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The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.
We have laid down a basic structure and foundation for the remaining presentations. It was essential to begin with the genome, which changed the course of teaching of biology and medicine in the 20th century, and introduced a central dogma of translation by transcription. Nevertheless, there were significant inconsistencies and unanswered questions entering the twenty first century, accompanied by vast improvements in technical advances to clarify these issues. We have covered carbohydrate, protein, and lipid metabolism, which function in concert with the development of cellular structure, organ system development, and physiology. To be sure, the progress in the study of the microscopic and particulate can’t be divorced from the observation of the whole. We were left in the not so distant past with the impression of the Sufi story of the elephant and the three blind men, who one at a time held the tail, the trunk, and the ear, each proclaiming that it was the elephant.
I introduce here a story from the Brazilian biochemist, Jose
Eduardo des Salles Rosalino, on a formativr experience he had with the Nobelist, Luis Leloir.
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 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 the assays with PK I, PKII and PKIII of M. Rouxii and using the Sutherland route to cAMP failed in this case. I then asked 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, had 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.
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 reading and discussing “What is life” with him he asked me if as a biochemist in exile, talking to another biochemist, I expressed myself fully. I had considered that Schrödinger would not have confronted Darlington & Haldane because he was in U.K. in exile. This might explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes, in a way that suggested that 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 to 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 been 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’s rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy).
Millions of years later, 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 discussions that follow are concerned with protein interactions and signaling.
Extracellular evaluation of intracellular flux in yeast cells
Larry H. Bernstein, MD, FCAP, Reviewer and Curator
Leaders in Pharmaceutical Intelligence
This is the fourth article in a series on metabolomics, which is a major development in -omics, integrating transcriptomics, proteomics, genomics, metabolic pathways analysis, metabolic and genomic regulatory control using computational mapping. In the previous two part presentation, flux analysis was not a topic for evaluation, but here it is the major focus. It is a study of yeast cells, and bears some relationship to the comparison of glycemia, oxidative phosphorylation, TCA cycle, and ETC in leukemia cell lines. In the previous study – system flux was beyond the scope of analysis, and explicitly stated. The inferences made in comparing the two lymphocytic leukemia cells was of intracellular metabolism from extracellular measurements. The study of yeast cells is aimed at looking at cellular effluxes, which is also an important method for studying pharmacological effects and drug resistance.
Metabolomic series
1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Metabolomics has emerged as a powerful tool in the
quantitative identification of physiological and disease-induced biological states.
Extracellular metabolome or metabolic profiling data, in particular,
can provide an insightful view of intracellular physiological states in a noninvasive manner.
Results
We used an updated genome-scale
metabolic network model of Saccharomyces cerevisiae, iMM904, to investigate
how changes in the extracellular metabolome can be used
to study systemic changes in intracellular metabolic states.
The iMM904 metabolic network was reconstructed based on
an existing genome-scale network, iND750,
and includes 904 genes and 1,412 reactions.
The network model was first validated by
comparing 2,888 in silico single-gene deletion strain growth phenotype predictions
to published experimental data.
Extracellular metabolome data measured
of ammonium assimilation pathways
in response to environmental and genetic perturbations
was then integrated with the iMM904 network
in the form of relative overflow secretion constraints and
a flux sampling approach was used to characterize candidate flux distributions allowed by these constraints.
Predicted intracellular flux changes were
consistent with published measurements
on intracellular metabolite levels and fluxes.
Patterns of predicted intracellular flux changes
could also be used to correctly identify the regions of
The inferred perturbations in intracellular reaction fluxes were further analyzed
using reporter metabolite and subsystem (i.e., metabolic pathway) approaches [30]
in order to identify dominant metabolic features that are collectively perturbed (Figure 2).
The sampling-based approach also has the additional benefit of
being less sensitive to inaccuracies in metabolite secretion profiles than
optimization-based methods and can effectively be used – in biofluid metabolome analysis.
integration of exometabolomic (EM) data
Figure 1.Schematic illustrating the integration of exometabolomic (EM) data with the constraint-based framework.
(A) Cells are subjected to genetic and/or environmental perturbations to secrete metabolite patterns unique to that condition.
(B) EM is detected, identified, and quantified.
(C) EM data is integrated as required secretion flux constraints to define allowable solution space.
(D) Random sampling of solution space yields the range of feasible flux distributions for intracellular reactions.
(E) Sampled fluxes were compared to sampled fluxes of another condition to determine
which metabolic regions were altered between the two conditions (see Figure 2).
(F) Significantly altered metabolic regions were identified.
sampling and scoring analysis to determine intracellular flux changes
Figure 2.Schematic of sampling and scoring analysis to determine intracellular flux changes.
(A) Reaction fluxes are sampled for two conditions.
(B & C) Sample of flux differences is calculated by selecting random flux values from each condition
to obtain a distribution of flux differences for each reaction.
(D) Standardized reaction Z-scores are determined, which represent
how far the sampled flux differences deviates from a zero flux change.
This study was divided into two parts and describes:
(i) the reconstruction and validation of an expanded S. cerevisiae metabolic network, iMM904; and
(ii) the systematic inference of intracellular metabolic states from
two yeast EM data sets using a constraint-based sampling approach.
The first EM data set compares wild type yeast to the gdh1/GDH2 (glutamate dehydrogenase) strain [31],
which indicated good agreement between predicted metabolic changes
of intracellular metabolite levels and fluxes [31,32].
The second EM data set focused on secreted amino acid measurements
from a separate study of yeast cultured in different ammonium and potassium concentrations [33].
We analyzed the EM data to gain further insight into
perturbed ammonium assimilation processes as well as
metabolic states relating potassium limitation and
ammonium excess conditions to one another.
The model-based analysis of both
separately published extracellular metabolome datasets
suggests a relationship between
glutamate,
threonine and
folate metabolism,
which are collectively perturbed when ammonium assimilation processes are broadly disrupted
The data provided are the model text files of the iMM904 metabolic network
that is compatible with the available COBRA Toolbox [13]. The model structure
can be loaded into Matlab using the ‘SimPhenyPlus’ format with GPR and compound information.
Minimization of Metabolic Adjustment (MoMA) [39], and
a linear version of MoMA (linearMoMA).
In the linearMoMA method, minimization of the quadratic objective function
of the original MoMA algorithm
was replaced by minimization of the corresponding 1-norm objective function
(i.e. sum of the absolute values of the differences of wild type FBA solution
and the knockout strain flux solution).
The computed results were then compared to growth phenotype data
(viable/lethal) from a previously published experimental gene deletion study [3].
The comparison between experimental and in silico deletion phenotypes involved
choosing a threshold for the predicted relative growth rate of
a deletion strain that is considered to be viable.
We used standard ROC curve analysis
to assess the accuracy of different prediction methods and models
across the full range of the viability threshold parameter,
results shown in Figure S2 [see Additional file 3].
The ROC curve plots the true viable rate against the false viable rate
allowing comparison of different models and methods
without requiring arbitrarily choosing this parameter a priori [40].
The optimal prediction performance corresponds to
the point closest to the top left corner of the ROC plot
(i.e. 100% true viable rate, 0% false viable rate).
Comparison of iMM904 and iLL672 gene deletion predictions and experimental data under minimal media conditions
Media
Model
Method
True viable
False viable
False lethal
True lethal
True viable %
False viable %
MCC
Glucose
iMM904 full
FBA
647
10
32
33
95.29
23.26
0.6
iMM904 full
linMOMA
644
10
35
33
94.85
23.26
0.58
iMM904 full
MOMA
644
10
35
33
94.85
23.26
0.58
iMM904 red
FBA
440
9
28
33
94.02
21.43
0.61
iMM904 red
linMOMA
437
9
31
33
93.38
21.43
0.6
iMM904 red
MOMA
437
9
31
33
93.38
21.43
0.6
iLL672 full
MOMA
433
9
35
33
92.52
21.43
0.57
Galactose
iMM904 full
FBA
595
32
36
59
94.29
35.16
0.58
iMM904 full
linMOMA
595
32
36
59
94.29
35.16
0.58
iMM904 full
MOMA
595
32
36
59
94.29
35.16
0.58
iMM904 red
FBA
409
12
33
56
92.53
17.65
0.67
iMM904 red
linMOMA
409
12
33
56
92.53
17.65
0.67
iMM904 red
MOMA
409
12
33
56
92.53
17.65
0.67
iLL672 full
MOMA
411
19
31
49
92.99
27.94
0.61
Glycerol
iMM904 full
FBA
596
43
36
47
94.3
47.78
0.48
iMM904 full
linMOMA
595
44
37
46
94.15
48.89
0.47
iMM904 full
MOMA
598
44
34
46
94.62
48.89
0.48
iMM904 red
FBA
410
20
34
46
92.34
30.3
0.57
iMM904 red
linMOMA
409
21
35
45
92.12
31.82
0.56
iMM904 red
MOMA
412
21
32
45
92.79
31.82
0.57
iLL672 full
MOMA
406
20
38
46
91.44
30.3
0.55
Ethanol
iMM904 full
FBA
593
45
29
55
95.34
45
0.54
iMM904 full
linMOMA
592
45
30
55
95.18
45
0.54
iMM904 full
MOMA
592
44
30
56
95.18
44
0.55
iMM904 red
FBA
408
21
27
54
93.79
28
0.64
iMM904 red
linMOMA
407
21
28
54
93.56
28
0.63
iMM904 red
MOMA
407
20
28
55
93.56
26.67
0.64
iLL672 full
MOMA
401
13
34
62
92.18
17.33
0.68
MCC, Matthews correlation coefficient (see Methods). Note that the iLL672 predictions were obtained directly from [3] and thus the viability threshold was not optimized using the maximum MCC approach.
we assumed that sampling of the optimal solution space was appropriate for this study.
In order to sample a physiologically reasonable range of flux distributions,
samples for four different oxygen uptake rates
(1, 2, 3, and 4 mmol/hr/gDW with 5 million steps each)
were combined in the final analysis.
Standardized scoring of flux differences between perturbation and control conditions
A Z-score based approach was implemented to quantify differences in flux samples between two conditions (Figure 2).
First, two flux vectors were chosen randomly,
one from each of the two samples to be compared and
the difference between the flux vectors was computed.
This approach was repeated to create a sample of 10,000 (n) flux difference vectors
for each pair of conditions considered (e.g. mutant or perturbed environment vs. wild type).
Based on this flux difference sample, the sample mean (μdiff,i) and standard deviation (σdiff,i)
between the two conditions was calculated for each reaction i. The reaction Z-score was calculated as:
reaction Z-score
which describes the sampled mean difference deviation
from a population mean change of zero (i.e. no flux difference
between perturbation and wild type).
Note that this approach allows accounting for uncertainty in the
flux distributions inferred based on the extracellular metabolite secretion constraints.
This is in contrast to approaches such as FBA or MoMA that would predict
a single flux distribution for each condition and thus potentially
overestimate differences between conditions.
The reaction Z-scores can then be further used in analysis
to identify significantly perturbed regions of the metabolic network
based on reporter metabolite [44] or subsystem [30] Z-scores.
These reporter regions indicate, or “report”, dominant perturbation features
at the metabolite and pathway levels for a particular condition.
The reporter metabolite Z-score for any metabolite j can be derived from the reaction Z-scores
of the reactions consuming or producing j (set of reactions denoted as Rj) as:
reporter z-score for any metabolite j
where Nj is the number of reactions in Rj and mmet,j is calculated as
distributional correction for m_met,j SQRT
To account and correct for background distribution, the metabolite Z-score was normalized
by computing μmet,Nj and σmet,,Nj corresponding to the mean mmet and
its standard deviation for 1,000 randomly generated reaction sets of size Nj.
Z-scores for subsystems were calculated similarly by considering the set of reactions Rk
that belongs to each subsystem k.
Hence, positive metabolite and subsystem scores indicate a significantly perturbed metabolic region
relative to other regions, whereas
a negative score indicate regions that are not perturbed
more significantly than what is expected by random chance.
Perturbation subnetworks of reactions and connecting metabolites were visualized using Cytoscape [45].
Results and discussion
Reconstruction and validation of iMM904 network iMM904 network content
A previously reconstructed S. cerevisiae network, iND750,
was used as the basis for the construction of the expanded iMM904 network.
Prior to its presentation here, the iMM904 network content was the basis for a consensus jamboree network that was recently published
but has not yet been adapted for FBA calculations [46].
The majority of iND750 content was carried over and
further expanded on to construct iMM904, which accounts for
904 genes,
1,228 individual metabolites, and
1,412 reactions of which
395 are transport reactions.
Both the number of gene-associated reactions and the number of metabolites
increased in iMM904 compared with the iND750 network.
Additional genes and reactions included in the network primarily expanded the
lipid,
transport, and
carbohydrate subsystems.
The lipid subsystem includes
new genes and
reactions involving the degradation of sphingolipids and glycerolipids.
Sterol metabolism was also expanded to include
the formation and degradation of steryl esters, the
storage form of sterols.
The majority of the new transport reactions were added
to connect network gaps between intracellular compartments
to enable the completion of known physiological functions.
We also added a number of new secretion pathways
based on experimentally observed secreted metabolites [31].
A number of gene-protein-reaction (GPR) relationships were modified
to include additional gene products that are required to catalyze a reaction.
For example, the protein compounds
thioredoxin and
ferricytochrome C
were explicitly represented as compounds in iND750 reactions, but
the genes encoding these proteins were not associated with their corresponding GPRs.
Other examples include glycogenin and NADPH cytochrome p450 reductases (CPRs),
which are required in the assembly of glycogen and
to sustain catalytic activity in cytochromes p450, respectively.
These additional proteins were included in iMM904 as
part of protein complexes to provide a more complete
representation of the genes and
their corresponding products necessary for a catalytic activity to occur.
Major modifications to existing reactions were in cofactor biosynthesis, namely in
quinone,
beta-alanine, and
riboflavin biosynthetic pathways.
Reactions from previous S. cerevisiae networks associated with
quinone,
beta-alanine, and
riboflavin biosynthetic pathways
were essentially inferred from known reaction mechanisms based on
reactions in previous network reconstructions of E. coli [2,47].
These pathways were manually reviewed
based on current literature and subsequently replaced by
reactions and metabolites specific to yeast.
Additional changes in other subsystems were also made, such as
changes to the compartmental location of a gene and
its corresponding reaction(s),
changes in reaction reversibility and cofactor specificity, and
the elucidation of particular transport mechanisms.
A comprehensive listing of iMM904 network contents as well as
a detailed list of changes between iND750 and iMM904 is included
[see Additional file 1].
Predicting deletion growth phenotypes
The updated genome-scale iMM904 metabolic network was validated
by comparing in silico single-gene deletion predictions to
in vivo results from a previous study used
to analyze another S. cerevisiae metabolic model, iLL672 [3].
This network was constructed based on the iFF708 network [22],
To validate the predicted results, reaction flux changes from both FBA and sampling methods were compared to differential intracellular metabolite level data measured from the same study. Intracellular metabolites involved in highly perturbed reactions (i.e. reactants and products) predicted from FBA and sampling analyses were identified and
compared to metabolites that were experimentally identified as significantly changed (p < 0.05) between mutant and wild-type. Statistical measures of recall, accuracy, and
precision were calculated and represent the predictive sensitivity, exactness, and reproducibility respectively. From the sampling analysis, a considerably larger number of
significantly perturbed reactions are predicted in the anaerobic case (505 reactions, or 70.7% of active reactions) than in aerobic (394 reactions, or 49.8% of active reactions). The top percentile of FBA flux changes equivalent to the percentage of significantly perturbed sampling reactions were compared to the intracellular data. Results from both analyses are summarized in Table 2. Sampling predictions were considerably higher in recall than FBA predictions for both conditions, with respective ranges of 0.83–1
compared to 0.48–0.96. Accuracy was also higher in sampling predictions; however, precision was slightly better in the FBA predictions as expected due to the smaller
number of predicted changes. Overall, the sampling predictions of perturbed intracellular metabolites are strongly consistent with the experimental data and significantly
outperforms that of FBA optimization predictions in accurately predicting differential metabolites involved in perturbed intracellular fluxes.
Table 2. Statistical comparison of the differential intracellular metabolite data set (p < 0.05) with metabolites involved in perturbed reactions predicted by FBA optimization and sampling analyses for aerobic and anaerobic gdh1/GDH2 mutant.
Table 2 Statistical comparison of the differential intracellular metabolite data set (p < 0.05)
with metabolites involved in perturbed reactions predicted by FBA optimization and
sampling analyses for aerobic and anaerobic gdh1/GDH2 mutant.
Aerobic
Anaerobic
Overall
FBA
Sampling
FBA
Sampling
FBA
Recall
0.48
0.83
0.96
1
0.71
0.91
Accuracy
0.55
0.62
0.64
0.64
0.6
0.63
Precision
0.78
0.69
0.64
0.63
0.68
0.66
Overall statistics indicate combined results of both conditions.
Figure 3.Perturbation reaction subnetwork of gdh1/GDH2 mutant under aerobic conditions.
The network illustrates a simplified subset of highly perturbedPerturbation subnetworks can be drawn to visualize predicted significantly perturbed intracellular reactions and illustrate their connection to the observed secreted metabolites in the aerobic and anaerobic gdh1/GDH2 mutants.
Perturbation reaction subnetwork of gdh1.GDH2 mutant under aerobic conditions.
Figure 3 shows an example of a simplified aerobic perturbation subnetwork consisting primarily of proximal pathways connected directly to a subset of major secreted
metabolites
glutamate,
proline,
D-lactate, and
2-hydroxybuturate.
Figure 4 displays anaerobic reactions with Z-scores of similar magnitude to the perturbed reactions in Figure 3. The same subset of metabolites is also present in the
larger anaerobic perturbation network and indicates that the NADPH/NADH balance perturbation induced by the gdh1/GDH2 manipulation has widespread effects
beyond just altering glutamate metabolism anaerobically.
Interestingly, it is clear that the majority of the secreted metabolite pathways involve connected perturbed reactions that broadly converge on glutamate.
Note that Figures 3 and 4 only show the subnetworks that consisted of two or more connected reactions for a number of secreted metabolites no contiguous perturbed pathway could be identified by the sampling approach. This indicates that the secreted metabolite pattern alone is not sufficient to determine which specific
production and secretion pathways are used by the cell for these metabolites.
Reactions connected to aerobically-secreted metabolites predicted from the sampling analysis of the gdh1/GDH2 mutant strain.
The major secreted metabolites
glutamate,
proline,
D-lactate, and
2-hydroxybuturate
were also detected in the anaerobic condition. Metabolite abbreviations are found in Additional file 1.
Perturbation reaction subnetwork of gdh1/GDH2 mutant under anaerobic conditions.
Perturbation reaction subnetwork of gdh1.GDH2 mutant under anaerobic conditions
Subnetwork illustrates the highly perturbed anaerobic reactions of similar Z-reaction magnitude to the reactions in Figure 3.
A significantly larger number of reactions indicates mutant metabolic effects are more widespread in the anaerobic environment.
The network shows that perturbed pathways converge on glutamate, the main site in which the gdh1/GDH2 modification was introduced, which
suggests that the direct genetic perturbation effects are amplified under this environment. Metabolite abbreviations are found in Additional file 1.
To further highlight metabolic regions that have been systemically affected by the gdh1/GDH2 modification, reporter metabolite and subsystem methods [30] were used to
summarize reaction scores around specific metabolites and in specific metabolic subsystems. The top ten significant scores for metabolites/subsystems associated with more
than three reactions are summarized in Tables 3 (aerobic) and 4 (anaerobic), with Z > 1.64 corresponding to p < 0.05 for a one-tailed distribution. Full data for all reactions,
reporter metabolites, and reporter subsystems is included [see Additional file 4].
Table 3. List of the top ten significant reporter metabolite and subsystem scores for the gdh1/GDH2 vs. wild type comparison in aerobic conditions.
Table 3
List of the top ten significant reporter metabolite and subsystem scores for the gdh1/GDH2 vs. wild type comparison in aerobic conditions.
Reporter metabolite
Z-score
No of reactions*
L-proline [c]
2.71
4
Carbon dioxide [m]
2.51
15
Proton [m]
2.19
51
Glyceraldehyde 3-phosphate [c]
1.93
7
Ubiquinone-6 [m]
1.82
5
Ubiquinol-6 [m]
1.82
5
Ribulose-5-phosphate [c]
1.8
4
Uracil [c]
1.74
4
L-homoserine [c]
1.72
4
Alpha-ketoglutarate [m]
1.71
8
Reporter subsystem
Z-score
No of reactions
Citric Acid Cycle
4.58
7
Pentose Phosphate Pathway
3.29
12
Glycine and Serine Metabolism
2.69
17
Alanine and Aspartate Metabolism
2.65
6
Oxidative Phosphorylation
1.79
8
Thiamine Metabolism
1.54
8
Arginine and Proline Metabolism
1.44
20
Other Amino Acid Metabolism
1.28
5
Glycolysis/Gluconeogenesis
0.58
14
Anaplerotic reactions
0.19
9
*Number of reactions categorized in a subsystem or found to be neighboring each metabolite
Perturbations under aerobic conditions largely consisted of pathways involved in mediating the NADH and NADPH balance. Among the highest scoring aerobic subsystems
are TCA cycle and pentose phosphate pathway – key pathways directly involved in the generation of NADH and NADPH. Reporter metabolites involved in these
subsystems –
glyceraldehyde-3-phosphate,
ribulose-5-phosphate, and
alpha-ketoglutarate – were also identified.
These results are consistent with flux and enzyme activity measurements
of the gdh1/GDH2 strain under aerobic conditions [32],
which reported significant reduction in the pentose phosphate pathway flux
with concomitant changes in other central metabolic pathways.
Levels of several TCA cycle intermediates (e.g. fumarate, succinate, malate) were also elevated
in the gdh1/GDH2 mutant according to the differential intracellular metabolite data.
Clustergram of top reporter metabolites – y in ammonium-toxic and potassium-limited conditions
Clustergram of top reporter metabolites (i.e. in yellow) in ammonium-toxic and potassium-limited conditions.
Amino acid perturbation patterns (shown in red) were shown to be consistently scored across conditions, indicating that potassium-limited environments K1 (lowest
concentration) and K2 (low concentration) elicited a similar ammonium detoxification response as ammonium-toxic environments N1 (high concentration) and N2
(highest concentration). Metabolites associated with folate metabolism (highlighted in green) are also highly perturbed in ammonium-toxic conditions. Metabolite
abbreviations are found in Additional file 1.
In addition to perturbed amino acids, a secondary effect notably appears at high ammonia levels in which metabolic regions related to folate metabolism are significantly affected. As highlighted in green in Figure 3, we predicted significantly perturbed key metabolites involved in the cytosolic folate cycle. These include tetrahydrofolate derivatives and other metabolites connected to the folate pathway, namely glycine and the methionine-derived methylation cofactors S-adenosylmethionine and S-adenosyl-homocysteine. Additionally, threonine was identified to be a key perturbed metabolite in excess ammonium conditions. These results further illustrate the close
connection between threonine biosynthesis, folate metabolism involving glycine derived from its threonine precursor, and nucleotide biosynthesis [51] that was discussed in
conjunction with the gdh1/GDH2 strain data. Taken together with the anaerobic gdh1/GDH2 data, the results consistently suggest highly perturbed threonine and folate
metabolism when amino acid-related pathways are broadly affected.
In both ammonium-toxic and potassium-limited environments, impaired cellular growth was observed, which can be attributed to high energetic costs of increased
ammonium assimilation to synthesize and excrete amino acids. However, under high ammonium environments, reporter metabolites related to threonine and folate
metabolism indicated that their perturbation, and thus purine supply, may be an additional factor in decreasing cellular viability as there is a direct relationship between
intracellular folate levels and growth rate [54]. Based on these results, we concluded that while potassiumlimited growth in yeast indeed shares physiological features with
growth in ammonium excess, its effects are not as detrimental as actual ammonium excess. The effects on proximal amino acid metabolic pathways are similar in both
environments as indicated by the secretion of the majority of amino acids. However, when our method was applied to analyze the physiological basis behind differences in
secretion profiles between low potassium and high ammonium conditions, ammonium excess was predicted to likely disrupt physiological ammonium assimilation processes,
which in turn potentially impacts folate metabolism and associated cellular growth.
Conclusion
The method presented in this study presents an approach to connecting intracellular flux states to metabolites that are excreted under various physiological conditions. We
showed that well-curated genome-scale metabolic networks can be used to integrate and analyze quantitative EM data by systematically identifying altered intracellular
pathways related to measured changes in the extracellular metabolome. We were able to identify statistically significant metabolic regions that were altered as a result of
genetic (gdh1/GD2 mutant) and environmental (excess ammonium and limited potassium) perturbations, and the predicted intracellular metabolic changes were consistent
with previously published experimental data including measurements of intracellular metabolite levels and metabolic fluxes. Our reanalysis of previously published EM data
on ammonium assimilation-related genetic and environmental perturbations also resulted in testable hypotheses about the role of threonine and folate pathways in mediating
broad responses to changes in ammonium utilization. These studies also demonstrated that the samplingbased method can be readily applied when only partial secreted
metabolite profiles (e.g. only amino acids) are available.
With the emergence of metabolite biofluid biomarkers as a diagnostic tool in human disease [55,56] and the availability of genome-scale human metabolic networks [1],
extensions of the present method would allow identifying potential pathway changes linked to these biomarkers. Employing such a method for studying yeast metabolism was possible as the metabolomic data was measured under controllable environmental conditions where the inputs and outputs of the system were defined. Measured metabolite biomarkers in a clinical setting, however, is far from a controlled environment with significant variations in genetic, nutritional, and environmental factors between different
patients. While there are certainly limitations for clinical applications, the method introduced here is a progressive step towards applying genome-scale metabolic networks
towards analyzing biofluid metabolome data as it 1) avoids the need to only study optimal metabolic states based on a predetermined objective function, 2) allows dealing with noisy experimental data through the sampling approach, and 3) enables analysis even with limited identification of metabolites in the data. The ability to establish potential
connections between extracellular markers and intracellular pathways would be valuable in delineating the genetic and environmental factors associated with a particular
disease.
Authors’ contributions
Conceived and designed the experiments: MLM MJH BOP. Performed experiments: MLM MJH. Analyzed the data: MLM MJH. Wrote the paper: MLM MJH BOP. All authors have read and approved the final manuscript.
Acknowledgements
We thank Jens Nielsen for providing the raw metabolome data for the mutant strain, and Jan Schellenberger and Ines Thiele for valuable discussions. This work was supported by NIH grant R01 GM071808. BOP serves on the scientific advisory board of Genomatica Inc.
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Prologue to Cancer – e-book Volume One – Where are we in this journey?
Author and Curator: Larry H. Bernstein, MD, FCAP
Consulting Reviewer and Contributor: Jose Eduardo de Salles Roselino, MD
LH Bernstein
LES Roselino
This is a preface to the fourth in the ebook series of Leaders in Pharmaceutical Intelligence, a collaboration of experienced doctorate medical and pharmaceutical professionals. The topic is of great current interest, and it entails a significant part of current medical expenditure by a group of neoplastic diseases that may develop at different periods in life, and have come to supercede infections or even eventuate in infectious disease as an end of life event. The articles presented are a collection of the most up-to-date accounts of the state of a now rapidly emerging field of medical research that has benefitted enormously by progress in immunodiagnostics, radiodiagnostics, imaging, predictive analytics, genomic and proteomic discovery subsequent to the completion of the Human Genome Project, advances in analytic methods in qPCR, gene sequencing, genome mapping, signaling pathways, exome identification, identification of therapeutic targets in inhibitors, activators, initiators in the progression of cell metabolism, carcinogenesis, cell movement, and metastatic potential. This story is very complicated because we are engaged in trying to evoke from what we would like to be similar clinical events, dissimilar events in their expression and classification, whether they are within the same or different anatomic class. Thus, we are faced with constructing an objective evidence-based understanding requiring integration of several disciplinary approaches to see a clear picture. The failure to do so creates a high risk of failure in biopharmaceutical development.
The chapters that follow cover novel and important research and development in cancer related research, development, diagnostics and treatment, and in balance, present a substantial part of the tumor landscape, with some exceptions. Will there ever be a unifying concept, as might be hoped for? I certainly can’t see any such prediction on the horizon. Part of the problem is that disease classification is a human construct to guide us, and so are treatments that have existed and are reexamined for over 2,000 years. In that time, we have changed, our afflictions have been modified, and our environment has changed with respect to the microorganisms within and around us, viruses, the soil, and radiation exposure, and the impacts of war and starvation, and access to food. The outline has been given. Organic and inorganic chemistry combined with physics has given us a new enterprise in biosynthetics that is and will change our world. But let us keep in mind that this is a human construct, just as drug target development is such a construct, workable with limitations.
What Molecular Biology Gained from Physics
We need greater clarity and completeness in defining the carcinogenetic process. It is the beginning, but not the end. But we must first examine the evolution of the scientific structure that leads to our present understanding. This was preceded by the studies of anatomy, physiology, and embryology that had to occur as a first step, which was followed by the researches into bacteriology, fungi, sea urchins and the evolutionary creatures that could be studied having more primary development in scale. They are still major objects of study, with the expectation that we can derive lessons about comparative mechanisms that have been passed on through the ages and have common features with man. This became the serious intent of molecular biology, the discipline that turned to find an explanation for genetics, and to carry out controlled experiments modelled on the discipline that already had enormous success in physics, mathematics, and chemistry. In 1900, when Max Planck hypothesized that the frequency of light emitted by the black body depended on the frequency of the oscillator that emitted it, it had important ramifications for chemistry and biology (See Appendix II and Footnote 1, Planck equation, energy and oscillation). The leading idea is to search below the large-scale observations of classical biology.
The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, provides a starting point, but the construct is undergoing revision in light of emerging novel roles for RNA and signaling pathways. The term, coined by Warren Weaver (director of Natural Sciences for the Rockefeller Foundation), who observed an emergence of significant change given recent advances in fields such as X-ray crystallography. Molecular biology also plays important role in understanding formations, actions, regulations of various parts of cellswhich can be used efficiently for targeting new drugs, diagnosis of disease, physiology of the Cell. The Nobel Prize in Physiology or Medicine in 1969 was shared by Max Delbrück, Alfred D. Hershey, Salvador E. Luria, whose work with viral replication gave impetus to the field. Delbruck was a physicist who trained in Copenhagen under Bohr, and specifically committed himself to a rigor in biology, as was in physics.
Dorothy Hodgkin protein crystallography
Rosalind Franlin crystallographer double helix
Max Delbruck molecular biology
Max Planck Quantum Physics
We then stepped back from classical (descriptive) physiology, with the endless complexity, to molecular biology. This led us to the genetic code, with a double helix model. It has recently been found insufficiently explanatory, with the recent construction of triplex and quadruplex models. They have a potential to account for unaccounted for building blocks, such as inosine, and we don’t know whether more than one model holds validity under different conditions . The other major field of development has been simply unaccounted for in the study of proteomics, especially in protein-protein interactions, and in the energetics of protein conformation, first called to our attention by the work of Jacob, Monod, and Changeux (See Footnote 2). Proteins are not just rigid structures stamped out by the monotonously simple DNA to RNA to protein concept. Nothing is ever quite so simple. Just as there are epigenetic events, there are posttranslational events, and yet more.
JP Changeux
The Emergence of Molecular Biology
I now return the discussion to the topic of medicine, the emergence of molecular biology and the need for convergence with biochemistry in the mid-20th century. Jose Eduardo de Salles Roselino recalls “I was previously allowed to make of the conformational energy as made by R Marcus in his Nobel lecture revised (J. of Electroanalytical Chemistry 438:(1997) p251-259. (See Footnote 1) His description of the energetic coordinates of a landscape of a chemical reaction is only a two-dimensional cut of what in fact is a volcano crater (in three dimensions) (each one varies but the sum of the two is constant. Solvational+vibrational=100% in ordinate) nuclear coordinates in abcissa. In case we could represent it by research methods that allow us to discriminate in one by one degree of different pairs of energy, we would most likely have 360 other similar representations of the same phenomenon. The real representation would take into account all those 360 representations together. In case our methodology was not that fine, for instance it discriminates only differences of minimal 10 degrees in 360 possible, will have 36 partial representations of something that to be perfectly represented will require all 36 being taken together. Can you reconcile it with ATGC? Yet, when complete genome sequences were presented they were described as though we will know everything about this living being. The most important problems in biology will be viewed by limited vision always and the awareness of this limited is something we should acknowledge and teach it. Therefore, our knowledge is made up of partial representations. If we had the entire genome data for the most intricate biological problems, they are still not amenable to this level of reductionism. But going from general views of signals andsymptoms we could get to the most detailed molecular view and in this case genome provides an anchor.”
“Warburg Effect” describes the preference of glycolysis and lactic acid fermentation rather than oxidative phosphorylation for energy production in cancer cells. Mitochondrial metabolism is an important and necessary component in the functioning and maintenance of the cell, and accumulating evidence suggests that dysfunction of mitochondrial metabolism plays a role in cancer. Progress has demonstrated the mechanisms of the mitochondrial metabolism-to-glycolysis switch in cancer development and how to target this metabolic switch.
glycolysis
Otto Warburg
Louis Pasteur
The expression “Pasteur effect” was coined by Warburg when inspired by Pasteur’s findings in yeast cells, when he investigated this metabolic observation (Pasteur effect) in cancer cells. In yeast cells, Pasteur had found that the velocity of sugar used was greatly reduced in presence of oxygen. Not to be confused, in the “Crabtree effect”, the velocity of sugar metabolism was greatly increased, a reversal, when yeast cells were transferred from the aerobic to an anaerobic condition. Thus, the velocity of sugar metabolism of yeast cells was shown to be under metabolic regulatory control in response to change in environmental oxygen conditions in growth. Warburg had to verify whether cancer cells and tissue related normal mammalian cells also have a similar control mechanism. He found that this control was also found in normal cells studied, but was absent in cancer cells. Strikingly, cancer cells continue to have higher anaerobic gycolysis despite the presence of oxygen in their culture media (See Footnote 3).
Taking this a step further, food is digested and supplied to cells In vertebrates mainly in the form of glucose, which is metabolized producing Adenosine Triphosphate (ATP) by two pathways. Glycolysis, occurs via anaerobic metabolism in the cytoplasm, and is of major significance for making ATP quickly, but in a minuscule amount (2 molecules). In the presence of oxygen, the breakdown process continues in the mitochondria via the Krebs’s cycle coupled with oxidative phosphorylation, which is more efficient for ATP production (36 molecules). Cancer cells seem to depend on glycolysis. In the 1920s, Otto Warburg first proposed that cancer cells show increased levels of glucose consumption and lactate fermentation even in the presence of ample oxygen (known as “Warburg Effect”). Based on this theory, oxidative phosphorylation switches to glycolysis which promotes the proliferation of cancer cells. Many studies have demonstrated glycolysis as the main metabolic pathway in cancer cells.
Albert Szent Gyogy (Warburg’s student) and Otto Meyerhof both studied striated skeletal muscle metabolism invertebrates, and they found those changes observed in yeast by Pasteur. The description of the anaerobic pathway was largely credited to Emden and Meyerhof. Whenever there is increase in muscle work, energy need is above what can be provided by blood supply, the cell metabolism changes from aerobic (where Acetyl CoA provides the chemical energy for aerobic production of ATP) to anaerobic metabolism of glucose. In this condition, glucose is obtained directly from its muscle glycogen stores (not from hepatic glycogenolysis). This is the sole source of chemical energy that is independent of oxygen supplied to the cell. It is a physiological change on muscle metabolism that favors autonomy. It does not depend upon the blood oxygen for aerobic metabolim or blood sources of carbon metabolites borne out from adipose tissue (free fatty acids) or muscle proteins (branched chain amino acids), or vascular delivery of glucose. On that condition, the muscle can perform contraction by its internal source of ATP and uses conversion of pyruvate to lactate in order to regenerate much-needed NAD (by hydride transfer from pyruvate) as a replacement for this mitochondrial function. This regulatory change, keeps glycolysis going at fast rate in order to meet ATP needs of the cell under low yield condition (only two or three ATP for each glucose converted into two lactate molecules). Therefore, it cannot last for long periods of time. This regulatory metabolic change is made in seconds, minutes and therefore happens with the proteins that are already presented in the cell. It does not requires the effect of transcription factors and/or changes in gene expression (See Footnote 1, 2).
In other types mammalian cells, like those from the lens of the eye (86% gycolysis + pentose shunt), and red blood cells (RBC)[both lacking mitochondria], and also in the deep medullary layer of the kidneys, for lack of mitochondria in the first two cases and normally reduced blood perfusion in the third – A condition required for the counter current mechanism and our ability to concentrate urine also have, permanent higher anaerobic metabolism. In the case of RBC, it includes the ability to produce in a shunt of glycolytic pathway 2,3 diphospho- glycerate that is required to place the hemogloblin macromolecule in an unstable equilibrium between its two forms (R and T – Here presented as simplified accordingly to the model of Monod, Wyman and Changeux. The final model would be even much complex (see for instance, H-W and K review Nature 2007 vol 450: p 964-972 )
Any tissue under a condition of ischemia that is required for some medical procedures (open heart surgery, organ transplants, etc) displays this fast regulatory mechanism (See Footnote 1, 2). A display of these regulatory metabolic changes can be seen in: Cardioplegia: the protection of the myocardium during open heart surgery: a review. D. J. Hearse J. Physiol., Paris, 1980, 76, 751-756 (Fig 1). The following points are made:
1- It is a fast regulatory response. Therefore, no genetic mechanism can be taken into account.
2- It moves from a reversible to an irreversible condition, while the cells are still alive. Death can be seen at the bottom end of the arrow. Therefore, it cannot be reconciled with some of the molecular biology assumptions:
A- The gene and genes reside inside the heart muscle cells but, in order to preserve intact, the source of coded genetic information that the cell reads and transcribes, DNA must be kept to a minimal of chemical reactivity.
B- In case sequence determines conformation, activity and function , elevated potassium blood levels could not cause cardiac arrest.
In comparison with those conditions here presented, cancer cells keep the two metabolic options for glucose metabolism at the same time. These cells can use glucose that our body provides to them or adopt temporarily, an independent metabolic form without the usual normal requirement of oxygen (one or another form for ATP generation). ATP generation is here, an over-simplification of the metabolic status since the carbon flow for building blocks must also be considered and in this case oxidative metabolism of glucose in cancer cells may be viewed as a rich source of organic molecules or building blocks that dividing cells always need.
JES Roselino has conjectured that “most of the Krebs cycle reaction works as ideal reversible thermodynamic systems that can supply any organic molecule that by its absence could prevent cell duplication.” In the vision of Warburg, cancer cells have a defect in Pasteur-effect metabolic control. In case it was functioning normally, it will indicate which metabolic form of glucose metabolism is adequate for each condition. What more? Cancer cells lack differentiated cell function. Any role for transcription factors must be considered as the role of factors that led to the stable phenotypic change of cancer cells. The failure of Pasteur effect must be searched for among the fast regulatory mechanisms that aren’t dependent on gene expression (See Footnote 3).
Extending the thoughts of JES Roselino (Hepatology 1992;16: 1055-1060), reduced blood flow caused by increased hydrostatic pressure in extrahepatic cholestasis decreases mitochondrial function (quoted in Hepatology) and as part of Pasteur effect normal response, increased glycolysis in partial and/or functional anaerobiosis and therefore blocks the gluconeogenic activity of hepatocytes that requires inhibited glycolysis. In this case, a clear energetic link can be perceived between the reduced energetic supply and the ability to perform differentiated hepatic function (gluconeogenesis). In cancer cells, the action of transcription factors that can be viewed as different ensembles of kaleidoscopic pieces (with changing activities as cell conditions change) are clearly linked to the new stable phenotype. In relation to extrahepatic cholestasis mentioned above it must be reckoned that in case a persistent chronic condition is studied a secondary cirrhosis is installed as an example of persistent stable condition, difficult to be reversed and without the requirement for a genetic mutation. (See Footnote 4).
The Rejection of Complexity
Most of our reasoning about genes was derived from scientific work in microorganisms. These works have provided great advances in biochemistry.
double helix
Triple helix
formation of triple helix
1- The “Gelehrter idea”: No matter what you are doing you will always be better off, in case you have a gene (In chapter 7 Principles of Medical Genetics Gelehrter and Collins Williams & Wilkins 1990).
2- The idea that everything could be found following one gene one enzyme relationship that works fine for our understanding of the metabolism, in all biological problems.
3- The idea that everything that explains biochemistry in microorganisms explains also for every living being (J Nirenberg).
4- The idea that biochemistry may not require that time should be also taken into account. Time must be considered only for genetic and biological evolution studies (S Luria. In Life- The unfinished experiment 1977 C Scribner´s sons NY).
5- Finally, the idea that everything in biology, could be found in the genome. Since all information in biology goes from DNA through RNA to proteins. Alternatively, are in the DNA, in case the strict line that includes RNA is not included.
This last point can be accepted in case it is considered that ALL GENETIC information is in our DNA. Genetics as part of life and not as its total expression.
For example, when our body is informed that the ambient temperature is too low or alternatively is too high, our body is receiving an information that arrives from our environment. This external information will affect our proteins and eventually, in case of longer periods in a new condition will cause adaptive response that may include conformational changes in transcription factors (proteins) that will also, produce new readings on the DNA. However, it is an information that moves from outside, to proteins and not from DNA to proteins. The last pathway, when transcription factors change its conformation and change DNA reading will follow the dogmatic view as an adaptive response (See Footnotes 1-3).
However, in case, time is taken into account, the first reactions against cold or warmer temperatures will be the ones that happen through change in protein conformation, activities and function before any change in gene expression can be noticed at protein level. These fast changes, in seconds, minutes cannot be explained by changes in gene expression and are strongly linked to what is needed for the maintenance of life.
“It is possible”, says Roselino, “desirable, to explain all these fast biochemical responses to changes in a living being condition as the sound foundation of medical practices without a single mention to DNA. In case a failure in any mechanism necessary to life is found to be genetic in its origin, the genome in context with with this huge set of transcription factors must be taken into account. This is the biochemical line of reasoning that I have learned with Houssay and Leloir. It would be an honor to see it restored in modern terms.”
More on the Mechanism of Metabolic Control
It was important that genomics would play such a large role in medical research for the last 70 years. There is also good reason to rethink the objections of the Nobelists James Watson and Randy Schekman in the past year, whatever discomfort it brings. Molecular biology has become a tautology, and as a result deranged scientific rigor inside biology.
Eatson and Crick
Randy-Schekman Berkeley
According to JES Roselino, “consider that glycolysis is oscillatory thanks to the kinetic behavior of Phosphofructokinase. Further, by its effect upon Pyruvate kinase through Fructose 1,6 diphosphate oscillatory levels, the inhibition of gluconeogenesis is also oscillatory. When the carbon flow through glycolysis is led to a maximal level gluconeogenesis will be almost completely blocked. The reversal of the Pyruvate kinase step in liver requires two enzymes (Pyruvate carboxylase (maintenance of oxaloacetic levels) + phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32)) and energy requiring reactions that most likely could not as an ensemble, have a fast enough response against pyruvate kinase short period of inhibition during high frequency oscillatory periods of glycolytic flow. Only when glycolysis oscillates at low frequency the opposite reaction could enable gluconeogenic carbon flow.”
In case it can be shown in a rather convincing way, the same reasoning could be applied to understand how simple replicative signals inducing Go to G1 transition in cells, could easily overcome more complex signals required for cell differentiation and differentiated function.
Perhaps the problem of overextension of the equivalence of the DNA and what happens to the organism is also related to the initial reliance on a single cell model to relieve the complexity (which isn’t fully the case).
For instance, consider this fragment:
“Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions.”
Cold Spring Harbour Symp. Quant. Biol. 1973 p 187-193 J.C Seidel and J Gergely – Investigation of conformational changes in Spin-Labeled Myosin Model for muscle contraction:
Huxley, A. F. 1971 Proc. Roy. Soc (London) (B) 178:1
Huxley, A.F and R. M. Simmons,1971. Nature 233:633
J.C Haselgrove X ray Evidence for a conformational Change in the Actin-containing filaments…Cold Spring Harbour Symp Quant Biol.1972 v 37: p 341-352
Only a very small sample indicating otherwise. Proteins were held as interacting macromolecules, changing their conformation in regulatory response to changes in the microenvironment (See Footnote 2). DNA was the opposite, non-interacting macromolecules to be as stable as a library must be.
The dogma held that the property of proteins could be read in DNA alone. Consequenly, the few examples quoted above, must be ignored and all people must believe that DNA alone, without environmental factors roles, controls protein amino acid sequence (OK), conformation (not true), activity (not true) and function (not true).
It appeared naively to be correct from the dogma to conclude from interpreting your genome: You have a 50% increased risk of developing the following disease (deterministic statement). The correct form must be: You belong to a population that has a 50% increase in the risk of….followed by – what you must do to avoid increase in your personal risk and the care you should take in case you want to have longer healthy life. Thus, genetics and non-genetic diseases were treated as the same and medical foundations were reinforced by magical considerations (dogmas) in a very profitable way for those involved besides the patient.
Footnotes:
There is a link of electricity with ions in biology and the oscillatory behavior of some electrical discharges. In addition, the oscillatory form of electrical discharged may have allowed Planck to relate high energy content with higher frequencies and conversely, low energy content in low frequency oscillatory events. One may think of high density as an indication of great amount of matter inside a volume in space. This helps the understanding of Planck’s idea as a high-density-energy in time for a high frequency phenomenon.
Take into account a protein that may have its conformation restricted by an S-S bridge. This protein also, may move to another more flexible conformation in case it is in HS HS condition when the S-S bridge is broken. Consider also that, it takes some time for a protein to move from one conformation for instance, the restricted conformation (S-S) to other conformations. Also, it takes a few seconds or minutes to return to the S-S conformation (This is the Daniel Koshland´s concept of induced fit and relaxation time used by him in order to explain allosteric behavior of monomeric proteins- Monod, Wyman and Changeux requires tetramer or at least, dimer proteins).
In case you have glycolysis oscillating in a frequency much higher than the relaxation time you could lead to the prevalence of high NADH effect leading to high HS /HS condition and at low glycolytic frequency, you could have predominance of S-S condition affecting protein conformation. In case you have predominance of NAD effect upon protein S-S you would get the opposite results. The enormous effort to display the effect of citrate and over Phosphofructokinase conformation was made by others. Take into account that ATP action as an inhibitor in this case, is a rather unusual one. It is a substrate of the reaction, and together with its action as activator F1,6 P (or its equivalent F2,6 P) is also unusual. However, it explains oscillatory behaviour of glycolysis. (Goldhammer , A.R, and Paradies: PFK structure and function, Curr. Top Cell Reg 1979; 15:109-141).
The results presented in our Hepatology work must be viewed in the following way: In case the hepatic (oxygenated) blood flow is preserved, the bile secretory cells of liver receive well-oxygenated blood flow (the arterial branches bath secretory cells while the branches originated from portal vein irrigate the hepatocytes. During extra hepatic cholestasis the low pressure, portal blood flow is reduced and the hepatocytes do not receive enough oxygen required to produce ATP that gluconeogenesis demands. Hepatic artery do not replace this flow since, its branches only join portal blood fluxes after the previous artery pressure is reduced to a low pressure venous blood – at the point where the formation of hepatic vein is. Otherwise, the flow in the portal vein would be reversed or, from liver to the intestine. It is of no help to take into account possible valves for this reasoning since minimal arterial pressure is well above maximal venous pressure and this difference would keep this valve in permanent close condition. In low portal blood flow condition, the hepatocyte increases pyruvate kinase activity and with increased pyruvate kinase activity Gluconeogenesis is forbidden (See Walsh & Cooper revision quoted in the Hepatology as ref 23). For the hemodynamic considerations, role of artery and veins in hepatic portal system see references 44 and 45 Rappaport and Schneiderman and Rappapaport.
Appendix I.
metabolic pathways
Signals Upstream and Targets Downstream of Lin28 in the Lin28 Pathway
1. Functional Proteomics Adds to Our Understanding
Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect. The crowded environments inside cells lead to the proteins shrinking. As these proteins interact with other molecules in the body and bring other proteins together, understanding of these processes is essential “as they play a major role in many processes in our body, for instance in the onset of cancer”, comments study coordinator Ben Schuler.
Measurements using the “molecular ruler”
“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.
With computer simulations the researchers have mimicked the behaviour of disordered proteins. They want to use them in future for more accurate predictions of their properties and functions.
Correcting test tube results
That’s why it’s important, according to Schuler, to monitor the proteins not only in the test tube but also in the organism. “This takes into account the fact that it is very crowded on the molecular level in our body as enormous numbers of biomolecules are crammed into a very small space in our cells”, says Schuler. The biochemists have mimicked this “molecular crowding” and observed that in this environment disordered proteins shrink, too.
Given these results many experiments may have to be revisited as the spatial organisation of the molecules in the organism could differ considerably from that in the test tube according to the biochemist from the University of Zurich. “We have, therefore, developed a theoretical analytical method to predict the effects of molecular crowding.” In a next step the researchers plan to apply these findings to measurements taken directly in living cells.
Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerantmarinemollusc, Littorina littorea
JL Lama , RAV Bell and KB Storey
Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions.
Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in K G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly.
All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.
Structural Basis for Isoform-Selective Inhibition in Nitric Oxide Synthase
TL. Poulos and H Li
In the cardiovascular system, the important signaling molecule nitric oxide synthase (NOS) converts L-arginine into L-citrulline and releases nitric oxide (NO). NO produced by endothelial NOS (eNOS) relaxes smooth muscle which controls vascular tone and blood pressure. Neuronal NOS (nNOS) produces NO in the brain, where it influences a variety of neural functions such as neural transmitter release. NO can also support the immune system, serving as a cytotoxic agent during infections. Even with all of these important functions, NO is a free radical and, when overproduced, it can cause tissue damage. This mechanism can operate in many neurodegenerative diseases, and as a result the development of drugs targeting nNOS is a desirable therapeutic goal.
However, the active sites of all three human isoforms are very similar, and designing inhibitors specific for nNOS is a challenging problem. It is critically important, for example, not to inhibit eNOS owing to its central role in controlling blood pressure. In this Account, we summarize our efforts in collaboration with Rick Silverman at Northwestern University to develop drug candidates that specifically target NOS using crystallography, computational chemistry, and organic synthesis. As a result, we have developed aminopyridine compounds that are 3800-fold more selective for nNOS than eNOS, some of which show excellent neuroprotective effects in animal models. Our group has solved approximately 130 NOS-inhibitor crystal structures which have provided the structural basis for our design efforts. Initial crystal structures of nNOS and eNOS bound to selective dipeptide inhibitors showed that a single amino acid difference (Asp in nNOS and Asn in eNOS) results in much tighter binding to nNOS. The NOS active site is open and rigid, which produces few large structural changes when inhibitors bind. However, we have found that relatively small changes in the active site and inhibitor chirality can account for large differences in isoform-selectivity. For example, we expected that the aminopyridine group on our inhibitors would form a hydrogen bond with a conserved Glu inside the NOS active site. Instead, in one group of inhibitors, the aminopyridine group extends outside of the active site where it interacts with a heme propionate. For this orientation to occur, a conserved Tyr side chain must swing out of the way. This unanticipated observation taught us about the importance of inhibitor chirality and active site dynamics. We also successfully used computational methods to gain insights into the contribution of the state of protonation of the inhibitors to their selectivity. Employing the lessons learned from the aminopyridine inhibitors, the Silverman lab designed and synthesized symmetric double-headed inhibitors with an aminopyridine at each end, taking advantage of their ability to make contacts both inside and outside of the active site. Crystal structures provided yet another unexpected surprise. Two of the double-headed inhibitor molecules bound to each enzyme subunit, and one molecule participated in the generation of a novel Zn site that required some side chains to adopt alternate conformations. Therefore, in addition to achieving our specific goal, the development of nNOS selective compounds, we have learned how subtle differences in and structure can control proteinligand interactions and often in unexpected ways.
Nitric oxide synthase
arginine-NO-citulline cycle
active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).
NO – muscle, vasculature, mitochondria
Figure: (A) Structure of one of the early dipeptide lead compounds, 1, that exhibits excellentisoform selectivity. (B, C) show the crystal structures of the dipeptide inhibitor 1 in the active site of eNOS (PDB: 1P6L) and nNOS (PDB: 1P6H). In nNOS, the inhibitor “curls” which enables the inhibitor R-amino group to interact with both Glu592 and Asp597. In eNOS, Asn368 is the homologue to nNOS Asp597.
Accounts in Chem Res 2013; 46(2): 390-98.
Jamming a Protein Signal
Interfering with a single cancer-promoting protein and its receptor can open this resistance mechanism by initiating autophagy of the affected cells, according to researchers at The University of Texas MD Anderson Cancer Center in the journal Cell Reports. According to Dr. Anil Sood and Yunfei Wen, lead and first authors, blocking prolactin, a potent growth factor for ovarian cancer, sets off downstream events that result in cell by autophagy, the process recycles damaged organelles and proteins for new use by the cell through the phagolysozome. This in turn, provides a clinical rationale for blocking prolactin and its receptor to initiate sustained autophagy as an alternative strategy for treating cancers.
Steep reductions in tumor weight
Prolactin (PRL) is a hormone previously implicated in ovarian, endometrial and other cancer development andprogression. When PRL binds to its cell membrane receptor, PRLR, activation of cancer-promoting cell signaling pathways follows. A variant of normal prolactin called G129R blocks the reaction between prolactin and its receptor. Sood and colleagues treated mice that had two different lines of human ovarian cancer, both expressing the prolactin receptor, with G129R. Tumor weights fell by 50 percent for mice with either type of ovarian cancer after 28 days of treatment with G129R, and adding the taxane-based chemotherapy agent paclitaxel cut tumor weight by 90 percent. They surmise that higher doses of G129R may result in even greater therapeutic benefit.
Next the team used the prolactin-mimicking peptide to treat cultures of cancer spheroids which sharply reduced their numbers, and blocked the activation of JAK2 and STAT signaling pathways.
Protein analysis of the treated spheroids showed increased presence of autophagy factors and genomic analysis revealed increased expression of a number of genes involved in autophagy progression and cell death. Then a series of experiments using fluorescence and electron microscopy showed that the cytosol of treated cells had large numbers of cavities caused by autophagy.
The team also connected the G129R-induced autophagy to the activity of PEA-15, a known cancer inhibitor. Analysis of tumor samples from 32 ovarian cancer patients showed that tumors express higher levels of the prolactin receptor and lower levels of phosphorylated PEA-15 than normal ovarian tissue. However, patients with low levels of the prolactin receptor and higher PEA-15 had longer overall survival than those with high PRLR and low PEA-15.
Source: MD Anderson Cancer Center
Chemists’ Work with Small Peptide Chains of Enzymes
Korendovych and his team designed seven simple peptides, each containing seven amino acids. They then allowed the molecules of each peptide to self-assemble, or spontaneously clump together, to form amyloids. (Zinc, a metal with catalytic properties, was introduced to speed up the reaction.) What they found was that four of the seven peptides catalyzed the hydrolysis of molecules known as esters, compounds that react with water to produce water and acids—a feat not uncommon among certain enzymes.
“It was the first time that a peptide this small self-assembled to produce an enzyme-like catalyst,” says Korendovych. “Each enzyme has to be an exact fit for its respective substrate,” he says, referring to the molecule with which an enzyme reacts. “Even after millions of years, nature is still testing all the possible combinations of enzymes to determine which ones can catalyze metabolic reactions. Our results make an argument for the design of self-assembling nanostructured catalysts.”
Source: Syracuse University
Here are three articles emphasizing the value of combinatorial analysis, which can be formed from genomic, clinical, and proteomic data sets.
Comparative analysis of differential network modularity in tissue specific normal and cancer protein interaction networks
F Islam , M Hoque , RS Banik , S Roy , SS Sumi, et al.
As most biological networks show modular properties, the analysis of differential modularity between normal and cancer protein interaction networks can be a good way to understand cancer more significantly. Two aspects of biological network modularity e.g. detection of molecular complexes (potential modules or clusters) and identification of crucial nodes forming the overlapping modules have been considered in this regard.
The computational analysis of previously published protein interaction networks (PINs) has been conducted to identify the molecular complexes and crucial nodes of the networks. Protein molecules involved in ten major cancer signal transduction pathways were used to construct the networks based on expression data of five tissues e.g. bone, breast, colon, kidney and liver in both normal and cancer conditions.
Cancer PINs show higher level of clustering (formation of molecular complexes) than the normal ones. In contrast, lower level modular overlapping is found in cancer PINs than the normal ones. Thus a proposition can be made regarding the formation of some giant nodes in the cancer networks with very high degree and resulting in reduced overlapping among the network modules though the predicted molecular complex numbers are higher in cancer conditions.
Islam et al. Journal of Clinical Bioinformatics 2013, 3:19-32
A new 12-gene diagnostic biomarker signature of melanoma revealed by integrated microarray analysis
Here we present an integrated microarray analysis framework, based on a genome-wide relative significance (GWRS) and genome-wide global significance (GWGS) model. When applied to five microarray datasets on melanoma published between 2000 and 2011, this method revealed a new signature of 200 genes. When these were linked to so-called ‘melanoma driver’ genes involved in MAPK, Ca2+, and WNT signaling pathways we were able to produce a new 12-gene diagnostic biomarker signature for melanoma (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, andWNT4).We have begun to experimentally validate a subset of these genes involved inMAPK signaling at the protein level, including CXCL13, COL11A1, PTPRF and SHC4 and found these to be overexpressed inmetastatic and primarymelanoma cells in vitro and in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin.
catalytic amyloid forming particle
8. PanelomiX: A threshold-based algorithm to create panels of biomarkers
The PanelomiX toolbox combines biomarkers and evaluates the performance of panels to classify patients better than singlemarkers or other classifiers. The ICBTalgorithm proved to be an efficient classifier, the results of which can easily be interpreted.
Here are two current examples of the immense role played by signaling pathways in carcinogenic mechanisms and in treatment targeting, which is also confounded by acquired resistance.
Triple-Negative Breast Cancer
epidermal growth factor receptor (EGFR or ErbB1) and
high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway
are both targeted in triple-negative breast cancer (TNBC).
activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs.
This study found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3.
The phosphorylation of HER3 and EGFR in response to these treatments
was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A).
MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and
the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors.
In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3
decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone.
Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and
this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody.
After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction).
Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.
Reference: Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer. JJ Tao, P Castel, N Radosevic-Robin, M Elkabets, et al. Sci. Signal., 25 March 2014; 7(318), p. ra29 http://dx.doi.org/10.1126/scisignal.2005125
10. Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells
The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF–ERK (extracellular signal–regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)–ERK pathway.
Reference: BRAF Inhibitors Induce Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells by Reactivating MEK and ERK Signaling. B Sanchez-Laorden, A Viros, MR Girotti, M Pedersen, G Saturno, et al., Sci. Signal., 25 March 2014; 7(318), p. ra30 http://dx.doi.org/10.1126/scisignal.2004815
Appendix II.
The world of physics in the twentieth century saw the end of determinism established by Newton. This is characterized by discrete laws that describe natural observations. These are in gravity and in eletricity. In an early phase of investigation, an era of galvanic or voltaic electricity represented a revolutionary break from the historical focus on frictional electricity. Alessandro Voltadiscovered that chemical reactions could be used to create positively charged anodes and negatively charged cathodes. In 1790, Prof. Luigi Alyisio Galvani of Bologna, while conducting experiments on “animal electricity“, noticed the twitching of a frog’s legs in the presence of an electric machine. He observed that a frog’s muscle, suspended on an iron balustrade by a copper hook passing through its dorsal column, underwent lively convulsions without any extraneous cause, the electric machine being at this time absent. Volta communicated a description of his pile to the Royal Society of London and shortly thereafter Nicholson and Cavendish (1780) produced the decomposition of water by means of the electric current, using Volta’s pile as the source of electromotive force.
Siméon Denis Poisson attacked the difficult problem of induced magnetization, and his results provided a first approximation. His innovation required the application of mathematics to physics. His memoirs on the theory of electricity and magnetism created a new branch of mathematical physics. The discovery of electromagnetic induction was made almost simultaneously and independently by Michael Faraday and Joseph Henry. Michael Faraday, the successor of Humphry Davy, began his epoch-making research relating to electric and electromagnetic induction in 1831. In his investigations of the peculiar manner in which iron filings arrange themselves on a cardboard or glass in proximity to the poles of a magnet, Faraday conceived the idea of magnetic “lines of force” extending from pole to pole of the magnet and along which the filings tend to place themselves. On the discovery being made that magnetic effects accompany the passage of an electric current in a wire, it was also assumed that similar magnetic lines of force whirled around the wire. He also posited that iron, nickel, cobalt, manganese, chromium, etc., are paramagnetic (attracted by magnetism), whilst other substances, such as bismuth, phosphorus, antimony, zinc, etc., are repelled by magnetism or are diamagnetic.
Around the mid-19th century, Fleeming Jenkin‘s work on ‘ Electricity and Magnetism ‘ and Clerk Maxwell’s ‘ Treatise on Electricity and Magnetism ‘ were published. About 1850 Kirchhoff published his laws relating to branched or divided circuits. He also showed mathematically that according to the then prevailing electrodynamic theory, electricity would be propagated along a perfectly conducting wire with the velocity of light. Herman Helmholtz investigated the effects of induction on the strength of a current and deduced mathematical equations, which experiment confirmed. In 1853 Sir William Thomson (later Lord Kelvin) predicted as a result of mathematical calculations the oscillatory nature of the electric discharge of a condenser circuit. Joseph Henry, in 1842 discerned the oscillatory nature of the Leyden jardischarge.
In 1864 James Clerk Maxwell announced his electromagnetic theory of light, which was perhaps the greatest single step in the world’s knowledge of electricity. Maxwell had studied and commented on the field of electricity and magnetism as early as 1855/6 when On Faraday’s lines of force was read to the Cambridge Philosophical Society. The paper presented a simplified model of Faraday’s work, and how the two phenomena were related. He reduced all of the current knowledge into a linked set of differential equations with 20 equations in 20 variables. This work was later published as On Physical Lines of Force in1861. In order to determine the force which is acting on any part of the machine we must find its momentum, and then calculate the rate at which this momentum is being changed. This rate of change will give us the force. The method of calculation which it is necessary to employ was first given by Lagrange, and afterwards developed, with some modifications, by Hamilton’s equations. Now Maxwell logically showed how these methods of calculation could be applied to the electro-magnetic field. The energy of a dynamical systemis partly kinetic, partly potential. Maxwell supposes that the magnetic energy of the field is kinetic energy, the electric energypotential. Around 1862, while lecturing at King’s College, Maxwell calculated that the speed of propagation of an electromagnetic field is approximately that of the speed of light. Maxwell’s electromagnetic theory of light obviously involved the existence of electric waves in free space, and his followers set themselves the task of experimentally demonstrating the truth of the theory. By 1871, he presented the Remarks on the mathematical classification of physical quantities.
A Wave-Particle Dilemma at the Century End
In 1896 J.J. Thomson performed experiments indicating that cathode rays really were particles, found an accurate value for their charge-to-mass ratio e/m, and found that e/m was independent of cathode material. He made good estimates of both the charge e and the mass m, finding that cathode ray particles, which he called “corpuscles”, had perhaps one thousandth of the mass of the least massive ion known (hydrogen). He further showed that the negatively charged particles produced by radioactive materials, by heated materials, and by illuminated materials, were universal. In the late 19th century, the Michelson–Morley experiment was performed by Albert Michelson and Edward Morley at what is now Case Western Reserve University. It is generally considered to be the evidence against the theory of a luminiferous aether. The experiment has also been referred to as “the kicking-off point for the theoretical aspects of the Second Scientific Revolution.” Primarily for this work, Albert Michelson was awarded theNobel Prize in 1907.
Wave–particle duality is a theory that proposes that all matter exhibits the properties of not only particles, which have mass, but also waves, which transfer energy. A central concept of quantum mechanics, this duality addresses the inability of classical concepts like “particle” and “wave” to fully describe the behavior of quantum-scale objects. Standard interpretations of quantum mechanics explain this paradox as a fundamental property of the universe, while alternative interpretations explain the duality as an emergent, second-order consequence of various limitations of the observer. This treatment focuses on explaining the behavior from the perspective of the widely used Copenhagen interpretation, in which wave–particle duality serves as one aspect of the concept of complementarity, that one can view phenomena in one way or in another, but not both simultaneously. Through the work of Max Planck, Albert Einstein, Louis de Broglie, Arthur Compton, Niels Bohr, and many others, current scientific theory holds that all particles also have a wave nature (and vice versa).
Beginning in 1670 and progressing over three decades, Isaac Newton argued that the perfectly straight lines of reflection demonstrated light’s particle nature, but Newton’s contemporaries Robert Hooke and Christiaan Huygens—and later Augustin-Jean Fresnel—mathematically refined the wave viewpoint, showing that if light traveled at different speeds in different, refraction could be easily explained. The resulting Huygens–Fresnel principle was supported by Thomas Young‘s discovery of double-slit interference, the beginning of the end for the particle light camp. The final blow against corpuscular theory came when James Clerk Maxwell discovered that he could combine four simple equations, along with a slight modification to describe self-propagating waves of oscillating electric and magnetic fields. When the propagation speed of these electromagnetic waves was calculated, the speed of light fell out. While the 19th century had seen the success of the wave theory at describing light, it had also witnessed the rise of the atomic theory at describing matter.
Matter and Light
In 1789, Antoine Lavoisier secured chemistry by introducing rigor and precision into his laboratory techniques. By discovering diatomic gases, Avogadro completed the basic atomic theory, allowing the correct molecular formulae of most known compounds—as well as the correct weights of atoms—to be deduced and categorized in a consistent manner. The final stroke in classical atomic theory came when Dimitri Mendeleev saw an order in recurring chemical properties, and created a table presenting the elements in unprecedented order and symmetry. Chemistry was now an atomic science.
Black-body radiation, the emission of electromagnetic energy due to an object’s heat, could not be explained from classical arguments alone. The equipartition theorem of classical mechanics, the basis of all classical thermodynamic theories, stated that an object’s energy is partitioned equally among the object’s vibrational modes. This worked well when describing thermal objects, whose vibrational modes were defined as the speeds of their constituent atoms, and the speed distribution derived from egalitarian partitioning of these vibrational modes closely matched experimental results. Speeds much higher than the average speed were suppressed by the fact that kinetic energy is quadratic—doubling the speed requires four times the energy—thus the number of atoms occupying high energy modes (high speeds) quickly drops off. Since light was known to be waves of electromagnetism, physicists hoped to describe this emission via classical laws. This became known as the black body problem. The Rayleigh–Jeans law which, while correctly predicting the intensity of long wavelength emissions, predicted infinite total energy as the intensity diverges to infinity for short wavelengths.
The solution arrived in 1900 when Max Planck hypothesized that the frequency of light emitted by the black body depended on the frequency of the oscillator that emitted it, and the energy of these oscillators increased linearly with frequency (according to his constanth, where E = hν). By demanding that high-frequency light must be emitted by an oscillator of equal frequency, and further requiring that this oscillator occupy higher energy than one of a lesser frequency, Planck avoided any catastrophe; giving an equal partition to high-frequency oscillators produced successively fewer oscillators and less emitted light. And as in the Maxwell–Boltzmann distribution, the low-frequency, low-energy oscillators were suppressed by the onslaught of thermal jiggling from higher energy oscillators, which necessarily increased their energy and frequency. Planck had intentionally created an atomic theory of the black body, but had unintentionally generated an atomic theory of light, where the black body never generates quanta of light at a given frequency with energy less than hν.
In 1905 Albert Einstein took Planck’s black body model in itself and saw a wonderful solution to another outstanding problem of the day: the photoelectric effect, the phenomenon where electrons are emitted from atoms when they absorb energy from light. Only by increasing the frequency of the light, and thus increasing the energy of the photons, can one eject electrons with higher energy. Thus, using Planck’s constant h to determine the energy of the photons based upon their frequency, the energy of ejected electrons should also increase linearly with frequency; the gradient of the line being Planck’s constant. These results were not confirmed until 1915, when Robert Andrews Millikan, produced experimental results in perfect accord with Einstein’s predictions. While the energy of ejected electrons reflected Planck’s constant, the existence of photons was not explicitly proven until the discovery of the photon antibunching effect When Einstein received his Nobel Prizein 1921, it was for the photoelectric effect, the suggestion of quantized light. Einstein’s “light quanta” represented the quintessential example of wave–particle duality. Electromagnetic radiation propagates following linear wave equations, but can only be emitted or absorbed as discrete elements, thus acting as a wave and a particle simultaneously.
Radioactivity Changes the Scientific Landscape
The turn of the century also features radioactivity, which later came to the forefront of the activities of World War II, the Manhattan Project, the discovery of the chain reaction, and later – Hiroshima and Nagasaki.
Marie Curie
Marie Skłodowska-Curie was a Polish and naturalized-French physicist and chemist who conducted pioneering research on radioactivity. She was the first woman to win a Nobel Prize, the only woman to win in two fields, and the only person to win in multiple sciences. She was also the first woman to become a professor at the University of Paris, and in 1995 became the first woman to be entombed on her own merits in the Panthéon in Paris. She shared the 1903 Nobel Prize in Physics with her husband Pierre Curie and with physicist Henri Becquerel. She won the 1911 Nobel Prize in Chemistry. Her achievements included a theory of radioactivity (a term that she coined, techniques for isolating radioactive isotopes, and the discovery of polonium and radium. She named the first chemical element that she discovered – polonium, which she first isolated in 1898 – after her native country. Under her direction, the world’s first studies were conducted into the treatment of neoplasms using radioactive isotopes. She founded the Curie Institutes in Paris and in Warsaw, which remain major centres of medical research today. During World War I, she established the first military field radiological centres. Curie died in 1934 due to aplastic anemia brought on by exposure to radiation – mainly, it seems, during her World War I service in mobile X-ray units created by her.
Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function
Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FACP, Clinical Pathologist and Biochemist
Apoptosis signaling pathways (Photo credit: AJC1)
This discussion is a followup on a series of articles elucidating the importance of NO, eNOS, iNOS, cardiovascular and vascular endothelium effects, and therapeutic targets.
This mechanism of action and signaling actions have been introduced so that we identify endocrine, paracrine, and such effects in the normal, stressed, and dysfunctional state. The size and breadth of this vital adaptive process is now further explored.
The title is short, befitting a subtitle. The full topic may be considered “Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer”.
Nitric Oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.
Uncoupling of aerobic glycolysis
Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic. High levels of NO can be produced by inducible nitric-oxide synthase in response to cytokine stimulation, primarily from macrophages, and elevated levels of NO is injurious to endothelium.Ccytochrome c release and caspase activation are involved in NO induced apoptosis. ROS also induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels. Mitochondria have been recognized to play a pivotal role in the signaling cascade of apoptosis leading to atherosclerosis-induced damage in endothelial cells.
The processes involved in the signaling pathways leading to apoptosis are complex but have some degree of convergence between cell types including those in the vasculature. Release of cytochrome c from mitochondria is a proapoptotic signal, which activates several downstream signaling events including formation of the apoptosome and activation of caspases. Ubiquinol cytochrome c reductase (complex III) is a site for ROS formation, and cytochrome c oxidase (complex IV) is a target for the interaction of NO in mitochondria.
The impact of the inhibition of mitochondrial protein synthesis is particularly important in NO-dependent cytotoxicity, and depends also on other factors such as glycolysis. These authors examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.
Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis. A Ramachandran, DR Moellering, E Ceaser, S Shiva, J Xu, and V Darley-Usmar. PNAS May 14, 2002: 99(10): 6643–6648 http://www.pnas.orgcgidoi10.1073pnas.102019899
Nitric oxide (NO) is a ubiquitous signaling molecule whose physiological roles mediated through the activation of the soluble guanylate cyclase are now clearly recognized. At physiological concentrations, NO also inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen, and recently we have suggested that the interplay between the two gases allows this enzyme to act as an oxygen sensor in cells. In addition, NO plays a variety of patho-physiological roles, some of which also may be the consequence of its action at a mitochondrial level. We have characterized the sequence of events that follow inhibition of complex IV by continuous exposure to NO.
The mitochondrion is a key organelle in the control of cell death. Nitric oxide (NO) inhibits complex IV in the respiratory chain and is reported to possess both proapoptotic and antiapoptotic actions. We investigated the effects of continuous inhibition of respiration by NO on mitochondrial energy status and cell viability. Serum-deprived human T cell leukemia (Jurkat) cells were exposed to NO at a concentration that caused continuous and complete (;85%) inhibition of respiration. Serum deprivation caused progressive loss of mitochondrial membrane potential (Dcm) and apoptotic cell death. In the presence of NO, Dcm was maintained compared to controls, and cells were protected from apoptosis. Similar results were obtained by using staurosporin as the apoptotic stimulus. As exposure of serum-deprived cells to NO progressed (>5 h), however, Dcm fell, correlating with the appearance of early apoptotic features and a decrease in cell viability. Glucose deprivation or iodoacetate treatment of cells in the presence of NO resulted in a collapse of Dcm, demonstrating involvement of glycolytic ATP in its maintenance. Under these conditions cell viability also was decreased. Treatment with oligomycin and or bongkrekic acid indicated that the maintenance of Dcm during exposure to NO is caused by reversal of the ATP synthase and other electrogenic pumps. Thus, blockade of complex IV by NO initiates a protective action in the mitochondrion to maintain Dcm; this results in prevention of apoptosis. It is likely that during cellular stress involving increased generation of NO this compound will trigger a similar sequence of events, depending on its concentration and duration of release. (mitochondrial membrane potential ; apoptosis ; necrosis)
Another study by this group shows that inhibition of respiration by exogenous nitric oxide (NO) in Jurkat cells leads to mitochondrial membrane hyperpolarization dependent on the utilization of glycolytic ATP by the F1Fo-ATPase and other transporters acting in reverse mode. This process also occurs in astrocytes, which are highly glycolytic cells, but not in neurons , which do not invoke glycolysis to maintain ATP concentrations. In addition, this hyperpolarization correlates with protection against apoptotic cell death. Others found an early phase of mitochondrial hyperpolarization after treatment of a variety of cells with different pro-apoptotic stimuli, which precedes the generation of free. At present, no satisfactory explanation has been proposed to explain the mechanism of hyperpolarization, the reasons why free radicals are released from the mitochondrion, or the connection of these phenomena with apoptosis.
The authors surmise that a pro-apoptotic stimulus, anti-Fas Ab, leads to release of endogenous NO from Jurkat cells in sufficient amounts to inhibit cell respiration and cause a hyperpolarization dependent on the reversal of the F1Fo-ATPase. Moreover, the reduction of the mitochondrial electron transport chain, after inhibition of cytochrome oxidase by NO, leads to generation of superoxide anion (O2). They suggest the process is a cellular defense response that may be overcome by pro-apoptotic mechanisms that occur in parallel.
Nitric oxide has been shown to render cells resistant to oxidative stress. Mechanisms proposed for the ability of nitric oxide to protect cells against oxidative stress include reactions of nitric oxide and the induction of adaptive responses that require protein synthesis. Nitric oxide forms iron complexes preventing the formation of strong oxidants. In addition, reactions of nitric oxide with lipid and or organic radicals protect against membrane peroxidation and peroxidative chemistry-induced cell injury. Exposure to low, nonlethal doses of nitric oxide induces adaptive responses that render cells resistant to lethal concentrations of nitric oxide and or peroxides, such as, the induction of hemoxygenase-1 (HO-1) and Mn superoxide dismutase. The up-regulation of HO-1 was accompanied by an increase in ferritin to account for the release of iron from HO-1, indicating a role of both iron heme and nonheme iron for peroxide-mediated cellular injury. Further, nitric oxide, by regulating critical mitochondrial functions such as respiration, membrane potential, and release of cytochrome c, is able to trigger defense mechanisms against cell death induced by pro-apoptotic stimuli.
This study investigates the potential contribution of nitric oxide’s ability to protect cells from oxidative stress, low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2. Spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 mM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria. ECV3041 eNOS cells with diminished mitochondria respiration are injured to the same extent as non-transfected ECV304 cells, and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide had a profound effect in cell metabolism, because ECV3041eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.
Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors of neurons. NO has also been implicated for such pathophysiological conditions as destruction of tumor cells by macrophages, rheumatoid arthritis, and focal brain ischemia. Some of these effects of NO are associated with hypoxic conditions. O2 radicals and ions that result from reactivity of NO are presumed to be involved in NO cytotoxicity. These investigators report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-Lglutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 mM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 5 6.6 mM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 5 2.5 mM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1a or HIF-1a-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1a to a DNA-binding form. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. K Sogawa, K Numayama-Tsuruta, M Ema, M Abe, et al. Proc. Natl. Acad. Sci. USA (Biochemistry) June 1998; 95:7368–7373. 1998. The National Academy of Sciences 0027-8424.98.957368-6. http:yywww.pnas.org.
The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism. Glycolysis and the Tricarboxylic Acid Cycle Are Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of Lotus japonicus[W][OA]. M Rocha, F Licausi, WL Arau´ jo, A Nunes-Nesi, et al. Plant Physiology Mar 2010; 152: 1501–1513. http://www.plantphysiol.org 2010 Amer Soc Plant Biologists
DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The PARP-1 activation leads to cell death. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation.
These findings suggest a sequence of events in which NAD+ depletion is a key event linking DNA damage to metabolic impairment and cell deathm. A similar scenario has been proposed by Zong et al. (2004), based on the finding that cell types that depend on aerobic glycolysis for ATP production exhibit a particularly high sensitivity to DNA damage and PARP-1 activation. In mature brain, glucose is normally the dominant metabolic substrate due to relatively slow transport of other metabolites across the blood– brain barrier. Oncein brain, glucose may be metabolized directly by neurons and glia or may be metabolized to lactate in glia and thelactate subsequently shuttled to neurons for oxidative metabolism (Dringen et al., 1993; Pellerin and Magistretti,1994; Wender et al., 2000; Dienel and Cruz, 2004). In either case, a block in glycolytic flux produced by NAD depletion will block energy metabolism in both neurons and glia in brain. Interestingly, the lactate shuttle hypothesis raises the possibility that activation of PARP-1 selectively in astroglia might also block energy metabolism in neurons.
Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO (Berkels et al., 1997)it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production. Nitric oxide and platelet energy metabolism. M Tomasiak, H Stelmach, T Rusak and J Wysocka. Acta Biochimica Polonica 2004; 51(3):789–803
These authors previously investigated the bioenergetic consequences of activating J774.A1 macrophages (MФ) with interferon (IFN)γ and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large
quantities of ATP. We now demonstrate, using TMRM fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨm) despite the complete inhibition of respiration. The maintenance of high ΔΨm is due to the utilization of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of FoF1-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MФ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨm and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the FoF1-ATP synthase. This collapse in ΔΨm was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspase 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation “glycolytically competent cells” such as MФ utilize significant amounts of the glycolytically-generated ATP to maintain ΔΨm and thereby prevent apoptosis.
Activated macrophages utilize glycolytic ATP to maintain mitochondrial membranepotential and prevent apoptotic cell death. A Garedew, SO Henderson, S Moncada. Cell Death and Differentiation. 2010. DOI : 10.1038/cdd.2010.27
The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.
Key points to take from this:
1. The role of NO in regulating cellular death is in many organs and central to this function is the stabilization of mitochondria through sufficient levels of NO. High levels of eNO leads to mitochondrial dysfunction that increases the dependence of ATP generated from glycolysis.
2. This is accompanied by inhibition of oxidative phosphorylation and stimulation of glycolysis, which brings the discussion to a different domain – cancer growth and Warburgh Effect.
3. This is accompanied by PPAR activation, cytoplasmic NAD+ depletion, and inhibition of glycolysis (critical in cells dependent on aerobic glycolysis), depletion of total energy production, and apoptosis.
4. Maintenance of high glycolytic generation of ATP is essential for cellular defense, but the oxygen consumption is uncoupled.
5. NO donors inhibiting mitochondrial respiration and cytochrome oxidase are similar to those stimulating glycolysis
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