

Furthermore, LY344864 increased

  • peroxisome proliferator–activated receptor (PPAR) coactivator 1-α, Cox1, and
  • NDUFB8 transcript levels and
  • mitochondrial DNA (mtDNA) copy number

in murine renal cortex, heart, and liver.

Finally, LY344864 accelerated recovery of renal function, as indicated by

  • decreased blood urea nitrogen and kidney injury molecule 1 and
  • increased mtDNA copy number

following ischemia/reperfusion-induced acute kidney injury (AKI).

In summary, these studies reveal that

  • the 5-HT1F receptor is linked to MB, 5-HT1F receptor agonism promotes MB in vitro and in vivo, and

5-HT1F receptor agonism promotes recovery from AKI injury.

Induction of MB through 5-HT1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Footnotes

  • Portions of this work have been presented previously: Garrett SM, Wills LP, and Schnellmann RG (2012) Serotonin (5-HT) 1F receptor agonism as a potential treatment for acceleration of recovery from acute kidney injury.American Society of Nephrology Annual Meeting; 2012 Nov 1–4; San Diego, CA.
  • dx.doi.org/10.1124/jpet.114.214700.

Ca2+ regulation of mitochondrial function in neurons.

Rueda CB1Llorente-Folch I1Amigo I1Contreras L1González-Sánchez P1Martínez-Valero P1Juaristi I1Pardo B1Del Arco A2Satrústegui J3

Biochim Biophys Acta. 2014 May 10. pii: S0005-2728(14)00126-1.
doi: 10.1016/j.bbabio.2014.04.010.

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca2+ signal or to Ca2+ itself.

[Na+]i, [Ca2+]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca2+ clearly showed that

  • Ca2+-stimulation of coupled respiration is required to maintain [ATP]i levels.

Ca2+ may regulate respiration by

  1. activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or
  2. after Ca2+ entry in mitochondria through the calcium uniporter (MCU).

Two Ca2+-regulated mitochondrial metabolite transporters are expressed in neurons,

  1. the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, with a Kd for Ca2+ activation of 300nM, and
  2. the ATP-Mg/Pi exchanger SCaMC-3/Slc25a23, with S0.5 for Ca2+ of 300nM and 3.4μM, respectively.

The lack of SCaMC-3 results in a smaller Ca2+-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas

  • the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca2+-dependent responses to all workloads (veratridine, K+-depolarization, carbachol).

The lack of ARALAR caused a reduction of about 65-70% in the response to the high workload imposed by veratridine, and

  • completely suppressed the OCR responses to moderate (K+-depolarization) and small (carbachol) workloads,
  • effects reverted by pyruvate supply.

For K+-depolarization, this occurs in spite of the presence of large [Ca2+]mit signals and increased reduction of mitochondrial NAD(P)H.

These results show that ARALAR-MAS is a major contributor of Ca2+-stimulated respiration in neurons

  • by providing increased pyruvate supply to mitochondria.

In its absence and under moderate workloads, matrix Ca2+ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions.

This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.    Copyright © 2014. Published by Elsevier B.V.

KEYWORDS:  ATP-Mg/Pi transporter; Aspartate–glutamate transporter; Calcium; Calcium-regulated transport; Mitochondrion; Neuronal respiration PMID: 24820519

 

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Ro SH1Nam M2Jang I1Park HW1Park H1Semple IA1Kim M1et al.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7849-54.
doi: 10.1073/pnas.1401787111.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans.

Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation.

Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation.

Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments.

Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

KEYWORDS: aging; homeostasis; mouse; β-adrenergic signaling      PMID: 24825887     PMCID:  PMC4040599

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans.

Yang F1Gao Y1Li Z2Chen L3Xia Z4Xu T5Qin Y6
Biochim Biophys Acta. 2014 May 15. pii: S0005-2728(14)00499-X.
doi: 10.1016/j.bbabio.2014.05.353.

How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown.

Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15°C).

At worms’ optimum growing temperature (20°C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction.

Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization.

At low temperature (15°C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV.

These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.

Copyright © 2014. Published by Elsevier B.V.

KEYWORDSC. elegans; EF4(LepA/GUF1); Mitochondrial dysfunction; Retrograde pathways; Translation    PMID:  24837196

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Chin RM1Fu X2Pai MY3Vergnes L4Hwang H5Deng G6Diep S2, et al.
Nature  2014 Jun 19;509(7505):397-401. doi: 10.1038/nature13264. 

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits.

Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans.

ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution.

Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan.

We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.

We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream.

Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction.

Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PMID: 24828042

 

 

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A Synthesis of the Beauty and Complexity of How We View Cancer


A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP

Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

 

This document has covered a broad spectrum of the research, translational biology, diagnostics (both laboratory and imaging methodologies), and treatments for a variety of cancers, mainly by organs, and selectively by the most common cancers seen in human populations. A number of observations stand out on review of all the material presented. 1. The most common cancers affecting humans is spread worldwide, with some variation by region. 2. Cancers within geographic regions may be expressed differently in relationship to population migrations, the incidence of specific environmental pollutants, occurrence of insect transmitted and sexually transmitted diseases (HIV, HCV, HPV), and possibly according to age, or relationship to ultraviolet or high dose radiation exposure. 3. Cancers are expressed within generally recognized age timelines. For example, acute lymphocytic leukemia and neuroblastoma in children under 10 years age; malignant giant cell tumor and osteosarcoma in the third and fourth decade; prostate cancer and breast cancer over age 40, and are more aggressive at an earlier age, both having a strong sex hormone dependence. 4. There is dispute about the effectiveness of screening for cancer with respect to what age, excessive risk in treatment modality, and the duration of progression free survival. Despite the evidence of several years potential life extension, a long term survival of 10 years is not the expected outcome. However, the quality of life in the remaining years is a valid point in favor of progress. 5. There has been a significant reduction in toxicity of treatment, but attention has been focused on a patient-centric decision process. 6. There has been a dramatic improvement in surgical approaches, post-surgical surveillance, and in diagnosis by invasive and noninvasive methods, especially in the combination of needle biopsy and imaging techniques. 7. There is significant variation within cancer cell types with respect to disease-free survival.

The work presented has several main components: First, there is the biology and mechanisms involved in carcinogenesis related to (1) mutations; (2) carcinogenesis; (3) cell regulatory mechanisms; (4) cell signaling pathways; (5) apoptosis (6) ubitination (7) mitochondrial dysfunction; (8) cell-cell interactions; (9) cell migration; (10) metastasis. Then there are large portions covering (1) imaging; (2) specific targeted therapy; (3) nanotechology-based therapy; (4) specific organ-type cancers; (5) genomics-based testing; (6) circulating cancer cells; (7) miRNAs; (8) siRNAs; (9) cancer immunology and (10) immunotherapy.

Classically, we refer to cancer development in terms of the germ cell layers – ectoderm, mesoderm, and endoderm. These are formative in embryonic development. The most active development occurs during embryonic development, with a high growth rate of cells and also a high utilization of energy. The cells utilize oxidation for energy in this period characterized by movement of cells in differentiation and organogenesis. This was observed to be unlike the cell metabolism in carcinogenesis, which is characterized by impaired mitochondrial function and reliance on lactate production for energy – termed anaerobic glycolysis, as investigated by Meyerhof, Embden, Warburg, Szent-Gyorgy, H. Krebs, Theorell, AV Hill, B Chance, P Mitchell, P Boyer, F Lippman, and others.

In addition, the body economy has been divided into two major metabolic compartments: fat and lean body mass (LBM), which is further denoted as visceral and structural. This denotes the gut, kidneys, liver, lung, pancreas, sexual organs, endocrines, brain and fat cells in one compartment, and skeletal muscle, bone and cardiovascular in another. LBM is calculated as fat free mass. Further, brown fat is distinguished from white fat. But this was a first layer of construction of the human body. One peels away this layer to find a second layer. For example, the gut viscera have an inner (outer) epithelial layer, a muscularis, and a deep epithelium, which has circulation and fat. There is also an interstitium between the gut epithelium and muscularis. The lung has an epithelium exposed to the airspaces, then capillaries, and then epithelium, designed for exchange of O2 and CO2, the source of heat generation. The pancreas has an endocrine portion in the islets that are embedded in an exocrine secretory organ. The sexual organs have a combination of glandular structures embedded in a mesothelium.

The structural compartment is entirely accounted for by the force of contraction. If this is purely anatomical, that is not really the case when one goes into the functioning substructures of these tissues – cytoplasm, endoplasmic reticulum (ribosomal), mitochondria, liposomes, chromatin apparatus, cell membrane and vesicles. Within and between these structures are the working and interacting mechanisms of the cell in its unique role. What ties these together was first thought to be found in the dogma following the discovery of the genetic code in 1953 that begat DNA to RNA to protein.

This led to many other discoveries that made it clear that it was only a first approximation. It did not account for noncoding DNA, which became unmasked with the culmination of the Human Genome Project and concurrent advances in genomics (mtDNA, mtRNA, siRNA, exosomes, proteomics, synthetic biology, predictive analytics, and regulatory pathways directed by signaling molecules. Here is a list of signaling pathways: 1. JAK-STAT 2. GPCR 3. Endocrine 4. Cytochemical 5. RTK 6. P13K 7. NF-KB 8. MAPK 9. Ubiquitin 10. TGF-beta 11. Stem cell These signaling pathways have become the basis for the discovery of inhibitors of signaling pathways (suppressors), as well as activators, as these have been considered as specific targets for selective therapy. (.See Figure below) Of course, extensive examination of these pathways has required that all such findings are validated based on the STRENGTH of their effect on the target and in the impact of suppression.

inhibitors of signaling pathways-1

http://www.SelleckChem.com

 

Let us continue this discussion elucidating several major points.  While the early observations that drove the interest in biochemical behavior of cancer cells has been displaced, it has not faded from view.

Bioenergetics of Cancer cells

Michael J. Gonzalez (Bioenergetic_Theory_of_Carcinigenesis. http://www.academia.edu/2224071/ Bioenergetic_Theory_of_Carcinigenesis) maintains that the altered energy metabolism of tumor cells provides a viable target for a non-toxic chemotherapeutic approach.  An increased glucose consumption rate  has been observed in malignant cells. Warburg (NobelLaureate in medicine) postulated that the respiratory process of malignant cells was impaired in the malignant transformation. Szent-Györgyi (Nobel in medicine) also viewed cancer as originating from insufficient oxygen utilization. Oxygen inhibits anaerobic  metabolism (fermentation and lactic acid production). Interestingly, during cell differentiation (where cell energy level is high) there is an increased cellular production of oxidation products that appear to provide physiological stimulation for changes in gene expression that may lead to a terminal differentiated state. The failure to maintain high ATP production (high cell energy levels) may be a consequence of inactivation of key enzymes, especially those related to the Krebs cycle and the electron transport system. A distorted mitochondrial function (transmembrane potential) may result.  This  aspect could be suggestive of an important mitochondrial involvement in the carcinogenic process in addition to presenting it as a possible therapeutic target for cancer. Intermediate metabolic correction of the mitochondria is postulated as a possible non-toxic therapeutic approach for cancer.

Fermentation is the anaerobic metabolic breakdown of glucose without net oxidation. Fermentation does not release all the available energy of glucose or need oxygen as part of its biochemical reactions ;  it merely allows glycolysis  (a process that yields two ATP per mole of glucose) to continue by replenishing reduced coenzymes and yields lactate as its final product. The first step in aerobic and anaerobic energy producing pathways, it occurs in the cytoplasm of cells, not in specialized organelles, and is found in all living organisms.  Cancer cells have a fundamentally different energy metabolism compared to normal cells, that  are obligate aerobes (oxygen-requiring cells)  meeting their energy needs with oxidative metabolic processes., while cancer cells do not  require oxygen for their survival. This increase in glycolytic  flux is a metabolic strategy of tumor cells to ensure growth and    survival  in  environments  with  low   oxygen concentrations.

Radoslav Bozov has commented that the process of genomic evolution cannot be fully revealed through comparative genomicsHe states that DNA would be entropic- favorable stable state going towards absolute ZERO temp. Themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. But stability emerges out of non-stable states, and never reaches absolute stability, for there would be neither feelings nor freedom.

Membrane potential(Vm)

Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of differention channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. (M Yang and WJ Brackenbury.

Membrane potential and cancer progression. Frontiers in Physiol.  2013(4); 185: 1.  http://dx.doi.org/10.3389/fphys.2013.00185)

It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, yperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be avaluable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.

Perspective beyond Cancer Genomics: Bioenergetics of Cancer Stem Cells

Hideshi Ishii, Yuichiro Doki, and Masaki Mori
Yonsei Med J 2010; 51(5):617-621.  http://dx.doi.org/10.3349/ymj.2010.51.5.617   pISSN: 0513-5796, eISSN: 1976-2437

Although the notion that cancer is a disease caused by genetic and epigenetic alterations is now widely accepted, perhaps more emphasis has been given to the fact that cancr is a genetic disease. It should be noted that in the post-genome sequencing project period of the 21st century, the underlined phenomenon nevertheless could not be discarded towards the complete control of cancer disaster as the whole strategy, and in depth investigation of the factors associated with tumorigenesis is required for achieving it. Otto Warburg has won a Nobel Prize in 1931 for the discovery of tumor bioenergetics, which is now commonly used as the basis of positron emission tomography (PET), a highly sensitive noninvasive technique used in cancer diagnosis. Furthermore, the importance of the cancer stem cell (CSC) hypothesis in therapy-related resistance and metastasis has been recognized during the past 2 decades. Accumulating evidence suggests that tumor bioenergetics plays a critical role in CSC regulation; this finding has opened up a new era of cancer medicine, which goes beyond cancer genomics.

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase.

Gema Santamaría1,#, Marta Martínez-Diez1,#, Isabel Fabregat2 and José M. Cuezva1,*
Carcinogenesis 2006 27(5):925-935      http://dx.doi.org/10.1093/carcin/bgi315

There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that down-regulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay

staurosporine-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after staurosporine treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production, prevented the oxidation of cellular proteins and the modification of mitochondrial proteins, delaying the release of cyt c and the execution of cell death. The results in this work establish the down-regulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid reactive oxygen species-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as main energy provision pathway.

see also –

The tumor suppressor function of mitochondria: Translation into the clinics

José M. CuezvaÁlvaro D. OrtegaImke Willers, et al.  
Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease  Dec 2009;  1792(12): 1145–1158  http://dx.doi.org/10.1016/j.bbadis.2009.01.006

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life–death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial β-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.

Self-Destructive Behavior in Cells May Hold Key to a Longer Life

Carl Zimmer, MY Times  October 5, 2009

In recent years, scientists have found evidence of autophagy in preventing a much wider range of diseases. Many disorders, like Alzheimer’s disease, are the result of certain kinds of proteins forming clumps. Lysosomes can devour these clumps before they cause damage, slowing the onset of diseases.

Lysosomes may also protect against cancer. As mitochondria get old, they cast off charged molecules that can wreak havoc in a cell and lead to potentially cancerous mutations. By gobbling up defective mitochondria, lysosomes may make cells less likely to damage their DNA. Many scientists suspect it is no coincidence that breast cancer cells are often missing autophagy-related genes. The genes may have been deleted by mistake as a breast cell divided. Unable to clear away defective mitochondria, the cell’s descendants become more vulnerable to mutations.

Unfortunately, as we get older, our cells lose their cannibalistic prowess. The decline of autophagy may be an important factor in the rise of cancer, Alzheimer’s disease and other disorders that become common in old age. Unable to clear away the cellular garbage, our bodies start to fail.

If this hypothesis turns out to be right, then it may be possible to slow the aging process by raising autophagy. It has long been known, for example, that animals that are put on a strict low-calorie diet can live much longer than animals that eat all they can. Recent research has shown that caloric restriction raises autophagy in animals and keeps it high. The animals seem to be responding to their low-calorie diet by feeding on their own cells, as they do during famines. In the process, their cells may also be clearing away more defective molecules, so that the animals age more slowly.

Some scientists are investigating how to manipulate autophagy directly. Dr. Cuervo and her colleagues, for example, have observed that in the livers of old mice, lysosomes produce fewer portals on their surface for taking in defective proteins. So they engineered mice to produce lysosomes with more portals. They found that the altered lysosomes of the old experimental mice could clear away more defective proteins. This change allowed the livers to work better.

 

Essentiality of pyruvate kinase, oxidation, and phosphorylation

We can move to the next level with greater clarity. Yu et al. reported an important relationship between Pyruvate kinase M2 (PKM2) and the Warburg effect of cancer cells ( M Yu, et al. PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells. J Biol Chem (PMID: 24142698) http://dx.doi.org10.1074/jbc.M113.508226 ).  They found that PIM2 could directly phosphorylate PKM2 on the Thr454 residue, which resulted in an increase of PKM2 protein levels. PKM2 with a phosphorylation-defective mutation displayed a reduced effect on glycolysis compared to the wild-type, thereby co-activating HIF-1α and β-catenin, and enhanced mitochondria respiration and chemotherapeutic sensitivity of cancer cells. This indicated that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.

In another study of the effect of 3 homoplastic mtDNA mutations on oxidative metabolism of osteosarcoma cells, there was a difference proportional to the magnitude of the defect. (Iommarini L, et al. Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment. Hum Mol Genet. 2013 Nov 11. [Epub ahead of print]; PMID: 24163135 ).   Osteosarcoma cells carrying the most marked impairment of the gene encoding mitochondrial complex I  (CI) of oxidative phosphorylation displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction. The severe CI dysfunction was an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation.  The result suggested that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

An unrelated finding shares some agreement with what has been identified (Systematic isolation of context-dependent vulnerabilities in NSCLC. Cell, 24 Oct 2013; 155 (3): 552-566, http://dx.doi.org/10.1016/ j.cell.2013.09.041). They report  three distinct target/response-indicator pairings that are represented with significant frequencies (6%–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature.   This is depicted in the Figure below.  The authors noted a frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets.

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The forging of a cancer-metabolism link and twists in the chain (Biome 19th April 2013)

Ten years ago, Grahame Hardie and Dario Alessi discovered that the elusive upstream kinase required for the activation of AMP-activated protein kinase (AMPK) by metabolic stress that the Hardie lab had been pursuing in their research on the metabolic regulator AMPK was the tumor suppressor, LKB1, that the neighbouring Alessi lab was working on at the time. This finding represented the first clear link between AMPK and cancer.

The resulting paper [1], published in 2003 in what was then Journal of Biology (now BMC Biology), was one [1] of three [2, 3] connecting these two kinases and that helped to swell of a surge of interest in the metabolism of tumor cells that was just beginning at about that time and is still growing. (LKB1 and AMPK and the cancer-metabolism link – ten years after.  D Grahame Hardie, and Dario R Alessi.  BMC Biology 2013, 11:36.   http://dx doi.org.10.1186/1741-7007-11-36.)

 

In September 2003, both groups published a joint paper [1] in Journal of Biology (now BMC Biology) that identified the long-sought and elusive upstream kinase acting on AMP-activated protein kinase (AMPK) as a complex containing LKB1, a known tumor suppressor. Similar findings were reported at about the same time by David Carling and Marian Carlson [2] and by Reuben Shaw and Lew Cantley [3]; at the time of writing these three papers have received between them a total of over 2,000 citations. These findings provided a direct link between a protein kinase, AMPK, which at the time was mainly associated with regulation of metabolism, and another protein kinase, LKB1, which was known from genetic studies to be a tumor suppressor. While the idea that cancer is in part a metabolic disorder (first suggested by Warburg in the 1920s [4]) is well recognized today [5], this was not the case in 2003, and our paper perhaps contributed towards its renaissance.

The distinctive metabolic feature of tumor cells that enables them to meet the demands of unrestrained growth is the switch from oxidative generation of ATP to aerobic glycolysis – a phenomenon now well known as the Warburg effect. Operating this switch is one of the central functions of the AMP-activated protein kinase (AMPK) that has long been the focus of research in the Hardie lab. AMPK is an energy sensor that is allosterically tuned by competitive binding of ATP, ADP and AMP to sites on its g regulatory subunit (its portrait here, with AMP bound at two sites, was kindly provided by Bing Xiao and Stephen Gamblin). When phosphorylated by LKB1, AMPK responds to depletion of ATP by turning off anabolic reactions required for growth, and turning on catabolic reactions and oxidative phosphorylation – the reverse of the Warburg effect. In this light, it is not surprising that LKB1  is inactivated in some proportion of many different types of tumors.

AMPK as an energy sensor and metabolic switch

AMPK was discovered as a protein kinase activity that phosphorylated and inactivated two key enzymes of fatty acid and sterol biosynthesis: acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The ACC kinase activity was reported to be activated by 5’-AMP, and the HMGR kinase activity by reversible phosphorylation, but for many years the two activities were thought to be due to distinct enzymes. However, in 1987 the DGH laboratory showed that both were functions of a single protein kinase, which we renamed AMPK after its allosteric activator, 5’-AMP. It was subsequently found that AMPK regulated not only lipid biosynthesis, but also many other metabolic pathways, both by direct phosphorylation of metabolic enzymes, and through longer-term effects mediated by phosphorylation of transcription factors and co-activators. In general, AMPK switches off anabolic pathways that consume ATP and NADPH, while switching on catabolic pathways that generate ATP (Figure 1).

 

target proteins and metabolic pathways regulated by AMPK 1741-7007-11-36-1_1

 

Summary of a selection of target proteins and metabolic pathways regulated by AMPK. Anabolic pathways switched off by AMPK are shown in the top half of the ‘wheel’ and catabolic pathways switched on by AMPK in the bottom half. Where a protein target for AMPK responsible for the effect is known, it is shown in the inner wheel; a question mark indicates that it is not yet certain that the protein is directly phosphorylated. For original references see [54].

Key to acronyms: ACC1/ACC2, acetyl-CoA carboxylases-1/-2; HMGR, HMG-CoA reductase; SREBP1c, sterol response element binding protein-1c; CHREBP, carbohydrate response element binding protein; TIF-1A, transcription initiation factor-1A; mTORC1, mechanistic target-of-rapamycin complex-1; PFKFB2/3, 6-phosphofructo-2-kinase, cardiac and inducible isoforms; TBC1D1, TBC1 domain protein-1; SIRT1, sirtuin-1; PGC-1α, PPAR-γ coactivator-1α; ULK1, Unc51-like kinase-1.

Regulation of AMPK  1741-7007-11-36-3

 

Regulation of AMPK. AMPK can be activated by increases in cellular AMP:ATP or ADP:ATP ratio, or Ca2+ concentration. AMPK is activated >100-fold on conversion from a dephosphorylated form (AMPK) to a form phosphorylated at Thr172 (AMPK-P) catalyzed by at least two upstream kinases: LKB1, which appears to be constitutively active, and CaMKKβ, which is only active when intracellular Ca2+ increases. Increases in AMP or ADP activate AMPK by three mechanisms: (1) binding of AMP or ADP to AMPK, causing a conformational change that promotes phosphorylation by upstream kinases (usually this will be LKB1, unless [Ca2+] is elevated); (2) binding of AMP or ADP, causing a conformational change that inhibits dephosphorylation by protein phosphatases; (3) binding of AMP (and not ADP), causing allosteric activation of AMPK-P. All three effects are antagonized by ATP, allowing AMPK to act as an energy sensor.

AMPK and AMPK-related kinase (ARK) family  1741-7007-11-36-4

 

Members of the AMPK and AMPK-related kinase (ARK) family. All the kinases named in the figure are phosphorylated and activated by LKB1, although what regulates this phosphorylation is known only for AMPK. Alternative names are shown, where applicable.

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

 

 

Three possible mechanisms to explain how the AMPK-activating drugs metformin or phenformin might provide protection against cancer. (a) Metformin acts on the liver and other insulin target tissues by activating AMPK (and probably via other targets), normalizing blood glucose; this reduces insulin secretion from pancreatic β cells, reducing the growth-promoting effects of insulin (and high glucose) on tumor cells. Since metformin does not reduce glucose levels in normoglycemic individuals, this mechanism would only operate in insulin-resistant subjects. (b) Metformin or phenformin activates AMPK in pre-neoplastic cells, restraining their growth and proliferation and thus delaying the onset of tumorigenesis; this mechanism would only operate in cells where the LKB1-AMPK pathway was intact. (c) Metformin or phenformin inhibits mitochondrial ATP synthesis in tumor cells, promoting cell death. If the LKB1-AMPK pathway was down-regulated in the tumor cells, they would be more sensitive to cell death induced by the biguanides than surrounding normal cells.

Metformin and phenformin are biguanides that inhibit mitochondrial function and so deplete ATP by inhibiting its production . AMPK is activated by any metabolic stress that depletes ATP, either by inhibiting its production (as do hypoxia, glucose deprivation, and treatment with biguanides) or by accelerating its consumption (as does muscle contraction). By switching off anabolism and other ATP-consuming processes and switching on alternative ATP-producing catabolic pathways, AMPK acts to restore cellular energy homeostasis.

Findings that AMPK is activated in skeletal muscle during exercise and that it increases muscle glucose uptake and fatty acid oxidation led to the suggestion that AMPK-activating drugs might be useful for treating type 2 diabetes. Indeed, it turned out that AMPK is activated by metformin, a drug that had at that time been used to treat type 2 diabetes for over 40 years, and by phenformin , a closely related drug that had been withdrawn for treatment of diabetes due to side effects of lactic acidosis.

If only it were so simple. Effects of metformin on cancer in type 2 diabetics could be secondary to reduction in insulin levels, and although there is evidence for direct effects of AMPK activation on the development of tumors in mice, there is also recent evidence that tumors that become established without down-regulating LKB1 survive metformin better than those that have lost it – probably because metformin poisons the mitochondrial respiratory chain, depressing ATP levels, and cells in which AMPK can still be activated in response to the challenge do better than those in which it can’t.

In their review, Hardie and Alessi chart these  twists and turns, and point to the explosion of further possibilities opened up by the discovery, since their 2003 publication, of at least one other class of kinase upstream of AMPK (the CaM kinases), and at least a dozen other downstream targets of LKB1 (AMPK-related kinases, or ARKs) – not to mention the innumerable downstream targets of AMPK; all which make half their schematic illustrations look like hedgehogs.

Analysis of respiration  in human cancer

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective

Therapeutic  strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function.

(T Kaambre,V Chekulayev, I Shevchuk, et al. Metabolic control analysis of respiration  in human cancer tissue.  Frontiers Physiol 2013 (4); 151:  1. http://dx.doi.org/10.3389/fphys.2013.00151)

Our main goal is to demonstrate the applicability of MCA for in situ studies of energy

Metabolism in human breast and colorectal cancer cells as well as in normal tissues .We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolic control analysis of respiration in human cancer tissue.

Representative traces of change in the rate of oxygen consumption by permeabilized human colorectal cancer (HCC) fibers after their titration with increasing concentrations of mersalyl, an inhibitor of inorganic phosphate carrier (panel A). The values of respiration rate obtained were plotted vs. mersalyl concentration (panel B) and from the plot the corresponding flux control coefficient was calculated. Bars are ±SEM.

Oncologic diseases such as breast and colorectal cancers are still one of the main causes of premature death. The low efficiency of contemporary medicine in the treatment of these malignancies is largely mediated by a poor understanding of the processes involved in metastatic dissemination of cancer cells as well as the unique energetic properties of mitochondria from tumors. Current knowledge supports the idea that human breast and colorectal cancer cells exhibit increased rates of glucose consumption displaying Warburg phenotype,i.e.,elevated glycolysis even in the presence of oxygen (Warburg and Dickens, 1930; Warburg, 1956 ;Izuishietal., 2012). Notwithstanding,  there are some evidences that in these malignancies mitochondrial oxidative phosphorylation (OXPHOS) is the main source of ATP rather than glycolysis. Cancer cells have been classified according to their pattern of metabolic remodeling depending of the relative balance between aerobic glycolysis and OXPHOS (Bellanceetal.,2012). The first type of tumor cells is highly glycolytic, the second OXPHOS deficient and the third type of tumors dislay enhanced OXPHOS. Recent studies strongly sug gest  that cancer cells can utilize lactate, free fatty acids, ketone bodies, butyrate and glutamine as key respiratory substrate selic iting metabolic remodeling of normal surrounding cells toward aerobic glycolysis—“reverse Warburg”effect (Whitaker-Menezes et al.,2011;Salem et al.,2012;Sotgia et al.,2012;Witkiewicz et al., 2012).

In normal cells,the OXPHOS system is usually closely linked to phosphotransfer systems, including various creatine kinase(CK) isotypes,which ensure a safe operation of energetics over a broad functional range of cellular activities (Dzejaand Terzic,2003).  However, our current knowledge about the function of CK/creatine (Cr) system in human breast and colorectal cancer is insufficient. In some malignancies, for example sarcomas the CK/Cr system was shown to be strongly downregulated (Beraetal.,2008;Patraetal.,2008).  Our previous studies showed  that the mitochondrial-bound CK (MtCK) activity was significantly decreased in HL-1 tumor cells (Mongeetal.,2009), as compared to normal parent cardiac cells where the OXPHOS is the main ATP source of and the CK system is a main energy carrier. In the present study,we estimated the role of MtCK in maintaining energy homeostasis in human colorectal cancer cells. Understanding the control and regulation of energy metabolism requires analytical tools that take into account  the existing interactions between individual network components and their impact on systemic network function. Metabolic Control Analysis(MCA) is a theoretical framework relating the properties of metabolic systems to the kinetic characteristics of their individual enzymatic components (Fell,2005). An experimental approach of MCA has been already successfully applied to the studies of OXPHOS in isolated mitochondria (Tageretal.,1983; Kunzetal.,1999; Rossignoletal.,2000)  and in skinned muscle fibers (Kuznetsovetal.,1997;Teppetal.,2010).

Metabolic control analysis of respiration in human cancer tissue

Values of basal (Vo) and maximal respiration rate (Vmax, in the presence of 2 mM ADP) and apparent Michaelis Menten constant (Km) for ADP in permeabilized human breast and colorectal cancer samples as well as health tissue. – See more at: http://journal.frontiersin.org/Journal/10.3389/fphys.2013.00151/full#sthash.VBXPdodj.dpuf

Role of Uncoupling Proteins in Cancer

Adamo Valle, Jordi Oliver and Pilar Roca *
Cancers 2010; 2: 567-591;   http://dx.doi.org/10.3390/cancers2020567

Since Otto Warburg discovered that most cancer cells predominantly produce energy by glycolysis rather than by oxidative phosphorylation in mitochondria, much interest has been focused on the alterations of these organelles in cancer cells. Mitochondria have been shown to be key players in numerous cellular events tightly related with the biology of cancer. Although energy production relies on the glycolytic pathway in cancer cells, these organelles also participate in many other processes essential for cell survival and proliferation such as ROS production, apoptotic and necrotic cell death, modulation of oxygen concentration, calcium and iron homeostasis, and certain metabolic and biosynthetic pathways. Many of these mitochondrial-dependent processes are altered in cancer cells, leading to a phenotype characterized, among others, by higher oxidative stress, inhibition of apoptosis, enhanced cell proliferation, chemoresistance, induction of angiogenic genes and aggressive fatty acid oxidation. Uncoupling proteins, a family of inner mitochondrial membrane proteins specialized in energy-dissipation, has aroused enormous interest in cancer due to their relevant impact on such processes and their potential for the development of novel therapeutic strategies.

Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress UCPs are being investigated for a potential role in cancer. In this review we compile the latest evidence showing a link between uncoupling and the carcinogenic process, paying special attention to their involvement in cancer initiation, progression and drug chemoresistance.

The Warburg Effect

Uncoupling the Warburg effect from cancer

A Najafov and DR Alessi
Proc Nat Acad Sci                                      www.pnas.org/cgi/doi/10.1073/pnas.1014047107
A remarkable trademark of most tumors is their ability to break down glucose by glycolysis at a vastly higher rate than in normal tissues, even when oxygen is copious. This phenomenon, known as the Warburg effect, enables rapidly dividing tumor cells to generate essential biosynthetic building blocks such as nucleic acids, amino acids, and lipids from glycolytic intermediates to permit growth and duplication of cellular components during  division (1). An assumption dominating research in this area is that the Warburg effect is specific to cancer. Thus, much of the focus has been on uncovering mechanisms by which cancer-causing mutations influence metabolism to stimulate glycolysis.

This has lead to many exciting discoveries. For example, the p53 tumor suppressor can suppress glycolysis through its ability to control expression of key metabolic genes, such as phosphoglycerate mutase (2), synthesis of cytochrome C oxidase-2 (3), and TP53-induced glycolysis and apoptosis regulator (TIGAR) (4). Many cancer-causing mutations lead to activation of the Akt and mammalian target of rapamycin (mTOR) pathway that profoundly influences metabolism and expression of metabolic enzymes to promoteglycolysis (5).

Strikingly, all cancer cells but not nontransformed cells express a specific splice variant of pyruvate kinase, termed M2-PK, that is less active, leading to the build up of phosphoenolpyruvate (6). Recent work has revealed that reduced activity of M2-PK promotes a unique glycolytic pathway in which phosphoenolpyruvate is converted to pyruvate by a histidine-dependent phosphorylation of phosphoglycerate mutase, promoting assimilation of glycolytic products into biomass (7). However, despite these observations, one might imagine that the Warburg effect need not be specific for cancer and that any normal cell would need to stimulate glycolysis to generate sufficient biosynthetic materials to fuel expansion and division.

Recent work by Salvador Moncada’s group published in PNAS (8) and other recent work from the same group (9, 10) provides exciting evidence supporting the idea that the Warburg effect is also required for the proliferation of noncancer cells.

The key discovery was that the anaphase promoting complex/cyclosome-Cdh1(APC/C-Cdh1), a master regulator of the transition of G1 to S phase of the cell cycle, inhibits glycolysis in proliferating noncancer cells by mediating the degradation of two key metabolic enzymes, namely 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase isoform3 (PFKFB3) (9, 10) and glutaminase-(Fig. 1) (8).

Fig. 1. Mechanism by which APC_C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation

 

Fig.  Mechanism by which APC/C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation.

APC/C-Cdh1 E3 ligase recognizes KEN-box–containing metabolic enzymes, such as PFKFB3 and glutaminase-1 (GLS1), and ubiquitinates and targets them for proteasomal degradation. This inhibits glycolysis and glutaminolysis, leading to decrease in metabolites that can be assimilated into biomass, thereby suppressing proliferation.

PFKFB3 potently stimulates glycolysis by catalyzing the formation of fructose-2,6-bisphosphate, the allosteric activatorof 6-phosphofructo-1-kinase (11). Glutaminase-1 is the first enzyme in glutaminolysis, converting glutamine to lactate, yielding biosyntheticintermediates required for cell proliferation (12).

APC/C is a cell cycle-regulated E3 ubiquitin ligase that promotes ubiquitination of a distinct set of cell cycle proteins containing either a D-box (destruction box) or a KEN-box, named after the essential Lys-Glu-Asn motif required for APC recognition (13). Among its well-known substrates are crucial cell cycle proteins, such as cyclin B1, securin, and Plk1. By ubiquitinating and targeting its substrates to 26S proteasome-mediated degradation, APC/C regulates processes in late mitotic stage, exit  from mitosis, and several events in G1 (14). The Cdh1 subunit is the KENbox binding adaptor of the APC/C ligase and is essential for G1/S transition.

Importantly, APC/C-Cdh1 is inactivated at the initiation of the S-phase of the cell cycle when DNA and cellular organelles are replicated at the time of the greatest need for generation of biosynthetic materials. APC/C-Cdh1 is reactivated later at the mitosis/G1 phase of the cell cycle when there is a lower requirement for biomassgeneration.

Both PFKFB3 (9, 10) and glutaminase-1 (8) possess a KEN-box and are rapidly degraded in nonneoplastic lymphocytes during the cell cycle when APC/C-Cdh1 is active. Consistent with destruction being mediated by APC-C-Cdh1, ablation of the KEN-box prevents degradation of PFKFB3 (9, 10) and glutaminase-1 (8). Inhibiting the proteasomal-dependent degradation with the MG132 inhibitor

markedly increases levels of ubiquitinated PFKFB3 and glutaminase-1 (8). Moreover, overexpression of Cdh1 to activate APC/C-Cdh1 decreases levels of PFKFB3 as well as glutmaninase-1 and concomitantly inhibited glycolysis, as judged by decrease in lactate production. This effect is also observed when cells were treated with a glutaminase-1 inhibitor (6-diazo-5- oxo-L-norleucine) (8). The final evidence supporting the authors’ hypothesis is that proliferation and glycolysis is inhibited after shRNA-mediated silencing of either PFKFB3 or glutaminase-1 (8).

These results are interesting, because unlike most recent work in this area, Colombo et al. (8) link the Warburg effect to the machinery of the cell cycle that is present in all cells rather than to cancer driving mutations. Further work is required to properly define the overall importance of this pathway, which has thus far only been studied in a limited number of cells. It would also be of value to undertake a more detailed analysis of how the rate of glycolysis and other metabolic pathways vary during the cell cycle of normal and cancer cells…(see full 2 page article) at PNAS.

 

The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer

C Ruckenstuhl, S Buttner, D Carmona-Gutierre, et al.
PLoS ONE 2009; 4(2): e4592.  http://dx.doi.org/10.1371/journal.pone.0004592

Colonies of Saccharomyces cerevisiae, suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis, conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.

Conclusion/Significance: The Warburg effect might directly contribute to the initiation of cancer formation – not only by enhanced glycolysis – but also via decreased respiration in the presence of oxygen, which suppresses apoptosis.

 

PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells
Z Yu, L Huang, T Zhang, et al.
J Biol Chem 2013;                               http://dx.doi.org/10.1074/jbc.M113.508226

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.508226

Serine/threonine protein kinase PIM2, a known oncogene is a binding partner of pyruvate kinase M2 (PKM2), a key player in the Warburg effect of cancer cells.   PIM2 interacts with PKM2 and phosphorylates PKM2 on the Thr454 residue.

The phosphorylation of PKM2 increases glycolysis and proliferation in cancer cells.

The PIM2-dependent phosphoirylation of ZPKM2 is critical for regulating the Warburg effect in cancer.

 

Genome-Scale Metabolic Modeling Elucidates the Role of Proliferative Adaptation in Causing the Warburg Effect

Shlomi T, Benyamini T, Gottlieb E, Sharan R, Ruppin E
PLoS Comput Biol 2011; 7(3): e1002018.    http://dx.doi.org/10.1371/journal.pcbi.1002018
The Warburg effect – a classical hallmark of cancer metabolism – is a counter-intuitive phenomenon in which rapidly proliferating cancer cells resort to inefficient ATP production via glycolysis leading to lactate secretion, instead of relying primarily on more efficient energy production through mitochondrial oxidative phosphorylation, as most normal cells do.

The causes for the Warburg effect have remained a subject of considerable controversy since its discovery over 80 years ago, with several competing hypotheses. Here, utilizing a genome-scale human metabolic network model accounting for stoichiometric and enzyme solvent capacity considerations, we show that the Warburg effect is a direct consequence of the metabolic adaptation of cancer cells to increase biomass production rate. The analysis is shown to accurately capture a three phase metabolic behavior that is observed experimentally during oncogenic progression, as well as a prominent characteristic of cancer cells involving their preference for glutamine uptake over other amino acids.

 

The metabolic advantage of tumor cells

Maurice Israël and Laurent Schwartz

Additional article information

Abstract

1- Oncogenes express proteins of “Tyrosine kinase receptor pathways”, a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases.

2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas.

Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases.

3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A “bottleneck” between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis.

4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase)

5- IGFBP stops binding the IGF – IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability.

6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them…

Introduction

The metabolic network of biochemical pathways forms a system controlled by a few switches, changing the finality of this system. Specific substrates and hormones control such switches. If for example, glycemia is elevated, the pancreas releases insulin, activating anabolism and oxidative glycolysis, energy being required to form new substance or refill stores. If starvation decreases glycemia, glucagon and epinephrine activate gluconeogenesis and ketogenesis to form nutriments, mobilizing body stores. The different finalities of the system are or oriented by switches sensing the NADH/NAD+, the ATP/AMP, the cAMP/AMP ratios or the O2 supply… We will not describe here these metabolic finalities and their controls found in biochemistry books.

Many of the switches depend of the phosphorylation of key enzymes that are active or not. Evidently, there is some coordination closing or opening the different pathways. Take for example gluconeogenesis, the citrate condensation slows down, sparing OAA, which starts the gluconeogenic pathway. In parallel, one also has to close pyruvate kinase (PK); if not, phosphoenolpyruvate would give back pyruvate, interrupting the pathway. Hence, the properties of key enzymes acting like switches on the pathway specify the finality of the system. Our aim is to show that tumor cells invent a new specific finality, with mixed glycolysis and gluconeogenesis features. This very special metabolism gives to tumor cells a selective advantage over normal cells, helping the tumor to develop at the detriment of the rest of the body.

I Abnormal metabolism of tumors, a selective advantage

The initial observation of Warburg 1956 on tumor glycolysis with lactate production is still a crucial observation [1]. Two fundamental findings complete the metabolic picture: the discovery of the M2 pyruvate kinase (PK) typical of tumors [2] and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade [35].

A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors over-express the high affinity hexokinase 2, which strongly interacts with the mitochondrial ANT-VDAC-PTP complex. In this position, close to the ATP/ADP exchanger (ANT), the hexokinase receives efficiently its ATP substrate [6,7]. As long as hexokinase occupies this mitochondria site, glycolysis is efficient. However, this has another consequence, hexokinase pushes away from the mitochondria site the permeability transition pore (PTP), which inhibits the release of cytochrome C, the apoptotic trigger [8]. The site also contains a voltage dependent anion channel (VDAC) and other proteins. The repulsion of PTP by hexokinase would reduce the pore size and the release of cytochrome C. Thus, the apoptosome-caspase proteolytic structure does not assemble in the cytoplasm. The liver hexokinase or glucokinase, is different it has less interaction with the site, has a lower affinity for glucose; because of this difference, glucose goes preferentially to the brain.

Further, phosphofructokinase gives fructose 1-6 bis phosphate; glycolysis is stimulated if an allosteric analogue, fructose 2-6 bis phosphate increases in response to a decrease of cAMP. The activation of insulin receptors in tumors has multiple effects, among them; a decrease of cAMP, which will stimulate glycolysis.

Another control point is glyceraldehyde P dehydrogenase that requires NAD+ in the glycolytic direction. If the oxygen supply is normal, the mitochondria malate/aspartate (MAL/ASP) shuttle forms the required NAD+ in the cytosol and NADH in the mitochondria. In hypoxic conditions, the NAD+ will essentially come via lactate dehydrogenase converting pyruvate into lactate. This reaction is prominent in tumor cells; it is the first discovery of Warburg on cancer.

At the last step of glycolysis, pyruvate kinase (PK) converts phosphoenolpyruvate (PEP) into pyruvate, which enters in the mitochondria as acetyl CoA, starting the citric acid cycle and oxidative metabolism. To explain the PK situation in tumors we must recall that PK only works in the glycolytic direction, from PEP to pyruvate, which implies that gluconeogenesis uses other enzymes for converting pyruvate into PEP. In starvation, when cells need glucose, one switches from glycolysis to gluconeogenesis and ketogenesis; PK and pyruvate dehydrogenase (PDH) are off, in a phosphorylated form, presumably following a cAMP-glucagon-adrenergic signal. In parallel, pyruvate carboxylase (Pcarb) becomes active. Moreover, in starvation, much alanine comes from muscle protein proteolysis, and is transaminated into pyruvate. Pyruvate carboxylase first converts pyruvate to OAA and then, PEP carboxykinase converts OAA to PEP etc…, until glucose. The inhibition of PK is necessary, if not one would go back to pyruvate. Phosphorylation of PK, and alanine, inhibit the enzyme.

Well, tumors have a PK and a PDH inhibited by phosphorylation and alanine, like for gluconeogenesis, in spite of an increased glycolysis! Moreover, in tumors, one finds a particular PK, the M2 embryonic enzyme [2,9,10] the dimeric, phosphorylated form is inactive, leading to a “bottleneck “. The M2 PK has to be activated by fructose 1-6 bis P its allosteric activator, whereas the M1 adult enzyme is a constitutive active form. The M2 PK bottleneck between glycolysis and the citric acid cycle is a typical feature of tumor cell glycolysis.

We also know that starvation mobilizes lipid stores from adipocyte to form ketone bodies, they are like glucose, nutriments for cells. Growth hormone, cAMP, AMP, activate a lipase, which provides fatty acids; their β oxidation cuts them into acetyl CoA in mitochondria and in peroxisomes for very long fatty acids; forming ketone bodies. Normally, citrate synthase slows down, to spare acetyl CoA for the ketogenic route, and OAA for the gluconeogenic pathway. Like for starvation, tumors mobilize lipid stores. But here, citrate synthase activity is elevated, condensing acetyl CoA and OAA [1113]; citrate increases, ketone bodies decrease. Consequently, ketone bodies will stop stimulating Pcarb. In tumors, the OAA needed for citrate synthase will presumably come from PEP, via reversible PEP carboxykinase or other sources. The quiescent Pcarb will not process the pyruvate produced by alanine transamination after proteolysis, leaving even more pyruvate to lactate dehydrogenase, increasing the lactate released by the tumor, and the NAD+ required for glycolysis.

Above the bottleneck, the massive entry of glucose accumulates PEP, which converts to OAA via mitochondria PEP carboxykinase, an enzyme requiring biotine-CO2-GDP. This source of OAA is abnormal, since Pcarb, another biotin-requiring enzyme, should have provided OAA. Tumors may indeed contain “morule inclusions” of biotin-enzyme [14] suggesting an inhibition of Pcarb, presumably a consequence of the maintained citrate synthase activity, and decrease of ketone bodies that normally stimulate Pcarb. The OAA coming via PEP carboxykinase and OAA coming from aspartate transamination or via malate dehydrogenase condenses with acetyl CoA, feeding the elevated tumoral citric acid condensation starting the Krebs cycle. Thus, tumors have to find large amounts of acetyl CoA for their condensation reaction; it comes essentially from lipolysis and β oxidation of fatty acids, and enters in the mitochondria via the carnitine transporter. This is the major source of acetyl CoA; since PDH that might have provided acetyl CoA remains in tumors, like PK, in the inactive phosphorylated form. The blockade of PDH [15] was recently reversed by inhibiting its kinase [16,17].

The key question is then to find out why NADH, a natural citrate synthase inhibitor did not switch off the enzyme in tumor cells. Probably, the synthesis of NADH by the dehydrogenases of the Krebs cycle and malate/aspartate shuttle, was too low, or the oxidation of NADH via the respiratory electron transport chain and mitochondrial complex1 (NADH dehydrogenase) was abnormally elevated. Another important point concerns PDH and α ketoglutarate dehydrogenase that are homologous enzymes, they might be regulated in a concerted way; when PDH is off, α ketoglutarate dehydrogenase might be also be slowed. Moreover, this could be associated to an upstream inhibition of aconinase by NO, or more probably to a blockade of isocitrate dehydrogenase, which favors in tumor cells, the citrate efflux from mitochondria, and the ATP citrate lyase route.

Normally, an increase of NADH inhibits the citrate condensation, favoring the ketogenic route associated to gluconeogenesis, which turns off glycolysis. Apparently, this regulation does not occur in tumors, since citrate synthase remains active. Moreover, in tumor cells, the α ketoglutarate not processed by
α ketoglutarate dehydrogenase converts to glutamate, via glutamate dehydrogenase, in this direction the reaction forms NAD+, backing up the LDH production. Other sources of glutamate are glutaminolysis, which increases in tumors [2].

The Figure Figure11 shows how tumors bypass the PK and PDH bottlenecks and evidently, the increase of glucose influx above the bottleneck, favors the supply of substrates to the pentose shunt, as pentose is needed for synthesizing ribonucleotides, RNA and DNA. The Figure Figure11 represents the stop below the citrate condensation. Hence, citrate quits the mitochondria to give via ATP citrate lyase, acetyl CoA and OAA in the cytosol of tumor cells. Acetyl CoA supports the synthesis of fatty acids and the formation of triglycerides. The other product of the ATP citrate lyase reaction, OAA, drives the transaminase cascade (ALAT and GOT transaminases) in a direction that consumes GLU and glutamine and converts in fine alanine into pyruvate and lactate plus NAD+. This consumes protein body stores that provide amino acids and much alanine (like in starvation).

The Figure Figure11 indicates that malate dehydrogenase is a source of NAD+ converting OAA into malate, which backs-up LDH. Part of the malate converts to pyruvate (malic enzyme) and processed by LDH. Moreover, malate enters in mitochondria via the shuttle and gives back OAA to feed the citrate condensation. Glutamine will also provide amino groups for the “de novo” synthesis of purine and pyrimidine bases particularly needed by tumor cells. The Figure Figure11 indicates that ASP shuttled out of the mitochondrial, joins the ASP formed by cytosolic transaminases, to feed the synthesis of pyrimidine bases via ASP transcarbamylase, a process also enhanced in tumor cells. In tumors, this silences the argininosuccinate synthetase step of the urea cycle [1820].

This blockade also limits the supply of fumarate to the Krebs cycle. The latter, utilizes the α ketoglutarate provided by the transaminase reaction, since α ketoglutarate coming via aconitase slows down. Indeed, NO and peroxynitrite increase in tumors and probably block aconitase. The Figure Figure11 indicates the cleavage of arginine into urea and ornithine. In tumors, the ornithine production increases, following the polyamine pathway. Ornithine is decarboxylated into putrescine by ornithine decarboxylase, then it captures the backbone of S adenosyl methionine (SAM) to form polyamines spermine then spermidine, the enzyme controlling the process is SAM decarboxylase. The other reaction product, 5-methlthioribose is then decomposed into methylthioribose and adenine, providing purine bases to the tumor. We shall analyze below the role of SAM in the carcinogenic mechanism, its destruction aggravates the process.

metabolic pathways 1476-4598-10-70-1
Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase,

In summary, it is like if the mechanism switching from gluconeogenesis to glycolysis was jammed in tumors, PK and PDH are at rest, like for gluconeogenesis, but citrate synthase is on. Thus, citric acid condensation pulls the glucose flux in the glycolytic direction, which needs NAD+; it will come from the pyruvate to lactate conversion by lactate dehydrogenase (LDH) no longer in competition with a quiescent Pcarb. Since the citrate condensation consumes acetyl CoA, ketone bodies do not form; while citrate will support the synthesis of triglycerides via ATP citrate lyase and fatty acid synthesis… The cytosolic OAA drives the transaminases in a direction consuming amino acid. The result of these metabolic changes is that tumors burn glucose while consuming muscle protein and lipid stores of the organism. In a normal physiological situation, one mobilizes stores for making glucose or ketone bodies, but not while burning glucose! Tumor cell metabolism gives them a selective advantage over normal cells. However, one may attack some vulnerable points.

Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, converting pyruvate to lactate, (Warburg effect) and NAD+ required for glycolysis. Cytosolic malate dehydrogenase also provides NAD+ (in OAA to MAL direction). Malate moves through the shuttle giving back OAA in the mitochondria. Below the PK-bottleneck, pyruvate dehydrogenase (PDH) is phosphorylated (second bottleneck). However, citrate condensation increases: acetyl-CoA, will thus come from fatty acids β-oxydation and lipolysis, while OAA sources are via PEP carboxy kinase, and malate dehydrogenase, (pyruvate carboxylase is inactive). Citrate quits the mitochondria, (note interrupted Krebs cycle). In the cytosol, ATPcitrate lyase cleaves citrate into acetyl CoA and OAA. Acetyl CoA will make fatty acids-triglycerides. Above all, OAA pushes transaminases in a direction usually associated to gluconeogenesis! This consumes protein stores, providing alanine (ALA); like glutamine, it is essential for tumors. The transaminases output is aspartate (ASP) it joins with ASP from the shuttle and feeds ASP transcarbamylase, starting pyrimidine synthesis. ASP in not processed by argininosuccinate synthetase, which is blocked, interrupting the urea cycle. Arginine gives ornithine via arginase, ornithine is decarboxylated into putrescine by ornithine decarboxylase. Putrescine and SAM form polyamines (spermine spermidine) via SAM decarboxylase. The other product 5-methylthioadenosine provides adenine. Arginine deprivation should affect tumors. The SAM destruction impairs methylations, particularly of PP2A, removing the “signaling kinase brake”, PP2A also fails to dephosphorylate PK and PDH, forming the “bottlenecks”. (Black arrows = interrupted pathways).

 II Starters for cancer metabolic anomaly

1. Lessons from oncogenes

Following the discovery of Rous sarcoma virus transmitting cancer [21], we have to wait the work of Stehelin [22] to realize that this retrovirus only transmitted a gene captured from a previous host. When one finds that the transmitted gene encodes the Src tyrosine kinase, we are back again to the tyrosine kinase signals, similar to those activated by insulin or IGF, which control carbohydrate metabolism, anabolism and mitosis.

An up regulation of the gene product, now under viral control causes tumors. However, the captured viral oncogene (v-oncogene) derives from a normal host gene the proto-oncogene. The virus only perturbs the expression of a cellular gene the proto-oncogene. It may modify its expression, or its regulation, or transmit a mutated form of the proto-oncogene. Independently of any viral infection, a similar tumorigenic process takes place, if the proto-oncogene is translocated in another chromosome; and transcribed under the control of stronger promoters. In this case, the proto-oncogene becomes an oncogene of cellular origin (c-oncogene). The third mode for converting a prot-oncogene into an oncogene occurs if a retrovirus simply inserts its strong promoters in front of the proto-oncogene enhancing its expression.

It is impressive to find that retroviral oncogenes and cellular oncogenes disturb this major signaling pathway: the MAP kinases mitogenic pathways. At the ligand level we find tumors such Wilm’s kidney cancer, resulting from an increased expression of insulin like growth factor; we have also the erbB or V-int-2 oncogenes expressing respectively NGF and FGF growth factor receptors. The receptors for these ligands activate tyrosine kinase signals, similarly to insulin receptors. The Rous sarcoma virus transmits the src tyrosine kinase, which activates these signals, leading to a chicken leukemia. Similarly, in murine leukemia, a virus captures and retransmits the tyrosine kinase abl. Moreover, abl is also stimulated if translocated and expressed with the bcr gene of chromosome 22, as a fusion protein (Philadelphia chromosome). Further, ahead Ras exchanging protein for GTP/GDP, and then the Raf serine-threonine kinases proto-oncogenes are known targets for oncogenes. Finally, at the level of transcription factors activated by MAP kinases, one finds cjun, cfos or cmyc. An avian leucosis virus stimulates cmyc, by inserting its strong viral promoter. The retroviral attacks boost the mitogenic MAP kinases similarly to inflammatory cytokins, or to insulin signals, that control glucose transport and gycolysis.

In addition to the MAP kinase mitogenic pathway, tyrosine kinase receptors activate PI3 kinase pathways; PTEN phosphatase counteracts this effect, thus acting as a tumor suppressor. Recall that a DNA virus, the Epstein-Barr virus of infectious mononucleose, gives also the Burkitt lymphoma; the effect of the virus is to enhance PI3 kinase. Down stream, we find mTOR (the target of rapamycine, an immune-suppressor) mTOR, inhibits PP2A phosphatase, which is also a target for the simian SV40 and Polyoma viruses. Schematically, one may consider that the different steps of MAP kinase pathways are targets for retroviruses, while the different steps of PI3 kinase pathway are targets for DNA viruses. The viral-driven enhanced function of these pathways mimics the effects of their prolonged activation by their usual triggers, such as insulin or IGF; one then expects to find an associated increase of glycolysis. The insulin or IGF actions boost the cellular influx of glucose and glycolysis. However, if the signaling pathway gets out of control, the tyrosine kinase phosphorylations may lead to a parallel PK blockade [35] explaining the tumor bottleneck at the end of glycolysis. Since an activation of enyme kinases may indeed block essential enzymes (PK, PDH and others); in principle, the inactivation of phosphatases may also keep these enzymes in a phosphorylated form and lead to a similar bottleneck and we do know that oncogenes bind and affect PP2A phosphatase. In sum, a perturbed MAP kinase pathway, elicits metabolic features that would give to tumor cells their metabolic advantage.

2. The methylation hypothesis and the role of PP2A phosphatase

In a remarkable comment, Newberne [23] highlights interesting observations on the carcinogenicity of diethanolamine [24] showing that diethanolamine decreased choline derivatives and methyl donors in the liver, like does a choline deficient diet. Such conditions trigger tumors in mice, particularly in the B6C3F1 strain. Again, the historical perspective recalled by Newberne’s comment brings us back to insulin. Indeed, after the discovery of insulin in 1922, Banting and Best were able to keep alive for several months depancreatized dogs, treated with pure insulin. However, these dogs developed a fatty liver and died. Unlike pure insulin, the total pancreatic extract contained a substance that prevented fatty liver: a lipotropic substance identified later as being choline [25]. Like other lipotropes, (methionine, folate, B12) choline supports transmethylation reactions, of a variety of substrates, that would change their cellular fate, or action, after methylation. In the particular case concerned here, the removal of triglycerides from the liver, as very low-density lipoprotein particles (VLDL), requires the synthesis of lecithin, which might decrease if choline and S-adenosyl methionine (SAM) are missing. Hence, a choline deficient diet decreases the removal of triglycerides from the liver; a fatty liver and tumors may then form. In sum, we have seen that pathways exemplified by the insulin-tyrosine kinase signaling pathway, which control anabolic processes, mitosis, growth and cell death, are at each step targets for oncogenes; we now find that insulin may also provoke fatty liver and cancer, when choline is not associated to insulin.

We must now find how the lipotropic methyl donor controls the signaling pathway. We know that after the tyrosine kinase reaction, serine-threonine kinases take over along the signaling route. It is thus highly probable that serine-threonine phosphatases will counteract the kinases and limit the intensity of the insulin or insulin like signals. One of the phosphatases involved is PP2A, itself the target of DNA viral oncogenes (Polyoma or SV40 antigens react with PP2A subunits and cause tumors). We found a possible link between the PP2A phosphatase brake and choline in works on Alzheimer’s disease [26]. Indeed, the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyle, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated [27].

In Alzheimer’s disease, the poor methylation of PP2A is associated to an increase of homocysteine in the blood [26]. The result of the PP2A methylation failure is a hyperphosphorylation of Tau protein and the formation of tangles in the brain. Tau protein is involved in tubulin polymerization, controlling axonal flow but also the mitotic spindle. It is thus possible that choline, via SAM, methylates PP2A, which is targeted toward the serine-threonine kinases that are counteracted along the insulin-signaling pathway. The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes [28].

3. Cellular distribution of PP2A

In fact, the negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex. The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited [29]. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes. The carboxyl methylation of C-terminal leucine 309 of the catalytic C unit, permits to a dimeric form made of C and a structural unit A, to recruit one of the many regulatory units B, giving a great diversity of possible enzymes and effects. The different methylated ABC trimers would then find specific targets. It is consequently essential to have more information on methyl transferases and methyl esterases that control the assembly or disassembly of PP2A trimeric forms.

A specific carboxyl methyltransferase for PP2A [30] was purified and shown to be essential for normal progression through mitosis [31]. In addition, a specific methylesterase that demethylates PP2A has been purified [32]. Is seems that the methyl esterase cancels the action of PP2A, on signaling kinases that increase in glioma [33]. Evidently, the cellular localization of the methyl transferase (LCMT-1) and the phosphatase methyl esterase (PME-1) are crucial for controlling PP2A methylation and targeting. Apparently, LCMT-1 mainly localizes to the cytoplasm and not in the nucleus, where PME-1 is present, and the latter harbors a nuclear localization signal [34]. From these observations, one may suggest that PP2A gets its methyles in the cytoplasm and regulates the tyrosine kinase-signaling pathway, attenuating its effects.

A methylation deficit should then decrease the methylation of PP2A and boost the mitotic insulin signals as discussed above for choline deficiency, steatosis and hepatoma. At the nucleus, where PME-1 is present, it will remove the methyl, from PP2A, favoring the formation of dimeric AC species that have different targets, presumably proteins involved in the cell cycle. It is interesting to quote here the structural mechanism associated to the demethylation of PP2A. The crystal structures of PME-1 alone or in complex with PP2A dimeric core was reported [35] PME-1 binds directly to the active site of PP2A and this rearranges the catalytic triad of PME-1 into an active conformation that should demethylate PP2A, but this also seems to evict a manganese required for the phosphatase activity. Hence, demethylation and inactivation would take place in parallel, blocking mitotic actions.

However, another player is here involved, the so-called PTPA protein, which is a PP2A phosphatase activator. Apparently, this activator is a new type of cis/trans of prolyl isomerase, acting on Pro190 of the catalytic C unit isomerized in presence of Mg-ATP [36], which would then cancel the inactivation mediated by PME-1. Following the PTPA action, the demethylated phosphatase would become active again in the nucleus, and stimulate cell cycle proteins [37,38] inducing mitosis. Unfortunately, the ligand of this new prolyl isomerase is still unknown. Moreover, we have to consider that other enzymes such as cytochrome P450 have also demethylation properties.

In spite of deficient methylations and choline dehydrogenase pathway, tumor cells display an enhanced choline kinase activity, associated to a parallel synthesis of lecithin and triglycerides.

The hypothesis to consider is that triglycerides change the fate of methylated PP2A, by targeting it to the nucleus, there a methylesterase demethylates it; the phosphatase attacks new targets such as cell cycle proteins, inducing mitosis. Moreover, the phosphatase action on nuclear membrane proteins may render the nuclear membrane permeable to SAM the general methyl donor; promoters get methylated inducing epigenetic changes.

The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells.

In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism. This would decrease triglycerides, promote the methylation of PP2A and keep it in the cytosol, reestablishing the brake over signaling kinases.

Hypoxia is an essential issue to discuss

Many adequate “adult proteins” replace their fetal isoform: muscle proteins utrophine, switches to dystrophine; enzymes such as embryonic M2 PK [39] is replaced by M1. Hypoxic conditions seem to trigger back the expression of the fetal gene packet via HIF1-Von-Hippel signals. The mechanism would depend of a double switch since not all fetal genes become active after hypoxia. First, the histones have to be in an acetylated form, opening the way to transcription factors, this depends either of histone deacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch. Second, a more specific switch must be open, indicating the adult/fetal gene couple concerned, or more generally the isoform of a given gene that is more adapted to the specific situation. When the adult gene mutates, an unbound ligand may indeed indicate, directly or indirectly, the particular fetal copy gene to reactivate [40]. In anoxia, lactate is more difficult to release against its external gradient, leading to a cytosolic increase of up-stream glycolytic products, 3P glycerate or others. These products may then be a second signal controlling the specific switch for triggering the expression of fetal genes, such as fetal hemoglobin or the embryonic M2 PK; this takes place if histones (main switch) are in an acetylated form.

Growth hormone-IGF actions, the control of asymmetrical mitosis

When IGF – Growth hormone operate, the fatty acid source of acetyl CoA takes over. Indeed, GH stimulates a triglyceride lipase in adipocytes, increasing the release of fatty acids and their β oxidation. In parallel, GH would close the glycolytic source of acetyl CoA, perhaps inhibiting the hexokinase interaction with the mitochondrial ANT site. This effect, which renders apoptosis possible, does not occur in tumor cells. GH mobilizes the fatty acid source of acetyl CoA from adipocytes, which should help the formation of ketone bodies, but since citrate synthase activity is elevated in tumors, ketone bodies do not form.

Compounds for correcting tumor metabolism

The figure figure1 indicates interrupted and enhanced metabolic pathways in tumor cells.

In table table1,1, the numbered pathways represent possible therapeutic targets; they cover several enzymes. When the activity of the pathway is increased, one may give inhibitors; when the activity of the pathway decreases, we propose possible activators

Table - metabolic  targets

Table 1 Mol Cancer. 2011; 10 70. Published online Jun 7, 2011. doi  10.1186_1476-4598-10-70

The origin of Cancers by means of metabolic selection

The disruption of cells by internal or external compounds, releases substrates stimulating the tyrosine kinase signals for anabolism proliferation and stem cell repair, like for most oncogenes. If such signals are not limited, there is a parallel blockade of key metabolic enzymes by activated kinases or inhibited phosphatases. The result is a metabolism typical of tumor cells, which gives them a selective advantage; stabilized by epigenetic changes. A proliferation process, in which the two daughter cells divide, increases the tumor mass at the detriment of the body. Inevitable mutations follow.

Maurice Israël, et al. Mol Cancer. 2011;10:70-70.
Transcriptomics and Regulatory Processes

What are lncRNAs?

It was traditionally thought that the transcriptome would be mostly comprised of mRNAs, however advances in high-throughput RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNA is now known to make up the majority of transcribed RNAs and in addition to those that carry out well-known housekeeping functions (e.g. tRNA, rRNA etc), many different types of regulatory RNAs have been and continue to be discovered.

Long noncoding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins. Their expression is developmentally regulated and lncRNAs can be tissue- and cell-type specific. A significant proportion of lncRNAs are located exclusively in the nucleus. They are comprised of many types of transcripts that can structurally resemble mRNAs, and are sometimes transcribed as whole or partial antisense transcripts to coding genes. LncRNAs are thought to carry out important regulatory functions, adding yet another layer of complexity to our understanding of genomic regulation.

lncRNA-s   A summary of the various functions described for lncRNA

 

The evolution of genome-scale models of cancer metabolism
The importance of metabolism in cancer is becoming increasingly apparent with the identification of metabolic enzyme mutations and the growing awareness of the influence of metabolism on signaling, epigenetic markers, and transcription. However, the complexity of these processes has challenged our ability to make sense of the metabolic changes in cancer. Fortunately, constraint-based modeling, a systems biology approach, now enables one to study the entirety of cancer metabolism and simulate basic phenotypes. With the newness of this field, there has been a rapid evolution of both the scope of these models and their applications. (NE Lewis and AM.Abdel-Haleem. frontiers physiol  2013;4(237): 1   http://dx.doi.org/10.3389/fphys.2013.00237)

Here we review the various constraint-based models built for cancer metabolism and how their predictions are shedding new light on basic cancer phenotypes, elucidating pathway differences between tumors, and discovering putative anti-cancer targets. As the field continues to evolve, the scope of these genome-scale cancer models must expand beyond central metabolism to address questions related to the diverse processes contributing to tumor development and metastasis.

“One of the goals of cancer research is to ascertain the mechanisms of cancer.”These words, penned by Dulbecco (1986), began a treatise on how a mechanistic understanding of cancer requires a sequenced human genome. Now with the abundance of sequence data, we are finding diverse genetic changes among different cancers (Vogelstein et al.,2013). While we are cataloging these mutations, the associated mechanisms leading to phenotypic changes are often unclear since mutations occur in the context of complex biological networks. For example, mutations to isocitrate dehydrogenase lead to oncometabolite synthesis, which alters DNA methylation and ultimately changes gene expression and the balance of normal cell processes (Sasakietal.,2012). Furthermore, many different combinations of mutations can lead to cancer. Since the genetic heterogeneity between tumors can be large, the biomolecular mechanisms underlying tumor physiology can vary substantially.

This is apparent in metabolism, where tumors can differ in serine metabolism  dependence (Possematoetal., 2011) or TCA cycle function (Frezzaetal., 2011b). In addition, diverse mutations can alter NADPH synthesis by differentially regulat ing  signaling pathways, such as the AMPK pathway (Cairnsetal., 2011; Jeonetal., 2012). The challenges regarding complexity and heterogeneity in cancer metabolism are beginning to be addressed with the COnstraint-Based Reconstruction and Analysis (COBRA) approach (Hernández Patiñoetal., 2012; Sharma and König,  2013), an emerging field in systems biology.Specifically, it accounts for the complexity of the perturbed biochemical processes by using genome-scale metabolic network reconstructions (Duarteetal., 2007; Maetal., 2007;Thieleetal., 2013).

In a reconstruction, the stoichiometric chemical reactions in a cell are carefully annotated and stitched together into a large network, often containing thousands of reactions. Genes and enzymes associated with each reaction are also delineated. The networks are converted into computational models and analyzed using many algorithms (Lewisetal., 2012). COBRA approaches are also beginning to address heterogeneity in cancer by integrating experimental data with the reconstructions (Blazier and Papin, 2012; Hydukeetal., 2013)  to tailor the models to the unique gene expression profiles of general cancer tissue, and even individual cell lines and tumors. Here we describe the recent conceptual evolution that has occurred for constraint-based cancer modeling.

Targeting of  gene expression

Tumor Suppressor Genes and its Implications in Human Cancer

Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudson’s two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. (K Inoue, EA Fry and Pj Taneja. Recent Progress in Mouse Models for Tumor Suppressor Genes and its Implications in Human Cancer. Clinical Medicine Insights: Oncology2013:7 103–122). This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, β-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia.

Targeting cancer metabolism – aiming at a tumour’s sweet-spot
Neil P. Jones and Almut Schulze
Drug Discovery Today   January 2012

Targeting cancer metabolism has emerged as a hot topic for drug discovery. Most cancers have a high demand for metabolic inputs (i.e. glucose/glutamine), which aid proliferation and survival. Interest in targeting cancer metabolism has been renewed in recent years with the discovery that many cancer related (e.g. oncogenic and tumor suppressor) pathways have a profound effect on metabolism and that many tumors become dependent on specific metabolic processes. Considering the recent increase in our understanding of cancer metabolism and the increasing knowledge of the enzymes and pathways involved, the question arises: could metabolism be cancer’s Achilles heel?
During recent years, interest into the possible therapeutic benefit of targeting metabolic pathways in cancer has increased dramatically with academic and pharmaceutical groups actively pursuing this aspect of tumor physiology. Therefore, what has fuelled this revived interest in targeting cancer metabolism and what are the major advances and potential challenges faced in the race to develop new therapeutics in this area? This review will attempt to answer these questions and illustrate why we, and others, believe that targeting metabolism in cancer presents such a promising therapeutic rationale.

Oncogenes and cancer metabolism
Glycolysis  TCA cycle  Pentose phosphate pathway

 FIGURE 1

Schematic representation of the regulation of cancer metabolism pathways. Metabolic enzymes are regulated by signaling pathways involving oncogenes and tumor suppressors. Complex regulatory mechanisms, key pathway interactions and enzymes are shown along with key metabolic endpoints (shown in purple) necessary for proliferation and survival (biosynthetic intermediates and NADPH). Key oncogenic pathways are shown in green and key tumor suppressor pathways are shown in red. Mutant IDH (mIDH) pathway is listed but is only functional in cancers containing mIDH.

FIGURE 2

Schematic representation of key components of the pentose phosphate pathway (PPP). Key enzymes are shown in blue boxes and key intermediates in purple text/box outline. DNA damage can activate ATM which in turn activates G6PDH to upregulate nucleotide synthesis for DNA repair and NAPDH to combat reactive oxygen species. PPP is also regulated by the tumour suppressor p53. The PPP can function as two separate branches (oxidative and non-oxidative) or be coupled into a recycling pathway – the pentose phosphate shunt – for maximum NADPH production.

Serine biosynthesis

Another branch diverting from glycolysis recently implicated in cancer is the serine biosynthesis pathway which converts the glycolytic intermediate 3-phosphoglycerate into serine (Fig. 3). Serine is an amino acid and an important neurotransmitter but can also provide fuel for the synthesis of other amino acids and nucleotides. The serine biosynthesis pathway also provides another key metabolic intermediate, a-KG, from glutamate breakdown via the action of phosphoserine aminotransferase (PSAT1). This pathway couples glycolysis (via 3-phosphoglycerate) with glutaminolysis (via glutamate), thereby linking two metabolic pathways known to be activated in many cancers.

FIGURE 3

Schematic representation of the serine biosynthesis pathway. Synthesis of serine involves integration of metabolites from glycolysis and  glutaminolysis pathways  and generates a-ketoglutarate, a key biosynthetic intermediate, and serine. Serine has many essential uses in the cell including amino acid, phospholipid and nucleotide synthesis.

 

Silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1)

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressorgenes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely,demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action.  (Mayada Achour, et al. Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1.  Biochemical and Biophysical Research Communications 430 (2013) 208–212.    http://dx.doi.org/10.1016/j.bbrc.2012.11.087)

Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16INK4A  is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.

Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.  (Citation: Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

EGF Receptor

 Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction.

(CM Ardito,BM Gruner. ,EGF Receptor Is Required for KRAS-Induced Pancreatic Tumorigenesis.  http://dx.doi.org/10.1016/j.ccr.2012.07.024)

Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation

The almost universal lethality of PDA has led to the intense study of genetic mutations responsible for its formation and progression. The most common oncogenic mutations associated with all PDA stages are found in the KRAS gene, suggesting it as the primary initiator of pancreatic neoplasia. However, mutant Kras expression throughout the mouse pancreatic parenchyma shows that the oncogene remains largely indolent until secondary events, such as pancreatitis, unlock its transforming potential. We find KRAS requires an inside-outside-in signaling axis that involves ligand-dependent EGFR activation to initiate the signal transduction and cell biological changes that link PDA and pancreatitis. (Cancer Cell (2012); 22: 304–317).

HER4 (EGFR/ErbB, HER2/Neu, HER3)

 ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.

ErbB4 Possesses Multiple Functional Motifs and Mutations Have Been Engineered to Target These Motifs.

The organization of ErbB4 is as indicated in this schematic. The extracellular ligand-binding motifs reside in the amino-terminal region upstream of amino acid residue 651. The singlepass transmembrane domain consists of amino acid residues 652-675. The cytoplasmic tyrosine kinase domain consists of amino acid residues 713-989. The majority of cytoplasmic sites of tyrosine phosphorylation reside in amino acid residues 990-1308, most notably Tyr1056. Additional putative functional motifs include a TACE cleavage site, a gamma-secretase cleavage site, two LXXLL (steroid hormone receptor binding) motifs, a BH3 domain, three WW domain binding motifs, and a PDZ domain binding motif. Mutations that disrupt these motifs are noted. Finally, note the two locations of alternative transcriptional splicing, resulting in a total of four different splicing isoforms.

 

 

 

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavageby gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

(Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Resistance to Receptor Tyrosine Kinase Inhibition

Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and such cancers are often sensitive to specific inhibitors of the activated kinase. Two well-established examples of this paradigm include lung cancers with either EGFR mutations or ALK translocations. In these cancers, inhibition of the corresponding RTK leads to suppression of key downstream signaling pathways, such as the PI3K (phosphatidylinositol 3-kinase)/AKT and MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal–regulated kinase) pathways, resulting in cell growth arrest and death. Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers, resistance invariably develops, typically within 1 to 2 years. (MJ Niederst and JA Engelman. Sci Signal, 24 Sep 2013; 6(294), p. re6 .  http://dx.doi.org/10.1126/scisignal.2004652)

Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK. A second is activation of alternative RTKs that maintain the signaling of key downstream pathways despite sustained inhibition of the original drug target. Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples. In this mini-review, we summarize the concepts underlying RTK-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.

The TGF-β Pathway

Aberrations in the enzymes that modify ubiquitin moieties have been observed to cause a myriad of diseases, including cancer. Therefore a better understanding of these enzymes and their substrates will lead to the identification of prospective druggable targets. Here we discuss the role of ubiquitin modifying enzymes in the canonical TGF-β pathway highlighting the ubiquitin regulating enzymes, which may potentially be targeted by small molecule inhibitors. (Pieter Eichhorn. (DE) -Ubiquitination in The TGF-β Pathway. J Cancer Res Therap Oncol 2013; 1: 1-6).

TGF-β is a multifunctional cytokine that plays a key role in embryogenesis and adult tissue homoeostasis. TGF-β is secreted by a myriad of cell types triggering a varied array of cellular functions including apoptosis, proliferation, migration, endothelial and mesenchymal transition, and extracellular matrix production. Downstream TGFβ responses can also be modulated by other signalling pathways (i.e. PI3K, ERK, WNT, etc.) resulting in a complex web of TGF-β pathway activation or repression depending on the nature of the signal and cellular context. Apart from TGF-β mediated cell autonomous effects TGF-β can further play an important function in regulating tumour microenvironments effecting the interaction between stromal fibroblasts and tumour cells.
Due to the central role of TGF-β in cellular processes it is therefore unsurprising that loss of TGF-β pathway integrity is frequently observed in a variety of human diseases, including cancer. However, the TGF-β pathway plays a complex dual role in cancer. In normal epithelial cells and premalignant cells TGF-β acts a potent tumor suppressor eliciting a cytostatic response inhibiting tumor progression. Supporting this notion, inactivating mutations in members of the TGF-βpathway have been observed in a variety of cancers including pancreatic, colorectal, and head and neck cancer.

In contrast, during tumor progression the TGF-β antiproliferative function is lost, and in certain advanced cancers TGF-β becomes an oncogenic factor inducing cellular proliferation, invasion, angiogenesis, and immune suppression. As a consequence, the TGFβ pathway is currently considered a therapeutic target in advanced cancers and several anti- TGF-β agents in clinical trials have shown promising results. However, due to the complex dichotomous role of TGF-β in oncogenesis a detailed understanding of TGF-β biology is required in order to design successful therapeutic strategies to identify patient populations that will benefit most from these compounds.

G protein receptor

 G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers.

(D Leyva-Illades, S DeMorrow . Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management.  Cancer Management and Research 2013:5 147–155)

Signal transduction is essential for maintaining cellular homeostasis and to coordinate the activity of cells in all organisms. Proteins localized in the cell membrane serve as the interface between the outside and inside of the cell. G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are encoded by at least 800 genes in the human genome. GPCRs are also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors. GPCRs can detect an expansive array of extracellular signals or ligands that include photons, ions, odors, pheromones, hormones, and neurotransmitters. Nonsensory GPCRs (excluding light, odor, and taste receptors) have been classified into four families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and frizzled receptors. They have a peculiar structure that has been highly conserved over the course of evolution and are made up of an amino acid chain, the N-terminal of which is localized outside of the cellular membrane and the C-terminal in the cytoplasm. The amino acid chain spans the cellular membrane seven times and has three intracellular and three extracellular loops.

GPCRs are called that because they exert their actions by associating with a family of heterotrimeric proteins (made up of α, β, and γ subunits) that are capable of binding and hydrolyzing guanosine triphosphate (GTP).To date, 16 different α subunits, five β subunits, and 11 γ subunits have been described in mammalian tissues. When activated, these receptors undergo conformational changes that are mechanically transduced to the G proteins, which then initiate a cycle of activation and inactivationassociated with the binding and hydrolysis of GTP. Activated G proteins can then positively or negatively modulate ion channels (mainly potassium and calcium) or the second messenger generating enzymes (ie, adenylate cyclase and phospholipase C [PLC]) that allow the signal to be propagated to the interior of the cell to ultimately affect cell function.

 Matrix Metalloproteinases

Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all  components of the ECM. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.

(MATRIX METALLOPROTEINASES IN CANCER: PROGNOSTIC MARKERS AND THERAPEUTIC TARGETS.

Pia Vihinen and Veli-Matti Kahari.  Int. J. Cancer 2002;99: 157–166. http://dx.doi.org/10.1002/ijc.10329

Common properties of the MMPs include the requirement of zinc in their catalytic site for activity and their synthesis as inactive zymogens that generally need to be proteolytically cleaved to be active. Normally the MMPs are expressed only when and where needed for tissue remodeling accompanies various processes such as during embryonic development, wound healing, uterine and mammary involution, cartilage-to-bone transition during ossification, and trophoblast invasion into the endometrial stoma during placenta development. However, aberrant expression of various MMPs has been correlated with pathological conditions, such as periodontitis, rheumatoid arthritis, and tumor cell invasion and metastasis .

There are now over 20 members of the MMP family, and they can be subgrouped based on their structures. The minimal domain structure consists of a signal peptide, prodomain, and catalytic domain. The propeptide domain contains a conserved cysteine residue (the “cysteine switch”) that coordinates to the catalytic zinc to maintain inactivity. MMPs with only the minimal domain are referred to as matrilysins (MMP-7 and -26). The most common structures for secreted MMPs, including collagenases and stromelysins, have an additional hemopexin-like domain connected by a hinge region to the catalytic domain (MMP-1, -3, -8, -10, -12, -13, -19, and -20).

Terms: 1FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin

FIGURE 1 – Structure of human matrix metalloproteinases

 

FIGURE 1 – Structure of human matrix metalloproteinases. The signal peptide directs the proenzyme for secretion. The propeptide contains a conserved sequence (PRCGxPD), in which the cysteine forms a covalent bond (cysteine switch), with the catalytic zinc (Zn2_) to maintain the latency of proMMPs. Catalytic domain contains the highly conserved zinc binding site (HExGHxxGxxHS) in which Zn2_is coordinated by 3 histidines. The proline-rich hinge region links the catalytic domain to the hemopexin domain, which determines the substrate specificity of specific MMPs. The hemopexin domain is absent in matrilysin (MMP-7) and matrilysin-2 (endometase, MMP-26). Gelatinases  A and B (MMP-2 and MMP-9, respectively) contain 3 repeats of the fibronectin-type II domain inserted in the catalytic domain. MT1-, MT2-, MT3- and MT5-MMP contain a transmembrane domain and MT4- and MT6-MMPs contain a glycosylphosphatidylinositol (GPI) anchor in the C-terminus of the molecule, which attach these MMPs to the cell surface. MT-MMPs, MMP-11, MMP-23 and MMP-28 contain a furin cleavage site (RxKR) between the propeptide and catalytic domain, making these proenzymes susceptible to activation by intracellular furin convertases. MMP-23 contains an N-terminal signal anchor, which anchors proMMP-23 to the Golgi complex and has a different C-terminal domain instead of hemopexin-like domain.

The physiologic expression of MMP-13 in vivo is limited to situations, such as fetal bone development and fetal wound repair, in which rapid remodeling of collagenous ECM is required. MMP-13 is expressed in pathologic conditions, such as arthritis, chronic dermal and intestinal ulcers, chronic periodontal inflammation and atherosclerotic plaques. The expression of MMP-13 is detected in vivo in invasive malignant tumours, breast carcinomas, squamous cell carcinomas (SCCs) of the head and neck and vulva, malignant melanomas, chondrosarcomas and urinary bladder carcinomas.

Table I. Human MMPS, their chromosomal localization, substrates, exogenous activators, and activating capacity1
Enzyme Chromosomal location Substrates Activated by Activator of
  • FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin.

    …………..

Collagenases
 Collagenase-1 (MMP-1) 11q22.2-22.3 Collagen I, II, III, VII, VIII, X, aggregan, serpins, 2M MMP-3, -7, -10, plasmin kallikrein, chymase MMP-2
 Collagenase-2 (MMP-8) 11q22.2-22.3 Collagen I, II, III, aggregan, serpins, 2M MMP-3, -10, plasmin ND
 Collagenase-3 (MMP-13) 11q22.2-22.3 Collagen I, II, III, IV, IX, X, XIV, gelatin, FN, laminin, large tenascin aggrecan, fibrillin, osteonectin, serpins MMP-2, -3, -10, -14, -15, plasmin MMP-2, -9
Stromelysins
 Stromelysin-1 (MMP-3) 11q22.2-22.3 Collagen IV, V, IX, X, FN, elastin, gelatin, laminin, aggrecan, nidoge fibrillin*, osteonectin*, 1PI*, myelin basic protein*, OP, E-cadherin Plasmin, kallikrein, chymas tryptase MMP-1, -8, -9, -13
 Stromelysin-2 (MMP-10) 11q22.2-3 As MMP-3, except * Elastase, cathepsin G MMP-1, -7, -8, -9, -13
Stromelysin-like MMPs
 Stromelysin-3 (MMP-11) 22q11.2 Serine proteinase inhibitors, 1PI Furin ND
 Metalloelastase (MMP-12) 11q22.2-22.3 Collagen IV, gelatin, FN, laminin, vitronectin, elastin, fibrillin, 1-PI, myelin basic protein, apolipoprotein A ND ND
Matrilysins
 Matrilysin (MMP-7) 11q22.2-22.3 Elastin, FN, laminin, nidogen, collagen IV, tenascin, versican, 1PI, O E-cadherin, TNF- MMP-3, plasmin MMP-9
 Matrilysin-2 (MMP-26) 11q22.2 Gelatin, 1PI, synthetic MMP-substrates, TACE-substrate ND ND
Gelatinases
 Gelatinase A (MMP-2) 16q13 Gelatin, collagen I, IV, V, VII, X, FN, tenascin, fibrillin, osteonectin, Monocyte chemoattractant protein 3 MMP-1, -13, -14, -15, -16, -tryptase? MMP-9, -13
 Gelatinase B (MMP-9) 20q12-13 Gelatin, collagen IV, V, VII, XI, XIV, elastin, fibrillin, osteonectin 2 MMP-2, -3, 7, -13, plasmin, trypsin, chymotrypsin, cathepsin G ND
Membrane-type MMPs
 MT1-MMP (MMP-14) 14q12.2 Collagen I, II, III, gelatin, FN, laminin, vitronectin, aggrecan, tenasci nidogen, perlecan, fibrillin, 1PI, 2M, fibrin Plasmin, furin MMP-2, -13
 MT2-MMP (MMP-15) 16q12.2 FN, laminin, aggrecan, tenascin, nidogen, perlecan ND MMP-2, -13

 

MMP expression and activity are regulated at several levels. In most cases, MMPs are not synthesized until needed. Transcription can be induced by various signals including cytokines, growth factors, and mechanical stress. In certain cases, regulation of mRNA stability and translational efficiencyhave been reported. Because most MMPs are secreted as inactive zymogens, they need to be activated, usually by proteolytic cleavage of their NH2-terminal prodomains. Some MMPs are activated by other serine proteases such as plasmin and furin, whereas some of the MMPs can activate other members of their family. The most well characterized is the activation of pro-MMP-2 by MT1-MMP.

A number of MMPs have been strongly implicated in multiple stages of cancer progression including the acquisition of invasive and metastatic properties. Thus, efforts have been made for the past 20 years to develop MMPIs that can be used to halt the spread of cancer, which is what ultimately kills the person. However, initial clinical trials using first generation MMPIs proved to be disappointing . In the ensuing years, much has been learned about the roles of specific MMPs in the different processes of carcinogenesis and more specific MMPIs are being developed and brought to clinical trials.

However, the dosing and scheduling for optimal efficacy is not the same as required for conventional cytotoxic drugs because the MMPIs do not directly kill cancer cells, but instead target such processes as angiogenesis (the development of new blood vessels), invasion, and metastatic spread. (Matrix Metalloproteinases, Angiogenesis, and Cancer. Joyce E. Rundhaug.  Commentary re: A. C. Lockhart et al., Reduction of Wound Angiogenesis in Patients Treated with BMS-275291, a Broad Spectrum Matrix Metalloproteinase Inhibitor. Clin. Cancer Res., 2003; 9551–554).

 Role of p38 MAP Kinase Signal Transduction in Solid Tumors

HK Koul, M Pal, and S Koul. Genes & Cancer  2013 ; 4(9-10) 342–359.  http://dx.doi.org/10.1177/ 1947601913507951

Mitogen-activated protein kinases (MAPKs) mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the main subgroups, the p38 MAP kinases, has been implicated in a wide range of complex biologic processes, such as cell proliferation, cell differentiation, cell death, cell migration, and invasion. Dysregulation of p38 MAPK levels in patients are associated with advanced stages and short survival in cancer patients (e.g., prostate, breast, bladder, liver, and lung cancer). p38 MAPK plays a dual role as a regulator of cell death, and it can either mediate cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner. In addition to modulating cell survival, an essential role of p38 MAPK in modulation of cell migration and invasion offers a distinct opportunity to target this pathway with respect to tumor metastasis. The specific function of p38 MAPK appears to depend not only on the cell type but also on the stimuli and/or the isoform that is activated.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved among eukaryotes and have been implicated to play key roles in a number of biological processes, including cell growth, differentiation, apoptosis, inflammation, and responses to environmental stresses.

They are typically organized in 3-tiered architecture consisting of a MAPK, a MAPK activator (MAPK kinase), and a MAPKK activator (MAPKK kinase). The MAPK pathways can be regulated at multiple levels as well as via multiple mechanisms, of which the regulation of mitogen-activated protein kinase kinase kinase (MAPKKK/MAP3K) has been proved to be the most challenging due to the great diversity and versatility between different modules at this level. The complex array of growth factors and other ligands that can initiate intracellular cell signaling requires a very high level of coordination among the different proteins involved.

GTP cyclohydrolase (GCH1)

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth.

(G Picker, Hee-Young Lim, et al. Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages. Int. J. Cancer 2003; 132: 591–604 (2013)  http://dx.doi.org/10.1002/ijc.27706 )

We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and

endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

The Role of Stroma in Tumour-Host Co-Existence

Molnár et al.,  The Role of Stroma in Tumour-Host Co-Existence: Some Perspectives in Stroma-Targeted Therapy of Cancer   Biochem Pharmacol 2013, 2:1    http://dx.doi.org/10.4172/2167-0501.1000107

 Cancer grows at the expense of the host as a parasite or superparasite following the second law of thermodynamics (conservation of energy). When the cancer cell progresses via replication to the special state called “spheroid”, a new phase begins with its intimate interaction and development of responses from the stroma which together assist in the formation of a full blown cancer. Among the processes involved are the development of blood vessels and lymphatic channels which are essential for maintenance and further growth of the cancer mass. In this way the condition of “parasitism” is completed with simultaneous suppression of the immune response of the host to the histo-incompatability of the tumor mass. Stroma/parenchyma promotes cancer invasion by feeding cancer cells and inducing immune tolerance. The dynamic changes in composition of stroma and biological consequences as feeder of cancer cells and immune tolerance can give a perspective for rational drug design in anti-stromal therapy. There are differences between normal and cancer cells at subcellular level such as compartmentalzation and structure of cytoskeleton and energy distribution (that is low generally, but locally high in normal cells). In cancer cannibalism of normal cells, the growing cancer mass is a factor for progression and invasion.

Cancer cells have been shown to kill normal cells and the products of cell death used for progression of growth of the cancer cell. Serum and growth factors produced by tumor stroma also provide the needed nutrients and conditions for further tumor growth. Cancer cannot feed off other cancer cells and therefore grow poorly. Probably, although not yet proven, the inability of cancer to “parasitise” other cancer cell types is probably due to some kind of competition or interference. The tumor is in charge of its own development due to its induction proteinases, lipid mobilization factors and angiogenetic factors as well as its ability to negate immune responses of the host response to what is in essence a foreign body.

In our review co-existence of normal and cancer cells in tumor with the growth promoting factors, and the immune tolerance mediating factors produced in the stromal and cancer cells/tissues will be discussed with perspective of stroma targeted therapy.

The clinical significance of cell cannibalism is well defined and described in a large number of publications. The direction of process of cancer development is defined as the tumor invades the normal tissue which never occurs in the reverse direction. This suggests that the cancer cell strives to achieve the lowest energy level possible. Therefore the first of the development of a full blown cancer can be considered as the 2nd Thermodynamic principle  that explains, describes and drives the invading cancer into normal surrounding tissue.

From the normal living state, under particular conditions such as hypoxia, where ATP synthesis is decreased resulting in a switch to glycolytic pathways, cancer cells are selected from a fraction of the population [4]. Energetically, in the presence of electron transfer, by using high energy from respiration, the proliferating state is more stable than resting cells where a higher degree of protein stabilization occurs such as that needed for maintainance of the cytoskeleton of the cell. It was proposed that tumor-promotion might be controlled or modulated by small electronic currents originating from reactive oxygen species and transported through the cytoskeletal microfilament network of the cancer cell.

Aerobic glycolysis is the main energy producing process in cancer cells. Among many other aspects, recently the mitochondria have also been regarded as potential targets in the therapy of cancer. Several small molecules have been tested to restore their dysfunctional functions either by direct or indirect effects. Because of poorly functioning mitochondria, the electron transfer component of the respiration cycle is inefficient; therefore, cancer cells have smaller Gibbs energy than healthy cells. This means, that these cancer cells exists in a metastable state and are not able maintain normal cell structure.

Therefore, the cytoskeleton system is collapsed and dielectric bilayers are formed as a lower grade of cellular structure with decreased electron conductivity. Consequently, to halt cancer growth, one has to evaluate the process of cancer cell development in situ, where the primary tumor is growing as well as that of the metastatic cell that is invading surrounding or distal tissues. This affords one to suggest that the stroma is formed first during long term repeated oxidative stress, a process that is initially accompanied with inflammation due to an active immune response to the histoincompatability antigens present on the surface of the cancer cell. If the cancer cell evades the activity of killer T cells (Treg cells) by either secreting agents that reduce the response of the Treg cells or the immune system for whatever reason is ineffective (immunosuppressed states such as HIV/AIDS, pregnancy, transplantation  therapy, etc.), the formed cancer cells have the opportunity to initiate tumor development. Because of the limited capacity of its electron transfer cycle, cancer cells are essentially starving cells that require glycolytically useful substrates. These substrates are obtained from the killing of normal cells by agents secreted by the cancer cell and the products yielded from dead normal cells “eaten” (phagocytosed) by the starving cancer cell which is digested by the cancer cells lysosomal system. This autophagic process of cannibalism keeps the cancer cell alive and thriving and is known as cytophagy, i.e., cannibalism of normal cells. This type of autophagocytosis  results in a parasitic co-existence of tumor cells with normal cells and will determine the main pathway of interaction between the growing cancer tissue (tumor) and normal tissue where the cancer tissue gradually destroys normal tissues. This process obeys the second law of thermodynamics-conservation of energy within a defined system.

Treatments for Cancer

 Bosutinib: a SRC–ABL tyrosine kinase inhibitor for treatment of chronic myeloid leukemia. 

FE Rassi, HJ Khoury. Pharmacogenomics and Personalized Medicine  2013:6 57–62.

Bosutinib is one of five tyrosine kinase inhibitors commercially available in the United States for the treatment of chronic myeloid leukemia. This review of bosutinib summarizes the mode of action, pharmacokinetics, efficacy and safety data, as well as the patient-focused perspective through quality-of-life data. Bosutinib has shown considerable and sustained efficacy in chronic myeloid leukemia, especially in the chronic phase, with resistance or intolerance to prior tyrosine kinase inhibitors. Bosutinib has distinct but manageable adverse events. In the absence of T315I and V299L mutations, there are no absolute contraindications for the use of bosutinib in this patient population

Chronic myeloid leukemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the presence of a signature hybrid oncogene, the BCR–ABL. The Philadelphia chromosome (Ph+) results from a reciprocal translocation between chromosome 9 and chromosome 22 that juxtaposes the two genes BCR and ABL and drives the leukemogenesis in CML. The ABL gene encodes for a nonreceptor tyrosine kinase that becomes deregulated and constitutively active after the juxtaposition of BCR. BCR–ABL is central in controlling downstream pathways involved in cell proliferation, regulation of cellular adhesion, and apoptosis.The understanding of the importance of this kinase activity in the pathophysiology of CML led to the development of tyrosine kinase inhibitors (TKI) that specifically target BCR–ABL. These agents became the mainstay of modern therapy in CML. CML has a triphasic clinical course, and the majority of patients (∼80%) are diagnosed during the early phase or the chronic phase (CP). However, and without effective treatment, CML invariably progresses to the advanced phases of the disease – the accelerated phase (AP) and the blast phase (BP). BP CML is a lethal refractory secondary leukemia with a short predicted survival.

Comprehensive molecular portraits of human breast tumors

 The Cancer Genome Atlas Network

Nature. 2012 October 4; 490(7418): 61–70. http://dx.doi.org/10.1038/nature11412.

We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity.

Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at  > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.

Most molecular studies of breast cancer have focused on just one or two high information content platforms, most frequently mRNA expression profiling or DNA copy number analysis, and more recently massively parallel sequencing. Supervised clustering of mRNA expression data has reproducibly established that breast cancers encompass several distinct disease entities, often referred to as the intrinsic subtypes of breast cancer. The recent development of additional high information content assays focused on abnormalities in DNA methylation, microRNA expression and protein expression, provide further opportunities to more completely characterize the molecular architecture of breast cancer.

Synbiology contribution and Nanotechnology

Synthetic RNAs Designed to Fight Cancer

Xiaowei Wang and his colleagues at  Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  They have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  RNA plays an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.

“We are trying to merge two largely separate fields of RNA research and harness the advantages of both,” said Xiaowei Wang, assistant professor of radiation oncology and a research member of the Siteman Cancer Center.  The study appears in the December issue of the journal RNA.

“We designed an artificial RNA that is a combination of siRNA and microRNA,” Wang said “our artificial RNA simultaneously inhibits both cell migration and proliferation.”  For therapeutic purposes, “small interfering” RNAs, or siRNAs, are designed and assembled in a lab and can be made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby silencing a gene’s expression.

Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects: siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.  The siRNAs interfere with off-target genesthat closely complement their “seed region,” a section of the siRNA  that governs binding to a gene target. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now, we never tried to replace the seed region completely.”

Wang and his colleagues asked whether they could replace the siRNA’s seed region with the seed region from microRNA. Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce the toxic side effects caused by the artificial siRNA seed region. Plus, the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.

Wang’s group started with a bioinformatics approach, using a computer algorithm to design siRNA sequences against a common driver of cancer, a gene called AKT1 that encourages uncontrolled cell division. The program also selected siRNAs against AKT1 that had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus potentially reducing the cancer’s ability to spread.

A Neutralizing RNA Aptamer

 Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity.

(CL Esposito, D Passaro, et al. A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death. PLoS ONE 6(9): e24071. http://dx.doi.org/10.1371/journal.pone.0024071)

Here we generated, by a cell-Systematic Evolution of  Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.

In-Silico Molecular Docking Analysis of Cancer Biomarkers

Currently, in the research scenario for cancer, the identification of anti-cancer drugs using immuno-modulatory proteins and other molecular agents to initiate apoptosis in cancer cells and to inhibit the signaling pathways of cancer biomarkers as a drug targeted therapy, for cancer cell proliferation assays by the researchers. In-Silico analysis is used to recognize anticancer compounds as a future prospective for In-Vitro and In-Vivo analysis. A large number of herbal remedies (e.g. garlic, mistletoe) are used by cancer patients for treating the cancer and/or reducing the toxicities of chemotherapeutic drugs. Some herbal medicines have shown potentially beneficial effects on cancer progression and may ameliorate chemotherapy-induced toxicities.  (K. Gowri Shankar et al., In-Silico Molecular Docking Analysis of Cancer Biomarkers with Bioactive Compounds of Tribulus terrestris. Intl J NOVEL TRENDS PHARMAL SCI. 2013; 3(4).

Tribulus terrestris is mentioned in ancient Indian Ayurvedic medical texts dating back thousands of years. Tribulus terrestris has been widely used in the Ayurvedic system of medicine for the treatment of sexualdysfunction and various urinary disorders. The aim of the present study is to evaluate the interactions of some bioactive compounds of Tribulus terrestris for In-Silico anticancer analysis with cancer biomarkers as targets. The targeted biomarkers for analysis include NSE-Lung cancer, Follistatin-Prostrate cancer, GGT Hepatocellular carcinoma, Human Prostasin-Ovarian cancer.

GC-MS analysis of Tribulus terrestris whole plant methanol extract revealed the existence of the major compound like 3,7,11,15-tetramethylhexadec-2-en-1-ol, 1,2-Benzenedicarboxylic acid, disooctyl ester, 9,12,15-Octadecatrienoic acid, (z,z,z)-, 9,12-Octadecadienoic acid (z,z)-, Hexadecadienoic acid, ethyl ester, n-Hexadecadienoic acid, Octadecanoic acid, Phytol, α-Amyrin are chosen as ligands. Hence, by analyzing the minimum binding energy of the ligand binding complex with the receptors by dockinganalysis using AutoDock tools will show effective nature of inhibition of these receptors by the unique ligands. Based on the results low minimum binding energy ligands are identified and used as a future studies can be done for specific receptors  docking.

Anti-Cancerous Effect of4,4′-Dihydroxychalcone ((2E,2′E)-3,3′-(1,4-Phenylene) Bis (1-(4-hydroxyphenyl) Prop-2-en-1-one)) on T47D Breast Cancer Cell Line

Narges Mahmoodi, T Besharati-Seidani, N Motamed, and NO Mahmoodi*
Annual Research & Review in Biology 2014; 4(12): 2045-2052
SCIENCEDOMAIN international    www.sciencedomain.org

Aims: The majority of human breast tumors are estrogen receptor α (ERα) positive. However, not all of the ERα+ breast cancers respond to anti-estrogens drugs for those women who do respond, initial positive responses can be of short duration. Thus, more effective drugs are needed to enhance the efficacy of anti-estrogens drugs or to be used separately in a period of time. In view of potential cytotoxicity associated with silybin as polyhydroxy compounds a synthetic 4-hydroxychalcones (bis-phenol) was considered to explore its anti-carcinogenic effects in comparison to silybin on ERα+ breast cancer cell line.

Methodology: We have studied the inhibitory effect of 4,4′-dihydroxychalcone on the T47D breast cancer cell line by MTT test and the IC50s were estimated using Pharm PCS.

Results: The 4,4′-dihydroxychalcone showed significant dose- and time-dependent cell growth inhibitory effects on T47D breast cancer cells. The IC50 of 4,4′-dihydroxychalcone on T47D cells after 24 and 48 hours was 160.88+/1 μM, 62.20+/1 μM and for silybin was 373.42+/-1 μM,176.98+/1 μM respectively.

Conclusion: Our results strongly suggests that this premade synthetic 4,4′-dihydroxychalcone can promote anti carcinogenic actions on T47D cell line. All 4,4′-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC50 of 4,4′-dihydroxychalcone to silybin after 24 and 48 hours was 1: 2.3 and 1: 2.8 respectively.

Anticancer and multidrug resistance-reversal effects of solanidine analogs synthetized from pregnadienolone acetate.

István Zupkó, Judit Molnár, Borbála Réthy, Renáta Minorics, Eva Frank, et al.
Molecules (Impact Factor: 2.43). 01/2014; 19(2):2061-76.  http://dx.doi.org/10.3390/molecules19022061
Source: PubMed

ABSTRACT A set of solanidine analogs  with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.

Nutrition and Cancer

 Ascorbic Acid and Selenium Interaction: Its Relevance in Carcinogenesis

 Michael J. Gonzalez
Journal of Orthomolecular Medicine 1990; 5(2)

Ascorbic acid and selenium are two nutrients that seem to have a preventive potential in the process of carcinogenesis; because of a possible synergistic action that may produce an enhanced anticarcinogenic effect. Interaction between these nutrients have been reported. Results indicate that the protective effect of the inorganic form of selenium (Na Selenite) was nullified by ascorbic acid, whereas the chemopreventive action of the organic form (seleno-DL-methionine) was not affected.

A possibility exists that Selenite is reduced by ascorbic acid to elemental selenium and is therefore not available for tissue uptake. In experiments using Selenite; plasma and erythrocyte glutathione peroxidase enzyme activity was directly related to the level of ascorbic acid fed.

Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis

J W. Rensvold, Shao-En On, A Jeevananthan, et al.
Cell Rep. 2013 January 31; 3(1): .   http://dx.doi.org/10.1016/j.celrep.2012.11.029

Mitochondria are centers of metabolism and signaling whose content and function must adapt to
changing cellular environments. The biological signals that initiate mitochondrial restructuring
and the cellular processes that drive this adaptive response are largely obscure. To better define
these systems, we performed matched quantitative genomic and proteomic analyses of mouse
muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in
cellular iron homeostasis are highly coordinated with this process and that depletion of cellular
iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and
oxidative capacity. We further show that this process is universal across a broad range of cell
types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron
is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be
leveraged to explore posttranscriptional and posttranslational processes that are essential for
mitochondrial adaptation.

Avemar outshines new cancer ‘breakthrough’ drug

by Michael Traub
Townsend Letter / Oct, 2010

Many of us in the cancer research community were happy to hear about progress against metastatic melanoma reported this June at the annual meeting of the American Society of Clinical
Oncology (ASCO). since there has not been an improvement in overall survival from chemotherapy in over three decades.
Data from a phase III clinical trial of the experimental monoclonal antibody ipilimumab (pronounced “ep-eh-lim-uemab”) showed that patients with melanoma survived longer if they were taking ipilimumab than if they were not, regardless of whether they also were taking the other drug in the study, an experimental cancer vaccine. (1)

A Closer Look: How Big an Improvement, at What Cost to Patients?

Overall Survival: the ‘Gold Standard’ for Judging Cancer Therapies

Overall survival (OS) is the length of time that a patient actuallysurvives a cancer after treatment. It can also be measured as the percentage of patients surviving a specific time. It is the gold
standard by which the usefulness of a cancer treatment should be determined. Many things can help a patient, but the most important goal of doctors and patients is for the cancer patient to live longer, with a decent quality of life (QOL).

Among patients taking ipilimumab with or without the experimental vaccine, median overall survival was about 10 months. That is compared with 6.4 months’ overall survival among patients receiving the vaccine by itself. About 45.6% of patients taking ipilimumab survived one year, an improvement of some 7% over the 38% seen in some earlier studies. This very modest improvement in survival comes at quite a price.

Severe Side Effects in More Than One in Four Ipilimumab Patients Ipilimumab has some side effects that can be “both severe and long-lasting,” according to the study report. Among patients taking ipilimumab by itself (without the vaccine), 19.1% had side effects requiring hospitalization or invasive intervention, 3.8% died from the effects of the drug, and another 33.8% had life-threatening or disabling side effects. All totaled, 26.7% of the patients taking ipilimumab by itself– more than 1 in 4-had side effects that were severe, very severe, or fatal. Severe side effects included diarrhea, nausea, constipation, vomiting, abdominal pain, fatigue, cough, and headache. Vernon Sondak, MD, of the H. Lee Moffitt Cancer and Research Institute, said that “using the drug requires the medical team to be on guard to manage toxicity at all times.” But even with its severe side effects, the researchers said that the drug should be welcomed because it can increase median survival from 6.4 months to 10.1 months. That is because any lengthening of lives is welcome in a disease that hasn’t seen a new drug that can do that in many years.

Fermented Wheat Germ (Avemar) Improves Melanoma Survival Without Harsh Side Effects

But what if there already were such a treatment available-not a drug, but a safe, natural substance shown in clinical trials to have a remarkably similar ability to lengthen the lives of melanoma patients, without the severe side effects of the new drug?
What if the other substance had no significant side effects at all?
What if, instead of causing severe and sometimes fatal side effects, that other substance actually helped prevent and reduce serious side effects caused by chemotherapy and radiotherapy?
In fact, there is just such a treatment available. It is known as fermented wheat germ extract (FWGE) and by its trade name Avemar. It has been approved as a medical nutriment for cancer
patients in Europe for years and is available in the US as a dietary supplement. It has been compared to dacarbazine (DTIC), standard melanoma therapy, in a clinical trial with longer
follow-up than the ipilimumab trial. And with better results.

In 2008, data were published in the research journal Cancer Biotherapy and Radiopharmaceuticals from seven years’ follow-up on a trial at the N. N. Blokhin Cancer Center in Moscow,
Russia, involving 52 patients who had taken or not taken Avemar while taking dacarbazine for the year following surgical removal of their stage III melanoma tumors. (2) Patients who got only dacarbazine survived 44.7 months. Those who got Avemar along with their dacarbazine survived 66.2 months. This is an improvement in overall survival time of over 48%. In the Russian study,
just as it has in other studies, Avemar reduced side effects of the chemotherapy. Among those taking only dacarbazine, 11 % experienced severe (grade 3 or grade 4) side effects that required hospitalization or invasive intervention. None of the Avemar patients had grade 3 or 4 side effects. Since it is difficult to compare length of survival between the recent ipilimumab study and the Avemar melanoma study, because the ipilimumab study tested mostly stage 4 melanoma patients and the Avemar study tested mostly stage 3 melanoma patients, it is most instructive to look at
the percentage improvement in overall survival from adding either treatment to the regimen. Ipilimumab and Avemar both produced very similar improvements in OS (56% vs. 48%, respectively),

Avemar Ameliorates Conventional Treatment Side Effects

The improvement of survival and the amelioration of chemotherapy side effects by Avemar seen in the Russian melanoma study is typical of Avemar’s effects when used in treating other cancers, including in combination with chemotherapy or radiotherapy. Among 170 colorectal cancer patients in a 2003 study published in the British journal of Cancer, Avemar improved overall survival
and reduced metastasis and recurrences after surgery, chemotherapy, and radiotherapy. (3) Taking Avemar for six months during and after those conventional treatments resulted in a 61.8% reduction in the death rate among those patients, compared with those who received only the conventional treatment. Those taking Avemar experienced lower rates of recurrences and metastases
as well, even though most patients in the Avemar group came into the study with more advanced disease, had more radiation earlier, and had been diagnosed longer. Side effects of Avemar, as in
other Avemar trials., were rare, mild, and transient, with no serious adverse events occurring.

In a 2004 study published in the journal of Pediatric Hematology and Oncology, childhood cancer patients taking Avemar during and after conventional therapies had a 42.8% reduction in the
low white blood cell counts and high fever known as febrile neutropenia, which can be a life-threatening consequence of chemotherapy and radiation. (4) This and similar results with
Avemar in other cancers are consistent with animal studies showing that Avemar helps the immune system recover a full white blood cell count after chemotherapy and radiation faster
than would otherwise happen. This study also demonstrated the safety of Avemar for children.

Why Avemar Works in Many Different Kinds of Cancer

Extensive studies in cells and animals have shown how Avemar works. Perhaps its most important action is to restrict cancer cells’ use of glucose. (5) Cancer cells use up to 50 times more glucose
than normal cells, a phenomenon known as the Warburg effect. (6) They use those enormous amounts of glucose to make ribose, the backbone sugar of DNA, much faster than normal cells can. To
do this, they must use a different series of biochemical reactions (“pathway”) than normal cells. Avemar makes this very difficult for cancer cells to do, because it inhibits the activity of the key enzyme in that pathway, transketolase (TK). (7) With the TK pathway blocked, cancer cells cannot use large amounts of glucose to make DNA fast enough to support the proliferation that makes them so dangerous.(8-10)

In experiments in the US and abroad, scientists have learned that Avemar has these additional effects. It:

* lowers the levels of a DNA repair enzyme known as poly (ADPribose) polymerase (PARP).” With this effect, cancer cells are forced to self-destruct, preventing them from proliferating and
producing a synergistic cancer-cell killing effect when given with chemotherapy, which also works to damage cancer cells’ DNA;
* reduces the number of molecules on cancer cells that identify them as originating within the body (MHC-1 molecules). (12) With cancer cells stripped of that protection, the immune system,
which recognizes the cancer cells as abnormal, no longer gives them the pass given to cells originating in the body. The cancer cells are attacked by the immune system’s natural killer (NK)
cells and destroyed;
* increases levels of molecules called intercellular adhesion molecule-1 (ICAM-1) on the blood vessels of cancer tumors. (13). The increase helps immune system cells pass through the walls of the blood vessels supplying the tumor blood flow, moving directly into the tumor to attack its cancer cells; increases the activity of the primary anticancer cytokine, tumor necrosis factor alpha (TNF-a), and produces a synergistic effect in interaction with other anticancer cytokines. (14) Cytokines are substances produced by cells to act directly on other cells. TNF-a helps force cancer cells into the programmed death known as apoptosis and inhibits tumorigenesis, the process through which new tumors are formed;
* inhibits the activity of ribonucleotide reductase (RR), a key enzyme that cells must have to make new DNA so that each cancer cell can divide to make two more like it. (15) With DNA
production slowed, increases in cancer cell growth and replication are inhibited.

Antimetastatic and Immune-Boosting Effects Are Key to Survival

Because the biochemical changes listed above have consistently been shown in both animal and human studies to be directly linked to reducing cancer’s ability to metastasize and to
improving the immune system’s ability to fight cancer, scientists count them as among the most likely main causes of improved survival seen in cancer patients when Avemar is used alone or,
more often, as an adjuvant in addition to standard-of-care therapies such as chemotherapy, radiotherapy, or the combination of the two. (16-23)

Extending Life: How Long, Exactly, and At What Cost in Quality of Life?

Any improvement in advanced melanoma survival, no matter how small, is certainly an achievement. But ipilimumab had severe side effects requiring hospitalization or invasive intervention in
over one-quarter of patients treated with it. And it increased median survival only by 3-plus months. On the other hand, Avemar added to dacarbazine improved survival very markedly, with no severe side effects. If actually improving overall survival substantially without significant side effects means that a drug should be considered as the new standard of care for first-line therapy, then there is no need to wait for further results. Avemar has already demonstrated very significant improvement in survival over chemotherapy alone and has a safety profile unmatched by
conventional therapies.

Michael Traub, ND, FABNO, is in private practice and serves as a member of Oncology Association of Naturopathic Physicians board of examiners.
Notes
(1.) Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010 Jun 14.
(2.) Demidov LV. Manziuk LV, Kharkevitch GY, Pirogova NA,  Artamonova EV. Adjuvant fermented wheat germ extract (Avemar) nutraceutical improves survival of high-risk skin
melanoma patients; a randomized, pilot, phase ll clinical study with a 7-year follow-up. Cancer Biother Radiopharm. 2008 Aug. 23(4):477-482. Erratum in: Cancer Biother Radiopharm. 2008
Oct;2315):669.
(3.) Jakab F, Shoenfeld Y, Balogh A. et al. A medical nutriment has supportive value in the treatment of colorectal cancer. Br J Cancer. 2001 Aug 4;89(3):465-9.
(4.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients, J Pediatr Hematol Oncol. 2004
Oct;26(10):631-635.
(5.) Boros I.G, Lapis K, Szende B, et al. Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma
cells. Pancreas. 2001 Aug:23(2):141-147.
(6.) Warburg, O. On the origin of cancer cells. Science. 1956 Feb 24; 123(31 91):309-314.
(7.) Boros LG, Lee VVN, Go VL., A metabolic hypothesis of cell growth and death in pancreatic cancer, Pancreas. 2002 Jan;
24:(1):26 33.
(8.) Boros LG, Lapis K, Szende B, et al. Op cit.
(9.) Comin-Anduix B, Boros LG, Marin S, et al. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase
activation in Jurkat T-cell leukemia tumor cells. J Biol Chem. 2002 Nov 29;277 (48):46408-46414. Epub 2002 Sep 25.
(23.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients. J Pediatr Hematol Oncol. 2004 Oct;
26(10):631-635.

by Michael Traub, ND, FABNO
COPYRIGHT 2010 The Townsend Letter Group
COPYRIGHT 2010 Gale, Cengage Learning

Nanotechnology in Cancer Drug Delivery and Selective Targeting

Nanoparticles are rapidly being developed and trialed to overcome several limitations of traditional drug delivery systems and are coming up as a distinct therapeutics for cancer treatment. Conventional chemotherapeutics possess some serious side effects including damage of the immune system and other organs with rapidly proliferating cells due to nonspecific targeting, lack of solubility, and inability to enter the core of the tumors resulting in impaired treatment with reduced dose and with low survival rate.

Nanotechnology has provided the opportunity to get direct access of the cancerous cells selectively with increased drug localization and cellular uptake. Nanoparticles can be programmed for recognizing the cancerous cells and giving selective and accurate drug delivery avoiding interaction with the healthy cells. This review focuses on cell recognizing ability of nanoparticles by various strategies having unique identifying properties that distinguish them from previous anticancer therapies. It also discusses specific drug delivery by nanoparticles inside the cells illustrating many successful researches and how nanoparticles remove the side effects of conventional therapies with tailored cancer treatment.

(Kumar Bishwajit Sutradhar and Md. Lutful Amin. Hindawi Publ. Corp.  2014, Article ID 939378, 12 pages

http://dx.doi.org/10.1155/2014/939378)

Cancer, the uncontrolled proliferation of cells where apoptosis is greatly disappeared, requires very complex process of treatment. Because of complexity in genetic and phenotypic levels, it shows clinical diversity and therapeutic resistance. A variety of approaches are being practiced for the treatment of cancer each of which has some significant limitations and side effects. Cancer treatment includes surgical removal, chemotherapy, radiation, and hormone therapy. Chemotherapy, a  very common treatment, delivers anticancer drugs systemically to patients for quenching the uncontrolled proliferation of cancerous cells. Unfortunately, due to nonspecific targeting by anticancer agents, many side effects occur and poor drug delivery of those agents cannot bring out the desired outcome in most of the cases. Cancer drug development involves a very complex procedure which is associated with advanced polymer chemistry and electronic engineering.

The main challenge of cancer therapeutics is to differentiate the cancerous cells and the normal body cells. That is why the main objective becomes engineering the drug in such a way as it can identify the cancer cells to diminish their growth and proliferation. Conventional chemotherapy fails to target the cancerous cells selectively without interacting with the normal body cells. Thus they cause serious side effects including organ damage resulting in impaired  treatment with lower dose and ultimately low survival rates.

Nanotechnology is the science that usually deals with the size range from a few nanometers (nm) to several hundrednm, depending on their intended use. It has been the area of interest over the last decade for developing precise drug delivery systems as it offers numerous benefits to overcome the limitations of conventional formulations . It is very promising both in cancer diagnosis and treatment since it can enter the tissues at molecular level.

Cisplatin-incorporated nanoparticles of poly(acrylic acid-co-methyl methacrylate) copolymer

K Dong Lee, Young-Il Jeong,  DH Kim,  Gyun-Taek Lim,  Ki-Choon Choi.  Intl J Nanomedicine 2013:8 2835–2845.

Although cisplatin is extensively used in the clinical field, its intrinsic toxicity limits its clinical use. We investigated nanoparticle formations of poly(acrylic acid-co-methyl methacrylate) (PAA-MMA) incorporating cisplatin and their antitumor activity in vitro and in vivo.

Methods: Cisplatin-incorporated nanoparticles were prepared through the ion-complex for­mation between acrylic acid and cisplatin. The anticancer activity of cisplatin-incorporated nanoparticles was assessed with CT26 colorectal carcinoma cells.

Results: Cisplatin-incorporated nanoparticles have small particle sizes of less than 200 nm with spherical shapes. Drug content was increased according to the increase of the feeding amount of cisplatin and acrylic acid content in the copolymer. The higher acrylic acid content in the copolymer induced increase of particle size and decrease of zeta potential. Cisplatin-incorporated nanoparticles showed a similar growth-inhibitory effect against CT26 tumor cells in vitro. However, cisplatin-incorporated nanoparticles showed improved antitumor activity against an animal tumor xenograft model.

Conclusion: We suggest that PAA-MMA nanoparticles incorporating cisplatin are promising carriers for an antitumor drug-delivery system.

Researchers Say Molecule May Help Overcome Cancer Drug Resistance
By Estel Grace Masangkay

A group of researchers from the University of Delaware has discovered that a deubiquitinase (DUB) complex, USP1-UAF1, may present a key target in helping fight resistance to platinum-based anticancer drugs. The research team’s findings were published online in Nature Chemical Biology.

Zhihao Zhuang, associate professor in the Department of Chemistry and Biochemistry at UD, and his team studied a DNA damage tolerance mechanism called translesion synthesis (TLS). Enzymes known as TLS polymerases synthesize DNA over damaged nucleotide bases, followed by replication after lesion. The enzymes have been linked with building cancer cell resistance to certain cancer drugs including cisplatin. Cisplatin is used in treatment of ovarian, bladder, and testicular cancers which have spread.

“Cancer drugs like cisplatin work by damaging DNA and thereby preventing cancer cells from replicating the genomic DNA and dividing. However, cancer cells quickly develop resistance to cisplatin, and we and other researchers suspect that a polymerase known as Pol η is involved in overcoming cisplatin-induced lesions,” Professor Zhuang said.

The team found that USP1-UAF1 may play a crucial role in regulating DNA damage response. A new molecule ML323 can be used to inhibit processes such as translesion synthesis. Zhuang said, “Using ML323, we studied the cellular response to DNA damage and revealed new insights into the role of deubiquitination in both the TLS pathway and another one called the Fanconi anemia, or FA, pathway. We’re very encouraged by the fact that a single molecule is effective at inhibiting the USP1-UAF1 DUB complex and disrupting two essential DNA damage tolerance pathways.”

A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

Cui-yan Han,  Li-ling Yue, Ling-yu Tai,  Li Zhou  et al.  Intl J Nanomedicine 2013:8 1541–1549

The discovery of suitable ligands that bind to cancer cells is important for drug delivery specifically targeted to tumors. Monoclonal antibodies and fragments that serve as ligands have specific targets. Natural ligands have strong mitogenic and neoangiogenic activities. Currently, small pep­tides are pursued as targeting moieties because of their small size, low immunogenicity, and their ability to be incorporated into certain delivery vectors.

The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. The epidermal growth factor receptor (EGFR) is a transmembrane protein on the cell surface that is overexpressed in a wide variety of human cancers. EGFR is an effective tumor-specific target because of its significant functions in tumor cell growth, differentiation, and migration. EGFR-targeted small molecule peptides such as YHWYGYTPQNVI have been successfully identified using phage display library screening; by contrast, the peptide LARLLT has been generated using computer-assisted design (CAD).

These peptides can be conjugated to the surfaces of liposomes that are then delivered selectively to tumors by the specific and efficient binding of these peptides to cancer cells that express high levels of EGFR.

In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control.

Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors.

We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.

Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

SR Sarker  Y Aoshima,   R Hokama  T Inoue  et al. Intl J Nanomedicine 2013:8 1361–1375.

Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.

 Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000.

 We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular.

Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.

The gene delivery efficiency of amino acid-based cationic assemblies is influ­enced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment

H Kaneda, T Yoshida,  I Okamoto.   Cancer Management and Research 2013:5 91–101.

The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatmentbased on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.

Non-small-cell lung cancer (NSCLC) has a poor prognosis and remains the leading cause of death related to cancer worldwide. For most individuals with advanced, metastatic NSCLC, cytotoxic chemotherapy is the mainstay of treatment on the basis of the associated moderate improvement in survival and quality of life. However, the outcome of chemotherapy in such patients has reached a plateau in terms of overall response rate (25%–35%) and overall survival (OS; 8–10 months). This poor outcome, even for patients with advanced NSCLC who respond to such chemotherapy, has motivated a search for new therapeutic approaches.

Recent years have seen rapid progress in the development of new treatment strat­egies for advanced NSCLC, in particular the introduction of molecularly targeted therapiesand appropriate patient selection. First, the most important change has been customization of treatment according to patient selection based on the genetic profile of the tumor. Small-molecule tyrosine kinase inhibitors (TKIs) that target the epidermal growth-factor receptor (EGFR), such as gefitinib and erlotinib, are especially effective in the treatment of NSCLC patients who harbor activating EGFR mutations.

Surgical Nanorobotics using nanorobots made from advanced DNA origami and Synthetic Biology

Ido Bachelet’s moonshot to use nanorobotics for surgery has the potential to change lives globally. But who is the man behind the moonshot?

Ido graduated from the Hebrew University of Jerusalem with a PhD in pharmacology and experimental therapeutics. Afterwards he did two postdocs; one in engineering at MIT and one in synthetic biology in the lab of George Church at the Wyss Institute at Harvard.

Now, his group at Bar-Ilan University designs and studies diverse technologies inspired by nature.

They will deliver enzymes that break down cells via programmable nanoparticles.

Delivering insulin to tell cells to grow and regenerate tissue at the desired location.

Surgery would be performed by putting the programmable nanoparticles into saline and injecting them into the body to seek out remove bad cells and grow new cells and perform other medical work.

 

http://2.bp.blogspot.com/-bnAE6hL2RIE/Uy0wFB8pYPI/AAAAAAAAubM/BeSpFC4vLu0/s1600/screenshot-by-nimbus+(3).png

 

Robots killing and suppressing cancer cells

 

http://1.bp.blogspot.com/-LGsE1msGIrw/Uy0vKGoaQ3I/AAAAAAAAubE/2E1_lcAspao/s1600/screenshot-by-nimbus+(2).png

 

Robots delivering payload

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0

http://4.bp.blogspot.com/-kkfXlMyPRCI/Uy0wkYPMvBI/AAAAAAAAubU/0AQPpJpM5E4/s1600/screenshot-by-nimbus+(4).png

Molecular building blocks

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=236

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=283

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=287

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=292

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=333

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=397

http://2.bp.blogspot.com/-gCHiyZ2MBHg/Uy0ySRKw_II/AAAAAAAAubg/BeneEQ5bY-U/s1600/screenshot-by-nimbus+(5).png

 

Robot blocks neuron

http://4.bp.blogspot.com/-cbYNJnN_w7U/Uy0yrqyqebI/AAAAAAAAubo/b42r4WRMr8k/s1600/screenshot-by-nimbus+(6).png

 

automation of robotic surgery

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=470

Nanoparticles with computational logic has already been done

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=501

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=521

http://1.bp.blogspot.com/-rSyRzo7p50w/Uy0y5teQkDI/AAAAAAAAubw/8cxZ4t0WNHw/s1600/screenshot-by-nimbus+(7).png

 

 robotic algorithm

 

Load an ensemble of drugs into many particles for programmed release based on situation that is found in the body

http://1.bp.blogspot.com/-kc99CbOQYLs/Uy0zgUG13KI/AAAAAAAAub4/j6nM7hAVxUg/s1600/screenshot-by-nimbus+(8).png

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=572

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=577

 

robotic lung cancer Rx

 

chemotherapy regimen

 

Chemoprevention in Model Experiments

Effects of Two Disiloxanes ALIS-409 and ALIS-421 on Chemoprevention in Model Experiments

H TOKUDA,…. L AMARAL and J MOLNAR.ANTICANCER RESEARCH 33: 2021-2028 (2013).

ALIS

 

Figure 1. Chemical structures of ALIS-409 and ALIS-421.

Morpholino-disiloxane (ALIS-409) and piperazinodisiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of

tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate early antigen (EBV-EA) expression induced by tetradecanoylphorbolacetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than

ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study.

 

 

 

 

 

 

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