Posts Tagged ‘signaling pathways’

Deep Learning for In-silico Drug Discovery and Drug Repurposing: Artificial Intelligence to search for molecules boosting response rates in Cancer Immunotherapy: Insilico Medicine @John Hopkins University

Reporter: Aviva Lev-Ari, PhD, RN

Insilico Medicine –>>> transcriptome-based pathway perturbation analysis

Insilico Medicine, Inc. is a bioinformatics company located at the Emerging Technology Centers at the Johns Hopkins University Eastern campus in Baltimore with R&D resources in Belgium, Russia, and Poland hiring talent through hackathons and competitions. It utilizes advances in genomics, big data analysis and deep learning for in silico drug discovery and drug repurposing for aging and age-related diseases. The company pursues internal drug discovery programs in cancer, Parkinson’s, Alzheimer’s, sarcopenia and geroprotector discovery. Through its Pharmaceutical Artificial Intelligence division the company provides advanced machine learning services to biotechnology, pharmaceutical, and skin care companies.


Brief company video: https://www.youtube.com/watch?v=l62jlwgL3v8.

Insilico Medicine develops a new approach to concomitant cancer immunotherapy

Artificial intelligence to search for molecules boosting response rates in cancer immunotherapy





  • Some of the most promising drugs for cancer therapy called checkpoint inhibitors often result in complete remissions, however, a majority of patients fail cancer immunotherapy with antibodies targeting immune checkpoints, such as CTLA-4 or programmed death-1 (PD-1).
  • Insilico Medicine developed a set of pathway-based signatures of response to popular checkpoint inhibitors
  • Using these markers and a deep learned drug scoring engine Insilico Medicine identified 12 leads that may help increase response to cancer immunotherapy and is seeking industry partnerships to test these leads

Thursday, July 14, 2016, Baltimore, MD — Recent advances in cancer immunotherapy demonstrated complete remission in multiple tumor types including melanoma and lung cancers. Almost every major pharmaceutical company operating in oncology space started multiple programs in immuno-oncology with thousands of clinical trials underway. Immuno-oncology is now a very broad field ranging from treatment of a patient with an engineered antibody to genome editing of patient’s immune cells. Genetic mutations accruing from the inherent genomic instability of tumor cells present neo-antigens that are recognized by the immune system. Cross-presentation of tumor antigens at the immune synapse between antigen-presenting dendritic cells and T lymphocytes can potentially activate an adaptive antitumor immune response, however, tumors continuously evolve to counteract and ultimately defeat such immune surveillance by co-opting and amplifying mechanisms of immune tolerance to evade elimination by the immune system. This prerequisite for tumor progression is enabled by the ability of cancers to produce negative regulators of immune response.

Cancer immunotherapy is currently focused on targeting immune inhibitory checkpoints that control T cell activation, such as CTLA-4 and PD-1. Monoclonal antibodies that block these immune checkpoints (commonly referred to as immune checkpoint inhibitors) can unleash antitumor immunity and produce durable clinical responses in a subset of patients with advanced cancers, such as melanoma and non-small-cell lung cancer. However, these immunotherapeutics are currently constrained by their inability to induce clinical responses in the vast majority of patients and the frequent occurrence of immune-related adverse events. A key limitation of checkpoint inhibitors is that they narrowly focus on modulating the immune synapse but do not address other key molecular determinants that may also be responsible for immune dysfunction.

Immunoresistance often ensues as a result of the concomitant activation of multiple, often overlapping signaling pathways. Therefore, inhibition of multiple, cross-talking pathways involved in survival control with combination therapy is usually more effective in decreasing the likelihood that cancer cells will develop therapeutic resistance than with single agent therapy. While research efforts are now focused on identifying new inhibitory mechanisms that could be targeted to achieve responses in patients with refractory cancers and provide durable and adaptable cancer control, there are outstanding challenges in determining what combination of immunotherapies and conventional therapies will prove beneficial against each tumor type.

“Immunotherapy is the most promising area in oncology resulting in cures, but we need to identify effective combinations of both established methods and new drugs developed specifically to boost response rates. At Insilico Medicine we developed a new method for screening, scoring and personalizing small molecules that may boost response rates to PD-1, PD-L1, CTLA4 and other checkpoint inhibitors. We can identify effective combinations of both established methods and new drugs developed specifically to boost response rates to immunotherapy”, said Artem Artemov, director of computational drug repurposing at Insilico Medicine.

Insilico Medicine, Inc. is one of the leaders in transcriptome-based pathway perturbation analysis. It is also a pioneer in applying cutting edge artificial intelligence techniques to biological and medical data analysis, particularly focused on in silico screening for new compounds against cancer and known drugs which can be repurposed against different cancers. One of the major programs currently ongoing at Insilico Medicine is evaluation of the transcriptional responses to multiple checkpoint inhibitors and analyzing the pathway-level differences in patients who respond and fail to respond to clinically approved checkpoint inhibitors. This novel computational approach is aimed at identifying new drug candidates which can be used in combination with immunotherapy to unleash durable antitumor effect against several types of cancers.

Recently, scientists at Insilico Medicine performed a large in silico screening of compounds that can be administered in combination with anti-PD1 immunotherapy to increase response rates. The researchers collected transcriptomic data from tumors of patients who either responded or failed to respond to standard immunotherapy, using both publically available and internally generated data. Next, they used differential pathway activation analysis and deep learning based approaches to identify transcriptomic signatures predicting the success of immunotherapy in a particular tumor type.

Finally, they analyzed drug-induced transcriptomic effects to screen for the drugs that can robustly drive transcriptomes of tumor cells from non-responsive state to the state responsive to immunotherapy. In other words, researchers developed approach that can predict whether drug of interest would induce a transcriptional signature that characterizes those patients that respond to cancer immunotherapy in non-respondents. This method allows personalizing these drugs to individual patients and specific checkpoint inhibitors. Among the top-scoring drugs, they found several compounds known to increase response rates in combination with cancer immunotherapy. One of the top-scoring compounds included a naturally-occurring substance marketed as a natural product.

The current list of top-scoring leads that may increase response rates to checkpoint inhibitors included 12 small molecules identified using signaling pathway perturbation analysis and annotated using a deeply learned drug scoring system. Insilico Medicine is currently open for partnerships which will allow further testing and validation of the discovered compounds ex vivo on cell cultures established from tumors which respond and failed to respond to immunotherapy, as well as in mice with patient-derived tumor xenografts. This approach may greatly reduce the costs of preclinical trials and significantly shorten the timeframe from a drug prediction to validation and marketing. The compounds, after preclinical and clinical validation, may improve cancer care and dramatically increase the lifespan of cancer patients.

A panel of leads for concomitant immunotherapy is part of a large number of leads developed using DeepPharma™, artificially-intelligent drug discovery engine, which includes a large number of molecules predicted to be effective antineoplastic agents, metabolic regulators, CVD and CNS lead, senolytics and ED drugs. Recently Insilico Medicine published several seminal papers demonstrating proof of concept of utilizing deep learning techniques to predict pharmacological properties of small molecules using transcriptional response data utilizing deep neural networks for biomarker development. “Deep Learning Applications for Predicting Pharmacological Properties of Drugs and Drug Repurposing Using Transcriptomic Data,” a paper published in Molecular Pharmaceuticals received the American Chemical Society Editors’ Choice Award. Another recent collaboration with Biotime, Inc resulted in a launch of Embryonic.AI, deep learned predictor of differentiation state of the sample.



Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.



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Recent Research On SMAD4 In Pancreatic Cancer

Curator: David Orchard-Webb, PhD


Deleted in Pancreatic Cancer, locus 4 (DPC4) officially known as SMAD4 is a component of the Transforming Growth Factor Beta (TGFß) pathway with tumour suppressive properties. As its name suggests it is frequently lost in pancreatic cancer, although through a variety of mechanisms in addition to gene deletion. The loss of SMAD4 is important in the progression of pancreatic intraepithelial neoplasia (PanIN) towards pancreatic ductal adenocarcinoma (PDAC). The expression of SMAD4 can suppress metastasis, angiogenesis, and cancer stem-like cell generation. SMAD4 can promote cancer cell apoptosis through a recently described mechanism involving a lethal epithelial to mesenchymal transition (EMT). SMAD4 status has a predictive role in pancreatic cancer personalised medicine. This curation categorises recent publications of note regarding SMAD4.


Role of SMAD4 in neoplastic progression towards PDAC


Garcia-Carracedo, Dario, Chih-Chieh Yu, Nathan Akhavan, Stuart A. Fine, Frank Schönleben, Naoki Maehara, Dillon C. Karg, et al. ‘Smad4 Loss Synergizes with TGFα Overexpression in Promoting Pancreatic Metaplasia, PanIN Development, and Fibrosis’. Edited by Ilse Rooman. PLOS ONE 10, no. 3 (24 March 2015): e0120851. doi:10.1371/journal.pone.0120851.


Norris, A M, A Gore, A Balboni, A Young, D S Longnecker, and M Korc. ‘AGR2 Is a SMAD4-Suppressible Gene That Modulates MUC1 Levels and Promotes the Initiation and Progression of Pancreatic Intraepithelial Neoplasia’. Oncogene 32, no. 33 (15 August 2013): 3867–76. doi:10.1038/onc.2012.394.


Leung, Lisa, Nikolina Radulovich, Chang-Qi Zhu, Dennis Wang, Christine To, Emin Ibrahimov, and Ming-Sound Tsao. ‘Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis’. Edited by Hidayatullah G Munshi. PLoS ONE 8, no. 12 (27 December 2013): e84366. doi:10.1371/journal.pone.0084366.


Mechanism of SMAD4 deactivation


Xia, Xiang, Kundong Zhang, Gang Cen, Tao Jiang, Jun Cao, Kejian Huang, Chen Huang, Qian Zhao, and Zhengjun Qiu. ‘MicroRNA-301a-3p Promotes Pancreatic Cancer Progression via Negative Regulation of SMAD4’. Oncotarget 6, no. 25 (28 August 2015): 21046–63. doi:10.18632/oncotarget.4124.


Murphy, Stephen J., Steven N. Hart, Geoffrey C. Halling, Sarah H. Johnson, James B. Smadbeck, Travis Drucker, Joema Felipe Lima, et al. ‘Integrated Genomic Analysis of Pancreatic Ductal Adenocarcinomas Reveals Genomic Rearrangement Events as Significant Drivers of Disease’. Cancer Research 76, no. 3 (1 February 2016): 749–61. doi:10.1158/0008-5472.CAN-15-2198.


Sawai, Yugo, Yuzo Kodama, Takahiro Shimizu, Yuji Ota, Takahisa Maruno, Yuji Eso, Akira Kurita, et al. ‘Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations’. Cancer Research 75, no. 16 (15 August 2015): 3292–3301. doi:10.1158/0008-5472.CAN-14-3028.


Demagny, Hadrien, and Edward M De Robertis. ‘Point Mutations in the Tumor Suppressor Smad4/DPC4 Enhance Its Phosphorylation by GSK3 and Reversibly Inactivate TGF-β Signaling’. Molecular & Cellular Oncology 3, no. 1 (2 January 2016): e1025181. doi:10.1080/23723556.2015.1025181.


Foster, David. ‘BxPC3 Pancreatic Cancer Cells Express a Truncated Smad4 Protein upon PI3K and mTOR Inhibition’. Oncology Letters, 28 January 2014. doi:10.3892/ol.2014.1833.


Hao, Jun, Shuyu Zhang, Yingqi Zhou, Cong Liu, Xiangui Hu, and Chenghao Shao. ‘MicroRNA 421 Suppresses DPC4/Smad4 in Pancreatic Cancer’. Biochemical and Biophysical Research Communications 406, no. 4 (25 March 2011): 552–57. doi:10.1016/j.bbrc.2011.02.086.


SMAD4 effects on cell motility


Zhang, Xueying, Junxia Cao, Yujun Pei, Jiyan Zhang, and Qingyang Wang. ‘Smad4 Inhibits Cell Migration via Suppression of JNK Activity in Human Pancreatic Carcinoma PANC‑1 Cells’. Oncology Letters, 7 April 2016. doi:10.3892/ol.2016.4427.


Kang, Ya ’an, Jianhua Ling, Rei Suzuki, David Roife, Xavier Chopin-Laly, Mark J. Truty, Deyali Chatterjee, et al. ‘SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium’. Edited by Neil A. Hotchin. PLoS ONE 9, no. 9 (29 September 2014): e107948. doi:10.1371/journal.pone.0107948.


Chen, Yu-Wen, Pi-Jung Hsiao, Ching-Chieh Weng, Kung-Kai Kuo, Tzu-Lei Kuo, Deng-Chyang Wu, Wen-Chun Hung, and Kuang-Hung Cheng. ‘SMAD4 Loss Triggers the Phenotypic Changes of Pancreatic Ductal Adenocarcinoma Cells’. BMC Cancer 14, no. 1 (2014): 1. https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-14-181.


SMAD4 effects on angiogenesis


Zhou, Zhichao, Juming Lu, Jingtao Dou, Zhaohui Lv, Xi Qin, and Jing Lin. ‘FHL1 and Smad4 Synergistically Inhibit Vascular Endothelial Growth Factor Expression’. Molecular Medicine Reports 7, no. 2 (February 2013): 649–53. doi:10.3892/mmr.2012.1202.


SMAD4 mediated repression of cancer stem-like cells


Hoshino, Yukari, Jun Nishida, Yoko Katsuno, Daizo Koinuma, Taku Aoki, Norihiro Kokudo, Kohei Miyazono, and Shogo Ehata. ‘Smad4 Decreases the Population of Pancreatic Cancer–Initiating Cells through Transcriptional Repression of ALDH1A1’. The American Journal of Pathology 185, no. 5 (2015): 1457–1470. http://www.sciencedirect.com/science/article/pii/S0002944015000802.


SMAD4 mediated growth inhibition/ apoptosis induction


David, Charles J., Yun-Han Huang, Mo Chen, Jie Su, Yilong Zou, Nabeel Bardeesy, Christine A. Iacobuzio-Donahue, and Joan Massagué. ‘TGF-β Tumor Suppression through a Lethal EMT’. Cell 164, no. 5 (February 2016): 1015–30. doi:10.1016/j.cell.2016.01.009.


Wang, Qi, Juanjuan Li, Wei Wu, Ruizhe Shen, He Jiang, Yuting Qian, Yanping Tang, et al. ‘Smad4-Dependent Suppressor Pituitary Homeobox 2 Promotes PPP2R2A-Mediated Inhibition of Akt Pathway in Pancreatic Cancer’. Oncotarget 7, no. 10 (8 March 2016): 11208–22. doi:10.18632/oncotarget.7158.


Poorly characterised targets of SMAD4


Fullerton, Paul T., Chad J. Creighton, and Martin M. Matzuk. ‘Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling’. Molecular Endocrinology (Baltimore, Md.) 29, no. 10 (October 2015): 1440–53. doi:10.1210/me.2015-1102.


Li, Lei, Zhaoshen Li, Xiangyu Kong, Dacheng Xie, Zhiliang Jia, Weihua Jiang, Jiujie Cui, et al. ‘Down-Regulation of MicroRNA-494 via Loss of SMAD4 Increases FOXM1 and β-Catenin Signaling in Pancreatic Ductal Adenocarcinoma Cells’. Gastroenterology 147, no. 2 (August 2014): 485–497.e18. doi:10.1053/j.gastro.2014.04.048.


Drugs that restore SMAD4


Lin, Sheng-Zhang, Jin-Bo Xu, Xu Ji, Hui Chen, Hong-Tao Xu, Ping Hu, Liang Chen, et al. ‘Emodin Inhibits Angiogenesis in Pancreatic Cancer by Regulating the Transforming Growth Factor-Β/drosophila Mothers against Decapentaplegic Pathway and Angiogenesis-Associated microRNAs’. Molecular Medicine Reports 12, no. 4 (October 2015): 5865–71. doi:10.3892/mmr.2015.4158.


Predictive value of SMAD4 status in personalised medicine


Whittle, Martin C., Kamel Izeradjene, P. Geetha Rani, Libing Feng, Markus A. Carlson, Kathleen E. DelGiorno, Laura D. Wood, et al. ‘RUNX3 Controls a Metastatic Switch in Pancreatic Ductal Adenocarcinoma’. Cell 161, no. 6 (June 2015): 1345–60. doi:10.1016/j.cell.2015.04.048.


Boone, Brian A., Shirin Sabbaghian, Mazen Zenati, J. Wallis Marsh, A. James Moser, Amer H. Zureikat, Aatur D. Singhi, Herbert J. Zeh, and Alyssa M. Krasinskas. ‘Loss of SMAD4 Staining in Pre-Operative Cell Blocks Is Associated with Distant Metastases Following Pancreaticoduodenectomy with Venous Resection for Pancreatic Cancer’. Journal of Surgical Oncology 110, no. 2 (August 2014): 171–75. doi:10.1002/jso.23606.


Herman, Joseph M., Katherine Y. Fan, Aaron T. Wild, Laura D. Wood, Amanda L. Blackford, Ross C. Donehower, Manuel Hidalgo, et al. ‘Correlation of Smad4 Status With Outcomes in Patients Receiving Erlotinib Combined With Adjuvant Chemoradiation and Chemotherapy After Resection for Pancreatic Adenocarcinoma’. International Journal of Radiation Oncology*Biology*Physics 87, no. 3 (November 2013): 458–59. doi:10.1016/j.ijrobp.2013.06.2039.


Other Related Articles Published In This Open Access Online Journal Include The Following:





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A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP


There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.


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Summary to Metabolomics

Summary to Metabolomics

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

This concludes a long step-by-step journey into rediscovering biological processes from the genome as a framework to the remodeled and reconstituted cell through a number of posttranscription and posttranslation processes that modify the proteome and determine the metabolome.  The remodeling process continues over a lifetime. The process requires a balance between nutrient intake, energy utilization for work in the lean body mass, energy reserves, endocrine, paracrine and autocrine mechanisms, and autophagy.  It is true when we look at this in its full scope – What a creature is man?

 Recommended Readings and Historical Perspectives

Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.[1] The metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes.[2] mRNA gene expression data and proteomic analyses reveal the set of gene products being produced in the cell, data that represents one aspect of cellular function. Conversely, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomic information to provide a better understanding of cellular biology.

The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry (GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur B. Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.

Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples.This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Efforts to utilize NMR for metabolomics have been influenced by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.

In 2005, the first metabolomics web database, METLIN, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute and contained over 10,000 metabolites and tandem mass spectral data. As of September 2012, METLIN contains over 60,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.

On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.

As late as mid-2010, metabolomics was still considered an “emerging field”. Further, it was noted that further progress in the field depended in large part, through addressing otherwise “irresolvable technical challenges”, by technical evolution of mass spectrometry instrumentation.

Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The word was coined in analogy with transcriptomics and proteomics; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute in 2005. In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome. They catalogued approximately 2500 metabolites, 1200 drugs and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete.

Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information, while the study of biofluids can give more generalized though less specialized information. Commonly used biofluids are urine and plasma, as they can be obtained non-invasively or relatively non-invasively, respectively. The ease of collection facilitates high temporal resolution, and because they are always at dynamic equilibrium with the body, they can describe the host as a whole.

Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size.
A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes.  By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. Metabolites of foreign substances such as drugs are termed xenometabolites. The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions.

Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”. The word origin is from the Greek μεταβολή meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.

There is a growing consensus that ‘metabolomics’ places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. ‘Metabonomics’ extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied.

Toxicity assessment/toxicology. Metabolic profiling (especially of urine or blood plasma samples) detects the physiological changes caused by toxic insult of a chemical (or mixture of chemicals).

Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes.

Nutrigenomics is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients.


Jose Eduardo des Salles Roselino

The problem with genomics was it was set as explanation for everything. In fact, when something is genetic in nature the genomic reasoning works fine. However, this means whenever an inborn error is found and only in this case the genomic knowledge afterwards may indicate what is wrong and not the completely way to put biology upside down by reading everything in the DNA genetic as well as non-genetic problems.

Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012

Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012

analysis of metabolomic data and differential metabolic regulation for fetal lungs, and maternal blood plasma

conformational changes leading to substrate efflux.img

conformational changes leading to substrate efflux.img

The cellular response is defined by a network of chemogenomic response signatures.

The cellular response is defined by a network of chemogenomic response signatures.

Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

 genome cartoon

genome cartoon

central dogma phenotype

central dogma phenotype

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Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism

Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism

Larry H, Bernstein, MD, FCAP, Author and Curator
and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/9/27/2014/Metformin,_thyroid-pituitary_ axis,_diabetes_mellitus,_and_metabolism

The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.

We start with a case study and a publication in the J Can Med Assoc.  Then we shall look
into key literature on these metabolic relationships.

Part I.  Metformin , Diabetes Mellitus, and Thyroid Function

Hypothyroidism, Insulin resistance and Metformin
May 30, 2012   By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.

My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of
seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.

A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).

Guess what? This is exactly what happens to Diabetes patients when Metformin is
introduced. http://en.wikipedia.org/wiki/Metformin
Suggested articles: http://www.springerlink.com/content/r81606gl3r603167/  and

Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.

The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activating

If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.

Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.

It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.

Point – direct T3 is kicking the closed -down metabolic process back into life,
just like Metformin does for insulin resistance.
There is serotonin resistance! http://www.ncbi.nlm.nih.gov/pubmed/17250776

Metformin Linked to Risk of Low Levels of Thyroid Hormone

CMAJ (Canadian Medical Association Journal) 09/22/2014

Metformin, the drug commonly for treating type 2 diabetes,

  • is linked to an increased risk of low thyroid-stimulating hormone
    (TSH) levels
  • in patients with underactive thyroids (hypothyroidism),

according to a study in CMAJ (Canadian Medical Association Journal).

Metformin is used to lower blood glucose levels

  • by reducing glucose production in the liver.

previous studies have raised concerns that

  • metformin may lower thyroid-stimulating hormone levels.

Study characteristics:

  1. Retrospective  long-term
  2. 74 300 patient who received metformin and sulfonylurea
  3. 25-year study period.
  4. 5689 had treated hypothyroidism
  5. 59 937 had normal thyroid function.

Metformin and low levels of thyroid-stimulating hormone in
patients with type 2 diabetes mellitus

Jean-Pascal Fournier,  Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay  +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda

CMAJ Sep 22, 2014,   http://dx.doi.org:/10.1503/cmaj.140688


  • metformin may lower thyroid-stimulating hormone (TSH) levels.


  • determine whether the use of metformin monotherapy, when compared with
    sulfonylurea monotherapy,
  • is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
  • in patients with type 2 diabetes mellitus.


  • Used the Clinical Practice Research Datalink,
  • identified patients who began receiving metformin or sulfonylurea monotherapy
    between Jan. 1, 1988, and Dec. 31, 2012.
  • 2 subcohorts of patients with treated hypothyroidism or euthyroidism,

followed them until Mar. 31, 2013.

  • Used Cox proportional hazards models to evaluate the association of low TSH
    levels with metformin monotherapy, compared with sulfonylurea monotherapy,
    in each subcohort.


  • 5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
    included in the subcohorts.

For patients with treated hypothyroidism:

  1. 495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
  2. 322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
    in the euthyroid group.
  • metformin monotherapy was associated with a 55% increased risk of low TSH
    in patients with treated hypothyroidism (incidence rate 0.0795/person-years
    vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
    interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
  • the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
  • No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).

Interpretation: The clinical consequences of this needs further investigation.


Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone
levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared
with sulfonylurea monotherapy, in patients with treated hypothyroidism or
euthyroidism and type 2 diabetes
Variable No. events
TSH levels
Person-years of
Incidence rate,
per 1000 person-years (95% CI)
Adjusted HR*(95% CI)
Patients with treated hypothyroidism, = 5689
= 762
18 503 35.8
1.00 1.00
= 4927
130 3 633 35.8
1.05 0.99
Euthyroid patients, = 59 937
= 7980
12 8 576 1.4
1.00 1.00
= 51 957
75 63 047 1.2
0.85 1.03


Part II. Metabolic Underpinning 
(Source: Wikipedia, AMPK and thyroid)

5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that

  1. together make a functional enzyme, conserved from yeast to humans.
  2. It is expressed in a number of tissues, including the liver, brain, and skeletal
  3. The net effect of AMPK activation is stimulation of
    1. hepatic fatty acid oxidation and ketogenesis,
    2. inhibition of cholesterol synthesis,
    3. lipogenesis, and triglyceride synthesis,
    4. inhibition of adipocyte lipolysis and lipogenesis,
    5. stimulation of skeletal muscle fatty acid oxidation and muscle
      glucose uptake, and
    6. modulation of insulin secretion by pancreatic beta-cells.

The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.

  • the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
    giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
  • The four CBS domains create two binding sites for AMP commonly referred to as
    Bateman domains. Binding of one AMP to a Bateman domain cooperatively
    increases the binding affinity of the second AMP to the other Bateman domain.
  • As AMP binds both Bateman domains the γ subunit undergoes a conformational
    change which exposes the catalytic domain found on the α subunit.
  • It is in this catalytic domain where AMPK becomes activated when
    phosphorylation takes place at threonine-172by an upstream AMPK kinase
    (AMPKK). The α, β, and γ subunits can also be found in different isoforms.

AMPK acts as a metabolic master switch regulating several intracellular systems

  1. the cellular uptake of glucose,
  2. the β-oxidation of fatty acids and
  3. the biogenesis of glucose transporter 4 (GLUT4) and
  4. mitochondria

The energy-sensing capability of AMPK can be attributed to

  • its ability to detect and react to fluctuations in the AMP:ATP ratio that take
    place during rest and exercise (muscle stimulation).

During muscle stimulation,

  • AMP increases while ATP decreases, which changes AMPK into a good substrate
    for activation.
  • AMPK activity increases while the muscle cell experiences metabolic stress
    brought about by an extreme cellular demand for ATP.
  • Upon activation, AMPK increases cellular energy levels by
    • inhibiting anabolic energy consuming pathways (fatty acid synthesis,
      protein synthesis, etc.) and
    • stimulating energy producing, catabolic pathways (fatty acid oxidation,
      glucose transport, etc.).

A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,

  • the mice ate less and lost weight.

When AMPK activity was pharmacologically raised (AICAR see below)

  • the mice ate more and gained weight.

Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to

  1. reduced glucose production in the liver and
  2. reduced insulin resistance in the muscle.

(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)

Triggering the activation of AMPK can be carried out provided two conditions are met.

First, the γ subunit of AMPK

  • must undergo a conformational change so as to
  • expose the active site(Thr-172) on the α subunit.

The conformational change of the γ subunit of AMPK can be accomplished

  • under increased concentrations of AMP.

Increased concentrations of AMP will

  • give rise to the conformational change on the γ subunit of AMPK
  • as two AMP bind the two Bateman domains located on that subunit.
  • It is this conformational change brought about by increased concentrations
    of  AMP that exposes the active site (Thr-172) on the α subunit.

This critical role of AMP is further substantiated in experiments that demonstrate

  • AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide
    ribotide (ZMP) which is derived fromthe familiar
  • 5-amino-4-imidazolecarboxamide riboside (AICAR)

AMPK is a good substrate for activation via an upstream kinase complex, AMPKK
AMPKK is a complex of three proteins,

  1. STE-related adaptor (STRAD),
  2. mouse protein 25 (MO25), and
  3. LKB1 (a serine/threonine kinase).

The second condition that must be met is

  • the phosphorylation/activation of AMPK on its activating loop at
    Thr-172of the α subunit
  • brought about by an upstream kinase (AMPKK).

The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the
pseudokinase STE-related adaptor protein (STRAD) has been identified as

  • the major upstream kinase responsible for phosphorylation of AMPK
    on its activating loop at Thr-172

Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex,

  • it can also be regulated by allosteric modulators which
  • directly increase general AMPK activity and
  • modify AMPK to make it a better substrate for AMPKK
  • and a worse substrate for phosphatases.

It has recently been found that 3-phosphoglycerate (glycolysis intermediate)

  • acts to further pronounce AMPK activation via AMPKK

Muscle contraction is the main method carried out by the body that can provide
the conditions mentioned above needed for AMPK activation

  • As muscles contract, ATP is hydrolyzed, forming ADP.
  • ADP then helps to replenish cellular ATP by donating a phosphate group to
    another ADP,

    • forming an ATP and an AMP.
  • As more AMP is produced during muscle contraction,
    • the AMP:ATP ratio dramatically increases,
  • leading to the allosteric activation of AMPK

For over a decade it has been known that calmodulin-dependent protein kinase
kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK,

  • but it was not the main AMPKK in liver.

CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-
ribofuranoside (AICAR) phosphorylation and activation of AMPK.

  • AICAR is taken into the celland converted to ZMP,
  • an AMP analogthat has been shown to activate AMPK.

Recent LKB1 knockout studies have shown that without LKB1,

  • electrical and AICAR stimulation of muscleresults in very little
    phosphorylation of AMPK and of ACC, providing evidence that
  • LKB1-STRAD-MO25 is the major AMPKK in muscle.

Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is

  • the upregulation of fatty acid oxidation.

Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and

  • Adiponectin also stimulates the uptake of glucose in skeletal muscle.

An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.

One of the key pathways in AMPK’s regulation of fatty acid oxidation is the

  • phosphorylation and inactivation of acetyl-CoA carboxylase.
  1. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
    (MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
  2. CPT-1 transports fatty acids into the mitochondria for oxidation.
  3. Inactivation of ACC results in increased fatty acid transport and oxidation.
  4. the AMPK induced ACC inactivation  and reduced conversion to MCA
    may occur as a result of malonyl-CoA decarboxylase (MCD)
  5. MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
    (reversal of ACC conversion of ACA to MCA)
  6. This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.

AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.

  1. It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
  2. acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
    with inhibition of CPT-1
  3. HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
  4. which then travels down several more metabolic steps to become cholesterol.

Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,

  • The decreasedthe concentration of glucose molecules creates a gradient for more
    glucose to be transported into the cell.
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.
  •  Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
    a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol. 277
    (1 Pt 1): E1–10. PMID 10409121.
  • Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
    carboxylase and activation of AMP-activated protein kinase in muscle
    during exercise”. J. Physiol. 270 (2 Pt 1): E299–304. PMID 8779952.
  • Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
    inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
    protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
  • Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
    “Effects of endurance training on activity and expression of AMP-activated
    protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
    Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
  • Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
    protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
    315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
  • Winder WW (September 2001). “Energy-sensing and signaling by
    AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
    1017–28. PMID 11509493
  • Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
    2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
    and deactivation of AMP-activated protein kinase”.  J. Biol. Chem.
    281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194


Part III. Pituitary-thyroid axis and diabetes mellitus
The Interface Between Thyroid and Diabetes Mellitus

Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant   Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function

Metformin acts primarily by

  • suppressing hepatic gluconeogenesis via activation of AMPK
  • It has the opposite effects on hypothalamic AMPK,
    • inhibiting activity of the enzyme.
  • the metformin effects on hypothalamic AMPK activity will
    • counteractT3 effects at the hypothalamic level.
  • AMPK therefore represents a direct target for dual regulation
    • in the hypothalamic partitioning of energy homeostasis.
  • metformin crossesthe blood–brain barrier and
    • levels in the pituitary gland are substantially increased.
  • It convincinglysuppresses TSH

A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.

These studies reveal a

  • suppressive effect of metformin on TSH secretion patterns in
    hypothyroid patients, an effect that is apparently
  • independent of T4 treatment and does not alter the TH profile.
  • A rebound of TSH secretion occurs at about 3 months following metformin

It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.

Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy 
M López,  L Varela,  MJ Vázquez,  S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine  29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207

Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either

  • whole-body hyperthyroidism or central administration of triiodothyronine
    (T3) decreases

    • the activity of hypothalamic AMP-activated protein kinase (AMPK),
    • increases sympathetic nervous system (SNS) activity and
    • upregulates thermogenic markers in brown adipose tissue (BAT).

Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.

This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.

Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke
The Scientific World Journal 2012; 2012, ID 475675, 10 pp

The liver is a major organ responsible for most functions of cellular metabolism and

  • a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
  • In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
    constitutes an intrahepatic energy sensor
  • regulating physiological energy dynamics by limiting anabolism and stimulating
    catabolism, thus increasing ATP availability.
  • This is achieved by mechanisms involving direct allosteric activation and
    reversible phosphorylation of AMPK, in response to signals such as

    • energy status,
    • serum insulin/glucagon ratio,
    • nutritional stresses,
    • pharmacological and natural compounds, and
    • oxidative stress status.

Reactive oxygen species (ROS) lead to cellular AMPK activation and

  • downstream signaling under several experimental conditions.

Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,

  • triggers the redox upregulation of cytoprotective proteins
    • affording preconditioning against ischemia-reperfusion (IR) liver injury.

Data discussed in this work suggest that T3-induced liver activation of AMPK

  • may be of importance in the promotion of metabolic processes
  • favouring energy supply for the induction and operation of preconditioning

These include

  1. antioxidant,
  2. antiapoptotic, and
  3. anti-inflammatory mechanisms,
  4. repair or resynthesis of altered biomolecules,
  5. induction of the homeostatic acute-phase response, and
  6. stimulation of liver cell proliferation,

which are required to cope with the damaging processes set in by IR.

The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.

In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.

In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).

Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.

In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an

  • intracellular energy sensor regulating physiological energy dynamics by
  • limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
    utilization, and
  • by stimulating catabolic processes, to increase ATP production.

Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status

Liver AMPK: Structure and Regulation

AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising

  1. a catalytic subunit α and
  2. two regulatory subunits β and γ .

The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas

  • the γ subunit has four cystathionine β-synthase (CBS) motifs, which
  • bind AMP or ATP in a competitive manner.

75675.fig.001 (not shown)

Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.

Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through

  • binding to the regulatory subunit-γ, which induces a conformational change in
    the kinase domain of subunit α that protects AMPK from dephosphorylation
    of Thr172, probably by protein phosphatase-2C.

Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either

(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;

(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;

(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.

Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit

  • over 1000-fold increase in AMPK activity, thus allowing
  • the liver to respond to small changes in energy status in a highly sensitive fashion.

In addition to rapid AMPK regulation through allosterism and reversible phosphorylation

  • long-term effects of AMPK activation induce changes in hepatic gene expression.

This was demonstrated for

(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),

  • whose Ser568 phosphorylation by activated AMPK
  • blocks its DNA binding capacity and glucose-induced gene transcription
  • under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
    protein-1c (SREBP-1c), whose mRNA and protein expression and those of
    its target gene for fatty acid synthase (FAS)
  • are reduced by metformin-induced AMPK activation,
  • decreasing lipogenesis and increasing fatty acid oxidation due to
    malonyl-CoA depletion;

(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.

This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying

  • the plasma glucose-lowering effects of adiponectin and metformin
  • through AMPK activation by upstream LKB1.

Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,

  1. inhibiting anabolic processes, and
  2. enhancing catabolic pathways in response to different signals, including
    1. energy status,
    2. serum insulin/glucagon ratio,
    3. nutritional stresses,
    4. pharmacological and natural compounds, and
    5. oxidative stress status

Reactive Oxygen Species (ROS) and AMPK Activation

The high energy demands required to cope with all the metabolic functions
of the liver are met by

  • fatty acid oxidation under conditions of both normal blood glucose levels and
    hypoglycemia, whereas
  • glucose oxidation is favoured in hyperglycemic states, with consequent
    generation of ROS.

Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling

Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production

T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus

  • determining enhancement in fuel consumption for oxidation processes
  • and ATP repletion.

T3 acts predominantly through nuclear receptors (TR) α and β, forming

  • functional complexes with retinoic X receptor that
  • bind to thyroid hormone response elements (TRE) to activate gene expression.

T3 calorigenesis is primarily due to the

  • induction of enzymes related to mitochondrial electron transport and ATP
    synthesis, catabolism, and
  • some anabolic processes via upregulation of genomic mechanisms.

The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3

(i) energy expenditure due to higher active cation transport,

(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and

(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells

In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.

Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),

  • catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
    and acyl-CoA oxidase (ACO),
  • catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
    induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).

Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn

  • augments T3 induction of CPT-Iα expression.

Interestingly, PGC-1α is induced by

  1. T3,
  2. AMPK activation, and
  3. ROS,

thus establishing potential links between

  • T3 action, ROS generation, and AMPK activation

with the onset of mitochondrial biogenesis and fatty acid β-oxidation.

Liver ROS generation leads to activation of the transcription factors

  1. nuclear factor-κB (NF-κB),
  2. activating protein 1 (AP-1), and
  3. signal transducer and activator of transcription 3 (STAT3)

at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of

  1. cytoprotective proteins (Figure 3(A)).

These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing

  1. redox homeostasis,
  2. promoting cell survival, and
  3. protecting the liver against ischemia-reperfusion injury.

T3 liver preconditioning also involves the activation of the

  1. Nrf2-Keap1 defense pathway
  • upregulating antioxidant proteins,
  • phase-2 detoxifying enzymes, and
  • multidrug resistance proteins, members of the ATP binding cassette (ABC)
    superfamily of transporters (Figure 3(B))

In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with

  • activation of protein kinase C and
  • attenuation of p38 and
  • c-Jun-N-terminal kinase activation ,

and in the kidney, in association with

  • heme oxygenase-1 upregulation.



Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.


genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003


Figure 3: Genomic signaling mechanisms in T3 calorigenesis and liver reactive oxygen
species (ROS) production leading to
(A) upregulation of cytokine expression in Kupffer cells and hepatocyte activation of genes
conferring cytoprotection,
(B) Nrf2 activation controling expression of antioxidant and detoxication proteins, and
(C) activation of the AMPK cascade regulating metabolic functions.

Abbreviations: AP-1, activating protein 1; ARE, antioxidant responsive element; CaMKKβ,
Ca2+-calmodulin-dependent kinase kinase-β; CBP, CREB binding protein; CRC, chromatin
remodelling complex; EH, epoxide hydrolase; HO-1, hemoxygenase-1; GC-Ligase,
glutamate cysteine ligase; GPx, glutathione peroxidase; G-S-T, glutathione-S-transferase;
HAT, histone acetyltransferase; HMT, histone arginine methyltransferase; IL1,
interleukin 1; iNOS, inducible nitric oxide synthase; LKB1, tumor suppressor LKB1 kinase;
MnSOD, manganese superoxide dismutase; MRPs, multidrug resistance proteins; NF-κB,
nuclear factor-κB; NQO1, NADPH-quinone oxidoreductase-1; NRF-1, nuclear respiratory
factor-1; Nrf2, nuclear receptor-E2-related factor 2; PCAF, p300/CBP-associated
factor; RXR, retinoic acid receptor; PGC-1, peroxisome proliferator-activated receptor-γ
coactivator-1; QO2, rate of O2 consumption; STAT3, signal transducer and activator
of transcription 3; TAK1, transforming-growth-factor-β-activated kinase-1; TNF-α, tumor
necrosis factor-α; TR, T 3 receptor; TRAP, T3-receptor-associated protein; TRE,  T3 responsive element; UCP, uncoupling proteins; (—), reported mechanisms;
(- - - -), proposed mechanisms.


T3 is a key metabolic regulator coordinating short-term and long-term energy needs,
with major actions on liver metabolism. These include promotion of

(i) gluconeogenesis and hepatic glucose production, and

(ii) fatty acid oxidation coupled to enhanced adipose tissue lipolysis, with

  • higher fatty acid flux to the liver and
  • consequent ROS production (Figure 2) and
  • redox upregulation of cytoprotective proteins

affording liver preconditioning (Figure 3).

Thyroid Hormone and AMPK Activation: Skeletal Muscle and Heart

In skeletal muscle, T3 increases the levels of numerous proteins involved in

  1. glucose uptake (GLUT4),
  2. glycolysis (enolase, pyruvate kinase, triose phosphate isomerase),
  3. fatty acid oxidation (carnitine palmitoyl transferase-1, mitochondrial thioesterase I),
    and uncoupling protein-3,

effects that are achieved through enhanced transcription of TRE-containing genes

Skeletal muscle AMPK activation is characterized by

(i) being a rapid and transient response,

(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,

(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and

(iv) stimulation of mitochondrial fatty acid β-oxidation.

T3-induced muscle AMPK activation was found to trigger two major downstream

signaling pathways, namely,

(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to

  • mitochondrial biogenesis,
  • fatty acid oxidation, and
  • gluconeogenesis and

(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which

  • in turn induces PGC-1α expression in liver tissue, thus
  • reinforcing mechanism (i).

These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.

In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.

Thyroid Hormone, AMPK Activation, and Liver Preconditioning

Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).

  1. enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
    treated rats over control values thatis significant in the time period of 1 to 48
    hours after hormone treatment
  2. Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
    decreased liver AMPK activation at 4 h to return to control values at 6 h
    after treatment

Activation of liver AMPK by T3 may be of relevance in terms of

  • promotion of fatty acid oxidation for ATP supply,
  • supporting hepatoprotection against IR injury (Figure 3(C)).

This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic

  • antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
  • oxidized biomolecules repair or resynthesis,
  • induction of the homeostatic acute-phase response, and
  • promotion of hepatocyte and Kupffer cell proliferation,

mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by

  • n-3 long-chain polyunsaturated fatty acids given alone or
  • combined with T3 at lower dosages, or
  • by iron supplementation,

constitutes protective strategies against hepatic IR injury.

Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.


Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.

Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of  hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.

Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.

E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
metabolic regulator,” Biochemical Society Transactions 2003; 31(1):

G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
2011; 25(18): 1895–1908.

Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
Journal of Biological Chemistry 1996;271(17): 10282–10290.

Xiao, R. Heath, P. Saiu et al., “Structural basis for AMP binding to mammalian AMP-
activated protein kinase,” Nature 2007; 449(7161): 496–500.


Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.
Abdulrahman RM1, Boon MRSips HCGuigas BRensen PCSmit JWHovens GC.
Author information 
Thyroid. 2014 Jan;24(1):78-87.  Epub 2013 Sep 25.  PMID: 23819433

Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.

The aim of this study was

  1. to investigate the mechanism of AMPK modulation in iodide uptake.
  2. to investigate the potential of the AMPK inhibitor compound C as an enhancer of
    iodide uptake by thyrocytes.

Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with

  • CRE activation (0.6- and 3.0-fold).

These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.

NIS expression and iodine uptake in thyrocytes

  • can be modulated by metformin and compound C.

These compounds exert their effect by

  • modulation of AMPK, which, in turn, regulates
  • the activation of the CRE element in the NIS promoter.

Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.

AMPK: Master Metabolic Regulator

© 1996–2013 themedicalbiochemistrypage.org, LLC | info
@ themedicalbiochemistrypage.org

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

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


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

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


central role of AMPK in the regulation of metabolism



AMP-activated protein kinase (AMPK) was first discovered as an activity that

AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.

More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.


Once activated, AMPK-mediated phosphorylation events

These events are rapidly initiated and are referred to as

  • short-term regulatory processes.

The activation of AMPK also exerts

  • long-term effects at the level of both gene expression and protein synthesis.

Other important activities attributable to AMPK are

  1. regulation of insulin synthesis and
  2. secretion in pancreatic islet β-cells and
  3. modulation of hypothalamic functions involved in the regulation of satiety.

How these latter two functions impact obesity and diabetes will be discussed below.

Regulation of AMPK

In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.

The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which

  • lies in the activation loop.

One kinase activator of AMPK is

  • Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ)
  • which phosphorylates and activates AMPK in response to increased calcium.

The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,

  • increased release of intracellular stores of Ca2+ create a subsequent demand for

Activation of AMPK in response to Ca fluxes

  • provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.

The active LKB1 kinase is actually a complex of three proteins:

  1. LKB1,
  2. Ste20-related adaptor (STRAD) and
  3. mouse protein 25 (MO25).

Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,

  • LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.

Loss of LKB1 activity in adult mouse liver leads to

  • near complete loss of AMPK activity and
  • is associated with hyperglycemia.

The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of

  • the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
    (PGC-1α), which drives gluconeogenesis.
  • Reduction in PGC-1α activity results in normalized blood glucose levels in
    LKB1-deficient mice.

The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.

The effects of AMP are two-fold:

  1. a direct allosteric activation and making AMPK a poorer substrate for

Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,

the cascade is ultrasensitive. This means that

  1. a very small rise in AMP levels can induce a dramatic increase in the activity of

The activity of adenylate kinase, catalyzing the reaction shown below, ensures that

  • AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ——> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by
phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content

  • represses AMPK activity and
  • this is likely the result of interaction between the GBD and glycogen,
  • the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
  • by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to

  • phospholipase C-β (PLC-β) and by
  • hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).

Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on

  • glucose and lipid metabolism,
  • gene expression and
  • protein synthesis.

These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.

The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).

The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.

Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through
glycolysis and gluconeogenesis.

In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).

It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.

Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.

Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.

Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.

As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels

  • requires the activity of LKB1 in the liver for this function.

Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit
AMPK activation:

  1. leptin and adiponectin have been shown to stimulate AMPK activation, whereas,
  2. resistininhibits AMPK activation.

Cardiac effects exerted by activation of AMPK also include

AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.

In platelets, insulin action leads to an increase in eNOS activity that is

  • due to its phosphorylation by AMPK.

Activation of NO production in platelets leads to

  • a decrease in thrombin-induced aggregation, thereby,
  • limiting the pro-coagulant effects of platelet activation.

The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome

Activation of AMPK leads to a reduction in the level of SREBP

  • a transcription factor &regulator of the expression of numerous
    lipogenic enzymes

Another transcription factor reduced in response to AMPK activation is

  • hepatocyte nuclear factor 4α, HNF4α
    • a member of the steroid/thyroid hormone superfamily.
    • HNF4α is known to regulate the expression of several liver and
      pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
  • Of clinical significance is that mutations in HNF4α are responsible for
    • maturity-onset diabetes of the young, MODY-1.

Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.

The target of the thiazolidinedione (TZD) class of drugs used to treat type 2 diabetes is
the peroxisome proliferator-activated receptor γPPARγ which

  • itself may be a target for the action of AMPK.

The transcription co-activator, p300, is phosphorylated by AMPK

  • which inhibits interaction of p300 with not only PPARγ but also
  • the retinoic acid receptor, retinoid X receptor, and
  • thyroid hormone receptor.

PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.

These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as
FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.

This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart.  I also note at this time that there
nongenetic points to be made here:

  1. The tissue specificity of isoenzymes
  2. The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
    effects on metabolism tied to AMPK
  3. The tie in of stress or ROS with fast reactions to protect harm to tissues
  4. The relationship of cytokine activation and release to the above metabolic events
  5. The relationship of effective and commonly used diabetes medications to AMPK
    mediated processes
  6. The preceding presentation is notable for the importance of proteomic and
    metabolomic invetigations in elucidation common chronic and nongenetic diseases


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Compilation of References in Leaders in Pharmaceutical Intelligence about proteomics, metabolomics, signaling pathways, and cell regulation

Compilation of References in Leaders in Pharmaceutical Intelligence about
proteomics, metabolomics, signaling pathways, and cell regulation

Curator: Larry H. Bernstein, MD, FCAP



  1. The Human Proteome Map Completed
    Reporter and Curator: Larry H. Bernstein, MD, FCAP
  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
    Author and Curator, Larry H Bernstein, MD, FCAP
  1. Synthesizing Synthetic Biology: PLOS Collections
    Reporter: Aviva Lev-Ari



  1. Extracellular evaluation of intracellular flux in yeast cells
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  2. Metabolomic analysis of two leukemia cell lines. I.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  3. Metabolomic analysis of two leukemia cell lines. II.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  4. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
    Reviewer and Curator, Larry H. Bernstein, MD, FCAP
  5. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator


Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
    Reviewer and Curator: Larry H. Bernstein, MD, FCAP
  2. Mitochondria: More than just the “powerhouse of the cell”
    Reviewer and Curator: Ritu Saxena
  3. Mitochondrial fission and fusion: potential therapeutic targets?
    Reviewer and Curator: Ritu saxena
  4. Mitochondrial mutation analysis might be “1-step” away
    Reviewer and Curator: Ritu Saxena
  5. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com
    Curator: Larry H. Bernstein, MD, FCAP
  6. Metabolic drivers in aggressive brain tumors
    Prabodh Kandal, PhD
  7. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
    Author and Curator: Aviva Lev-Ari, PhD, RD
  8. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
    Author and curator:Larry H Bernstein, MD, FCAP
  9. Therapeutic Targets for Diabetes and Related Metabolic Disorders
    Reporter, Aviva Lev-Ari, PhD, RD
  10. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator
  11. The multi-step transfer of phosphate bond and hydrogen exchange energy
    Curator:Larry H. Bernstein, MD, FCAP,
  12. Studies of Respiration Lead to Acetyl CoA
    Author and Curator: Larry H. Bernstein, MD, FCAP
  13. Lipid Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  14. Carbohydrate Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
  15. Prologue to Cancer – e-book Volume One – Where are we in this journey?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  16. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?
    Author and Curator: Larry H. Bernstein, MD, FCAP
  17. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K
    Author and Curator: Larry H. Bernstein, MD, FCAP
  18. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
    Author and Curator: Larry H. Bernstein, MD, FCAP
  19. Mitochondrial Metabolism and Cardiac Function
    Author and Curator: Larry H. Bernstein, MD, FCAP
  20. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
    Curator: Larry H. Bernstein, MD, FCAP
  21. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
    Author and Curator: SJ. Williams
  22. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
    Author and Curator: Larry H. Bernstein, MD, FCAP
  23. Overview of Posttranslational Modification (PTM)
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  24. Malnutrition in India, high newborn death rate and stunting of children age under five years
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  25. Update on mitochondrial function, respiration, and associated disorders
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  26. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
    Larry H. Bernstein, MD, FCAP, Curator
  27. Late Onset of Alzheimer’s Disease and One-carbon Metabolism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.
  28. Problems of vegetarianism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Signaling Pathways

  1. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
    Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN  https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-translational_medicine-part_2/
  2. Epilogue: Envisioning New Insights in Cancer Translational Biology
    Series C: e-Books on Cancer & Oncology
    Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant
  3. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter  Writer and Curator: Larry H Bernstein, MD, FCAP and Curator and Content Editor: Aviva Lev-Ari, PhD, RN
  4. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
    Author and Curator: Larry H Bernstein, MD, FCAP and Article Curator: Aviva Lev-Ari, PhD, RN
  5. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
    Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN
  6. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Larry H Bernstein, MD, FCAP, Author and Curator
  7. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
    Author and Curator: Larry H Bernstein, MD, FCAP
  8. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology
    Demet Sag, PhD, Author and Curator
  9. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase
    Demet Sag, PhD, Author and Curator
  10. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad
    Author and Curator: Demet Sag, PhD, CRA, GCP
  11. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute
    Reporter: Aviva Lev-Ari, PhD, RN
  12. Naked Mole Rats Cancer-Free
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  13. Amyloidosis with Cardiomyopathy
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  14. Liver endoplasmic reticulum stress and hepatosteatosis
    Larry H Bernstein, MD, FACP
  15. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  16. Nitric Oxide Function in Coagulation – Part II
    Curator and Author: Larry H. Bernstein, MD, FCAP
  17. Nitric Oxide, Platelets, Endothelium and Hemostasis
    Curator and Author: Larry H Bernstein, MD, FACP
  18. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
    Curator and Author: Larry H Bernstein, MD, FACP
  19. Nitric Oxide and Immune Responses: Part 1
    Curator and Author:  Aviral Vatsa PhD, MBBS
  20. Nitric Oxide and Immune Responses: Part 2
    Curator and Author:  Aviral Vatsa PhD, MBBS
  21. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
    Curator and Author: Larry H Bernstein, MD, FACP
  22. New Insights on Nitric Oxide donors – Part IV
    Curator and Author: Larry H Bernstein, MD, FACP
  23. Crucial role of Nitric Oxide in Cancer
    Curator and Author: Ritu Saxena, Ph.D.
  24. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
    Curator and Author: Larry H Bernstein, MD, FACP
  25. Nitric Oxide and Immune Responses: Part 2
    Author and Curator: Aviral Vatsa, PhD, MBBS
  26. Mitochondrial Damage and Repair under Oxidative Stress
    Author and Curator: Larry H. Bernstein, MD, FCAP
  27. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
    Curator and Author: Larry H Bernstein, MD, FACP
  28. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
    Curator and Author: Ziv Raviv, PhD, RN 04/06/2013
  29. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
    Curator and Author: Larry H Bernstein, MD, FACP
  30. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III
    Curator and Author: Larry H Bernstein, MD, FACP
  31. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I
    Curator and Author: Larry H Bernstein, MD, FACP


Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  2. RNA and the transcription the genetic code
    Larry H. Bernstein, MD, FCAP, Writer and Curator
  3. A Primer on DNA and DNA Replication
    Writer and Curator: Larry H. Bernstein, MD, FCAP
  4. Pathology Emergence in the 21st Century
    Author and Curator: Larry Bernstein, MD, FCAP
  5. RNA and the transcription the genetic code
    Writer and Curator, Larry H. Bernstein, MD, FCAP
  6. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP
    Author: Larry H Bernstein, MD, FCAP
  7. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies
    Author an Curator: Larry H Bernstein, MD, FCAP
  8. Silencing Cancers with Synthetic siRNAs
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  9. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points
    Reporter: Aviva Lev-Ari, PhD, RN
  10. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
  11. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis
    Reporter: Aviva Lev-Ari, PhD, RN
  12. CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
    Genomics Curator, Larry H Bernstein, MD, FCAP
  13. Big Data in Genomic Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
  14.  From Genomics of Microorganisms to Translational Medicine
    Author and Curator: Demet Sag, PhD
  15.  Summary of Genomics and Medicine: Role in Cardiovascular Diseases
    Author and Curator, Larry H Bernstein, MD, FCAP

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Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP


This is an added selection of articles in Leaders in Pharmaceutical Intelligence after the third portion of the discussion in a series of articles that began with signaling and signaling pathways. There are fine features on the functioning of enzymes and proteins, on sequential changes in a chain reaction, and on conformational changes that we shall return to.  These are critical to developing a more complete understanding of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Selected References to Signaling and Metabolic Pathwayspublished in this Open Access Online Scientific Journal, include the following:

Update on mitochondrial function, respiration, and associated disorders

Curator and writer: Larry H. Benstein, MD, FCAP


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

Cancer Volume One – Summary

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

Author: Larry H. Bernstein, MD, FCAP


Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP


 The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Author and Curator: Larry H Bernstein, MD, FCAP, 
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
And Curator: Aviva Lev-Ari, PhD, RN


Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Author and Curator: Larry H. Bernstein, MD, FCAP
Curator:  Stephen J. Williams, PhD
and Curator: Aviva Lev-Ari, PhD, RN


Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP


Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP


Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP


Advanced Topics in Sepsis and the Cardiovascular System  at its End Stage

Author: Larry H Bernstein, MD, FCAP


Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator: Larry H Bernstein, MD, FACP


Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP



Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP 


Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP


Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Reporter and Curator: Larry H Bernstein, MD, FACP



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, FCAP


Mitochondria and Cancer: An overview of mechanisms

Author and Curator: Ritu Saxena, Ph.D.


Mitochondria: More than just the “powerhouse of the cell”

Author and Curator: Ritu Saxena, Ph.D.


Overview of Posttranslational Modification (PTM)

Curator: Larry H. Bernstein, MD, FCAP


Ubiquitin Pathway Involved in Neurodegenerative Diseases

Author and curator: Larry H Bernstein, MD,  FCAP


Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Author: Larry H. Bernstein, MD, FCAP 


New Insights on Nitric Oxide donors – Part IV

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


Perspectives on Nitric Oxide in Disease Mechanisms [Kindle Edition]

Margaret Baker PhD (Author), Tilda Barliya PhD (Author), Anamika Sarkar PhD (Author), Ritu Saxena PhD (Author), Stephen J. Williams PhD (Author), Larry Bernstein MD FCAP (Editor), Aviva Lev-Ari PhD RN (Editor), Aviral Vatsa PhD (Editor)




Nitric oxide and its role in vascular biology

Signal transmission by a gas that is produced by one cell, penetrates through membranes and regulates the function of another cell represents an entirely new principle for signaling in biological systems.   All compounds that inhibit endothelium-derived relaxation-factor (EDRF) have one property in common, redox activity, which accounts for their inhibitory action on EDRF. One exception is hemoglobin, which inactivates EDRF by binding to it. Furchgott, Ignarro and Murad received the Nobel Prize in Physiology and Medicine for discovery of EDRF in 1998 and demonstrating that it might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta.  These investigators working independently demonstrated that NO is indeed produced by mammalian cells and that NO has specific biological roles in the human body. These studies highlighted the role of NO in cardiovascular, nervous and immune systems. In cardiovascular system NO was shown to cause relaxation of vascular smooth muscle cells causing vasodilatation, in nervous system NO acts as a signaling molecule and in immune system it is used against pathogens by the phagocytosis cells. These pioneering studies opened the path of investigation of role of NO in biology.

NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances.

Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to

  • reduction in eNOS production
  • reduction in eNOS enzymatic activity
  • reduced bioavailability of NO

Nitric oxide is one of the smallest molecules involved in physiological functions in the body. It is seeks formation of chemical bonds with its targets.  Nitric oxide can exert its effects principally by two ways:

  • Direct
  • Indirect

Direct actions, as the name suggests, result from direct chemical interaction of NO with its targets e.g. with metal complexes, radical species. These actions occur at relatively low NO concentrations (<200 nM)

Indirect actions result from the effects of reactive nitrogen species (RNS) such as NO2 and N2O3. These reactive species are formed by the interaction of NO with superoxide or molecular oxygen. RNS are generally formed at relatively high NO concentrations (>400 nM)

Although it can be tempting for scientists to believe that RNS will always have deleterious effects and NO will have anabolic effects, this is not entirely true as certain RNS mediated actions mediate important signalling steps e.g. thiol oxidation and nitrosation of proteins mediate cell proliferation and survival, and apoptosis respectively.

  • Cells subjected to NO concentration between 10-30 nM were associated with cGMP dependent phosphorylation of ERK
  • Cells subjected to NO concentration between 30-60 nM were associated with Akt phosphorylation
  • Concentration nearing 100 nM resulted in stabilisation of hypoxia inducible factor-1
  • At nearly 400 nM NO, p53 can be modulated
  • >1μM NO, it nhibits mitochondrial respiration


Nitric oxide signaling, oxidative stress,  mitochondria, cell damage

Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced.

NO itself in low concentrations have protective action on mitochondrial signaling of cell death.

The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes.

Oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, can cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload.  The normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease.

We reviewed the complex interactions and underlying regulatory balances/imbalances between the mechanism of vasorelaxation and vasoconstriction of vascular endothelium by way of nitric oxide (NO), prostacyclin, in response to oxidative stress and intimal injury.

Nitric oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function. The influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the resulting balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

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.

ROS 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.

NO and circulatory diseases

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers that characterize their phenotype.

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.

VESCs discovered that reside at the blood vessel wall endothelium are a small population of CD117+ ECs capable of self-renewal.  These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. A single VESC cell isolated from the endothelial population was able to generate functional blood vessels.

Among many important roles of Nitric oxide (NO), one of the key actions is to act as a vasodilator and maintain cardiovascular health. Induction of NO is regulated by signals in tissue as well as endothelium.

Chen et. al. (Med. Biol. Eng. Comp., 2011) developed a 3-D model consisting of two branched arterioles and nine capillaries surrounding the vessels. Their model not only takes into account of the 3-D volume, but also branching effects on blood flow.

The model indicates that wall shear stress changes depending upon the distribution of RBC in the microcirculations of blood vessels, lead to differential production of NO along the vascular network.

Endothelial dysfunction, the hallmark of which is reduced activity of endothelial cell derived nitric oxide (NO), is a key factor in developing atherosclerosis and cardiovascular disease. Vascular endothelial cells play a pivotal role in modulation of leukocyte and platelet adherence, thrombogenicity, anticoagulation, and vessel wall contraction and relaxation, so that endothelial dysfunction has become almost a synonym for vascular disease. A single layer of endothelial cells is the only constituent of capillaries, which differ from other vessels, which contain smooth muscle cells and adventitia. Capillaries directly mediate nutritional supply as well as gas exchange within all organs. The failure of the microcirculation leads to tissue apoptosis/necrosis.

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Signaling and Signaling Pathways

Curator: Larry H. Bernstein, MD, FCAP


https://pharmaceuticalintelligence.com/8-9-2014/Signaling and Signaling Pathways

This portion of the discussion is a series of articles on signaling and signaling pathways. Many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  I considered placing this after the discussion of proteins and how they play out their essential role, but this is quite a suitable place for a progression to what follows.  This is introduced by material taken from Wikipedia, which will be followed by a series of mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, with the later goal of separating views introduced by molecular biology and genomics from functional cellular dynamics that are not dependent on the classic view.  The work is vast, and this discussion does not attempt to cover it in great depth.  It is the first in a series.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Signal transduction

(From Wikipedia, the free encyclopedia)





Signal transduction occurs when an extracellular signaling[1] molecule activates a specific receptor located on the cell surface or inside the cell. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.[2] Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.[3] The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.[4]

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell.[5] For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.



The earliest MEDLINE entry for “signal transduction” dates from 1972.[6] Some early articles used the terms signal transmission and sensory transduction.[7][8] In 2007, a total of 48,377 scientific papers—including 11,211 e review papers—were published on the subject. The term first appeared in a paper’s title in 1979.[9][10] Widespread use of the term has been traced to a 1980 review article by Rodbell:[5][11] Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.[12]

Notch-mediated juxtacrine signal between adjacent cells.

Notch-mediated juxtacrine signal between adjacent cells.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation. This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.[13] Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.[14] Examples of signaling molecules include the hormone melatonin,[15] the neurotransmitter acetylcholine[16] and the cytokine interferon γ.[17]

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye,[20] and odorants binding to odorant receptors in the nasal epithelium.[21] Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Unicellular organisms may respond to environmental stimuli through the activation of signal transduction pathways. For example, slime molds secrete cyclic adenosine monophosphate upon starvation, stimulating individual cells in the immediate environment to aggregate,[22] and yeast cells use mating factors to determine the mating types of other cells and to participate in sexual reproduction.[23] Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.


Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.[24] These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to various second messenger molecules.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example, calcium ions bind to the EF hand domains of calmodulin, allowing it to bind and activate calmodulin-dependent kinase. PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

G protein-coupled

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ.[25] Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits. The dissociation exposes sites on the subunits that can interact with other molecules.[26] The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.[27] The total strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.

A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2; mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer development.[28]

Tyrosine and histidine kinase

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.[29] To perform signal transduction, RTKs need to form dimers in the plasma membrane;[30] the dimer is stabilized by ligands binding to the receptor. The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes. Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.[29]

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are members of the Ras, Rho, and Raf families, referred to collectively as small G proteins. They act as molecular switches usually tethered to membranes by isoprenyl groups linked to their carboxyl ends. Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate small G proteins that activate guanine nucleotide exchange factors such as SOS1. Once activated, these exchange factors can activate more small G proteins, thus amplifying the receptor’s initial signal. The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.[31]

Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes, fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a different protein or the kinase itself, thus activating the aspartate residue.[32]


integrin-mediated signal transduction

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).[33]

Integrins are produced by a wide variety of cells; they play a role in cell attachment to other cells and the extracellular matrix and in the transduction of signals from extracellular matrix components such as fibronectin and collagen. Ligand binding to the extracellular domain of integrins changes the protein’s conformation, clustering it at the cell membrane to initiate signal transduction. Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.[33] As shown in the picture to the right, cooperative integrin-RTK signalling determines the timing of cellular survival, apoptosis, proliferation, and differentiation.

Important differences exist between integrin-signalling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.[34]

Toll gate

When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and TRAM.[35][36][37] These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1[disambiguation needed], and IKKi that amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation.

Ligand-gated ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the cell membrane through which ions relaying signals can pass. An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels induces action potentials, such as those that travel along nerves, by depolarizing the membrane of post-synaptic cells, resulting in the opening of voltage-gated ion channels.

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+; it acts as a second messenger initiating signal transduction cascades and altering the physiology of the responding cell. This results in amplification of the synapse response between synaptic cells by remodelling the dendritic spines involved in the synapse.

Ion transporters and channels in mammalian choroidal epithelium

Ion transporters and channels in mammalian choroidal epithelium




Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.[24] These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance


intercellular signaling

intercellular signaling






membrane protein receptor binds with hormone

membrane protein receptor binds with hormone




The multiple protein-dependent steps in signal transduction

The multiple protein-dependent steps in signal transduction

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to various second messenger molecules.

Ca++ exchange

Ca++ exchange

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example, calcium ions bind to the EF hand domains of calmodulin, allowing it to bind and activate calmodulin-dependent kinase. PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

G protein-coupled

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

membrane_receptor_g protein

membrane_receptor_g protein




Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ.[25] Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits. The dissociation exposes sites on the subunits that can interact with other molecules.[26] The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.[27] The total strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.

A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2; mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer development.[28]

Tyrosine and histidine kinase

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.[29] To perform signal transduction, RTKs need to form dimers in the plasma membrane;[30] the dimer is stabilized by ligands binding to the receptor. The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes. Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.[29]


















As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are members of the Ras, Rho, and Raf families, referred to collectively as small G proteins. They act as molecular switches usually tethered to membranes by isoprenyl groups linked to their carboxyl ends. Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate small G proteins that activate guanine nucleotide exchange factors such as SOS1. Once activated, these exchange factors can activate more small G proteins, thus amplifying the receptor’s initial signal. The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.[31]

Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes, fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a different protein or the kinase itself, thus activating the aspartate residue.[32]



integrin-mediated signal transduction

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).[33]

Integrins are produced by a wide variety of cells; they play a role in cell attachment to other cells and the extracellular matrix and in the transduction of signals from extracellular matrix components such as fibronectin and collagen. Ligand binding to the extracellular domain of integrins changes the protein’s conformation, clustering it at the cell membrane to initiate signal transduction. Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.[33] As shown in the picture to the right, cooperative integrin-RTK signalling determines the timing of cellular survival, apoptosis, proliferation, and differentiation.

Platelet signaling pathways

Platelet signaling pathways







Protein ubiquitylation

Protein ubiquitylation





Important differences exist between integrin-signaling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.[34]

Toll gate

When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and TRAM.[35][36][37] These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1[disambiguation needed], and IKKi that amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation.











Ligand-gated ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the cell membrane through which ions relaying signals can pass. An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels induces action potentials, such as those that travel along nerves, by depolarizing the membrane of post-synaptic cells, resulting in the opening of voltage-gated ion channels.

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+; it acts as a second messenger initiating signal transduction cascades and altering the physiology of the responding cell. This results in amplification of the synapse response between synaptic cells by remodelling the dendritic spines involved in the synapse.

Ion transporters and channels in mammalian choroidal epithelium

Ion transporters and channels in mammalian choroidal epithelium


Intracellular receptors, such as nuclear receptors and cytoplasmic receptors, are soluble proteins localized within their respective areas. The typical ligands for nuclear receptors are lipophilic hormones like the steroid hormones testosterone and progesterone and derivatives of vitamins A and D. To initiate signal transduction, the ligand must pass through the plasma membrane by passive diffusion. On binding with the receptor, the ligands pass through the nuclear membrane into the nucleus, enabling gene transcription and protein production.



Signal Transduction

Signal Transduction


Activated nuclear receptors attach to the DNA at receptor-specific hormone-responsive element (HRE) sequences, located in the promoter region of the genes activated by the hormone-receptor complex. Due to their enabling gene transcription, they are alternatively called inductors of gene expression. All hormones that act by regulation of gene expression have two consequences in their mechanism of action; their effects are produced after a characteristically long period of time and their effects persist for another long period of time, even after their concentration has been reduced to zero, due to a relatively slow turnover of most enzymes and proteins that would either deactivate or terminate ligand binding onto the receptor.

Signal transduction via these receptors involves little proteins, but the details of gene regulation by this method are not well-understood. Nucleic receptors have DNA-binding domains containing zinc fingers and a ligand-binding domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. DNA sequences that match the receptor are usually hexameric repeats of any kind; the sequences are similar but their orientation and distance differentiate them. The ligand-binding domain is additionally responsible for dimerization of nucleic receptors prior to binding and providing structures for transactivation used for communication with the translational apparatus.





protein changes in biological mechanisms

protein changes in biological mechanisms


Steroid receptors are a subclass of nuclear receptors located primarily within the cytosol; in the absence of steroids, they cling together in an aporeceptor complex containing chaperone or heatshock proteins (HSPs). The HSPs are necessary to activate the receptor by assisting the protein to fold in a way such that the signal sequence enabling its passage into the nucleus is accessible. Steroid receptors, on the other hand, may be repressive on gene expression when their transactivation domain is hidden; activity can be enhanced by phosphorylation of serine residues at their N-terminal as a result of another signal transduction pathway, a process called crosstalk.

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Retinoic acid receptors are another subset of nuclear receptors. They can be activated by an endocrine-synthesized ligand that entered the cell by diffusion, a ligand synthesised from a precursor like retinol brought to the cell through the bloodstream or a completely intracellularly synthesised ligand like prostaglandin. These receptors are located in the nucleus and are not accompanied by HSPs; they repress their gene by binding to their specific DNA sequence when no ligand binds to them, and vice versa.

Certain intracellular receptors of the immune system are cytoplasmic receptors; recently identified NOD-like receptors (NLRs) reside in the cytoplasm of some eukaryotic cells and interact with ligands using a leucine-rich repeat (LRR) motif similar to TLRs. Some of these molecules like NOD2 interact with RIP2 kinase that activates NF-κB signaling, whereas others like NALP3 interact with inflammatory caspases and initiate processing of particular cytokines like interleukin-1β.[38][39]


Cell signaling

signaling pathjways map

signaling pathjways map

Cell signalling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. By understanding cell signalling, diseases may be treated effectively and, theoretically, artificial tissues may be created.

Traditional work in biology has focused on studying individual parts of cell signaling pathways. Systems biology research helps us to understand the underlying structure of cell signaling networks and how changes in these networks may affect the transmission and flow of information. Such networks are complex systems in their organization and may exhibit a number of emergent properties. Long-range allostery is often a significant component of cell signaling events.[1]

Enzyme_Model allosterism

Enzyme_Model allosterism


Signaling within, between, and among cells is subdivided into the following classifications:

  • Intracrine signals are produced by the target cell that stay within the target cell.
  • Autocrine signals are produced by the target cell, are secreted, and effect the target cell itself via receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as the emitting cell. An example of this are immune cells.
  • Juxtacrine signals target adjacent (touching) cells. These signals are transmitted along cell membranes via protein or lipid components integral to the membrane and are capable of affecting either the emitting cell or cells immediately adjacent.


calcium release calmodulin + ER

calcium release calmodulin + ER


Ca++ exchange

Ca++ exchange

Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

  • Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.
  • Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to reach all parts of the body.
Notch-mediated juxtacrine signal between adjacent cells.

Notch-mediated juxtacrine signal between adjacent cells.


Notch-mediated juxtacrine signal between adjacent cells.

Some cell–cell communication requires direct cell–cell contact. Some cells can form gap junctions that connect their cytoplasm to the cytoplasm of adjacent cells. In cardiac muscle, gap junctions between adjacent cells allows for action potential propagation from the cardiac pacemaker region of the heart to spread and coordinately cause contraction of the heart.

The notch signaling mechanism is an example of juxtacrine signaling (also known as contact-dependent signaling) in which two adjacent cells must make physical contact in order to communicate. This requirement for direct contact allows for very precise control of cell differentiation during embryonic development. In the worm Caenorhabditis elegans, two cells of the developing gonad each have an equal chance of terminally differentiating or becoming a uterine precursor cell that continues to divide. The choice of which cell continues to divide is controlled by competition of cell surface signals. One cell will happen to produce more of a cell surface protein that activates the Notch receptor on the adjacent cell. This activates a feedback loop or system that reduces Notch expression in the cell that will differentiate and that increases Notch on the surface of the cell that continues as a stem cell.[5]

Many cell signals are carried by molecules that are released by one cell and move to make contact with another cell. Endocrine signals are called hormones. Hormones are produced by endocrine cells and they travel through the blood to reach all parts of the body. Specificity of signaling can be controlled if only some cells can respond to a particular hormone. Paracrine signals such as retinoic acid target only cells in the vicinity of the emitting cell.[6] Neurotransmitters represent another example of a paracrine signal. Some signaling molecules can function as both a hormone and a neurotransmitter. For example, epinephrine and norepinephrine can function as hormones when released from the adrenal gland and are transported to the heart by way of the blood stream. Norepinephrine can also be produced by neurons to function as a neurotransmitter within the brain.[7] Estrogen can be released by the ovary and function as a hormone or act locally via paracrine or autocrine signaling.[8] Active species of oxygen and nitric oxide can also act as cellular messengers. This process is dubbed redox signaling.

Signaling Pathways

Cell Signaling Biology

Michael J. Berridge

Module 2

Cell Signaling Pathways
The nine membrane-bound adenylyl cyclases (AC1–AC9) have a similar domain structure. The single polypeptide has a tandem repeat of six transmembrane domains (TM) with TM1- -TM6 in one repeat and TM7- -TM12 in the other. Each TM cassette is followed by large cytoplasmic domains (C1 and C2), which contain the catalytic regions that convert ATP into cyclic AMP. As shown in the lower panel, the C1 and C2 domains come together to form a heterodimer. The ATP-binding site is located at the interface between these two domains. The soluble AC10 isoform lacks the transmembrane regions, but it retains the C1 and C2 domains that are responsible for catalysis
www.cellsignallingbiology.org  http://www.biochemj.org/csb/002/csb002.pdf



Elucidate Target-Specific Pathways With a Suite of Cellular Assays

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DiscoveRx® offers a comprehensive collection of cell-based pathway indicator assays designed to detect activation or inhibition of complex signal transduction pathways in response to compound treatment. Based on the proven PathHunter® technology, These biosensor cell lines allow you to measure distinct events within a variety of pathways involved in compound toxicity, cholesterol metabolism, antioxidant function, DNA damage and ER stress. – See more at: http://www.discoverx.com/targets/signaling-pathways#sthash.ZTb5UXVO.dpuf



inhibitors of signal transduction pathway

inhibitors of signal transduction pathway

Inhibitors of MAPK Signaling Pathway

Inhibitors of MAPK Signaling Pathway





Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Nrf2 signaling in ARE-mediated coordinated activation of defensive genes


Regulation of AMPK

Regulation of AMPK



metabolic pathways

metabolic pathways


On these resource pages you can find signaling pathway diagrams, research overviews, relevant antibody products, publications, and other research resources organized by topic. The pathway diagrams associated with these topics have been assembled by CST scientists and outside experts to provide succinct and current overviews of selected signaling pathways. Please send suggestions for developing new pathways to info@cellsignal.com. Protein nodes in each pathway diagram are linked to specific antibody product information or, optionally, to protein-specific listings in the PhosphoSitePlus® database of post-translational modifications.


PI3K / Akt Signaling Overview

 The serine/threonine kinase Akt/PKB exists as three isoforms in mammals. Akt1 has a wide tissue distribution, whereas Akt2 is found predominantly in muscle and fat cells and Akt3 is expressed in testes and brain. Akt regulates multiple biological processes including cell survival, proliferation, growth, and glycogen metabolism. Various growth factors, hormones, and cytokines activate Akt by binding their cognate receptor tyrosine kinase (RTK), cytokine receptor, or GPCR and triggering activation of the lipid kinase PI3K, which generates PIP3 at the plasma membrane. Akt binds PIP3 through its pleckstrin homology (PH) domain, resulting in translocation of Akt to the membrane. Akt is activated through a dual phosphorylation mechanism. PDK1, which is also brought to the membrane through its PH domain, phosphorylates Akt within its activation loop at Thr308. A second phosphorylation at Ser473 within the carboxy terminus is also required for activity and is carried out by the mTOR-rictor complex, mTORC2.

PTEN, a lipid phosphatase that catalyzes the dephosphorylation of PIP3, is a major negative regulator of Akt signaling. Loss of PTEN function has been implicated in many human cancers. Akt activity is also negatively regulated by the phosphatases PP2A and PHLPP, as well as by the chemical modulators wortmannin and LY294002, both of which are inhibitors of PI3K.

Activated Akt phosphorylates a large number of downstream substrates containing the consensus sequence RXRXXS/T. One of its primary functions is to promote cell growth and protein synthesis through regulation of the mTOR signaling pathway. Akt directly phosphorylates and activates mTOR, as well as inhibits the mTOR inhibitor proteins PRAS40 and tuberin (TSC2). Combined, these actions promote cell growth and G1 cell cycle progression through signaling via p70 S6 Kinase and inhibition of 4E-BP1.

Phosphofructokinase mechanism

Phosphofructokinase mechanism

GSK-3 is a primary target of Akt and inhibitory phosphorylation of GSK-3α (Ser21) or GSK-3β (Ser9) has numerous cellular effects such as promoting glycogen metabolism, cell cycle progression, regulation of wnt signaling, and formation of neurofibrillary tangles in Alzheimers disease. Akt promotes cell survival directly by its ability to phosphorylate and inactivate several pro-apoptotic targets, including Bad, Bim, Bax, and the forkhead (FoxO1/3a) transcription factors. Akt also plays an important role in metabolism and insulin signaling. Insulin receptor signaling through Akt promotes Glut4 translocation through activation of AS160 and TBC1D1, resulting in increased glucose uptake. Akt regulates glycolysis through phosphorylation of PFK and hexokinase, and plays a significant role in aerobic glycolysis of cancer cells, also known as the Warburg Effect.

Aberrant Akt signaling is the underlying defect found in several pathologies. Akt is one of the most frequently activated kinases in human cancer as constitutively active Akt can promote unregulated cell proliferation. Abnormalities in Akt2 signaling can result in diabetes due to defects in glucose homeostasis. Akt is also a key player in cardiovascular disease through its role in cardiac growth, angiogenesis, and hypertrophy.


  1. Robey RB, Hay N (2009) Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis. Cancer Biol. 19(1), 25–31.
  2. Zhang S, Yu D (2010) PI(3)king apart PTEN’s role in cancer. Cancer Res. 16(17), 4325–30.
  3. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Rev. Mol. Cell Biol. 12(1), 21–35.
  4. Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biophys. Acta 1813(11), 1978–86.
  5. Kloet DE, Burgering BM (2011) The PKB/FOXO switch in aging and cancer. Biophys. Acta 1813(11), 1926–37.
  6. Hers I, Vincent EE, Tavars JM (2011) Akt signalling in health and disease. Signal. 23(10), 1515–27.
  7. Wang H, Zhang Q, Wen Q, Zheng Y, Lazarovici P, Philip L, Jiang H, Lin J, Zheng W (2012) Proline-rich Akt substrate of 40kDa (PRAS40): a novel downstream target of PI3k/Akt signaling pathway. Signal. 24(1), 17–24.
  8. Dazert E, Hall MN (2011) mTOR signaling in disease. Opin. Cell Biol. 23(6), 744–55.
  9. Bayley JP, Devilee P (2012) The Warburg effect in 2012. Curr Opin Oncol 24(1), 62–7.


mTOR Signaling Pathway

Akt mTOR pathway

Akt mTOR pathway

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes. mTOR complex 1 (mTORC1) is composed of mTOR, Raptor, GβL (mLST8), and Deptor and is partially inhibited by rapamycin. mTORC1 integrates multiple signals reflecting the availability of growth factors, nutrients, or energy to promote either cellular growth when conditions are favorable or catabolic processes during stress or when conditions are unfavorable. Growth factors and hormones (e.g. insulin) signal to mTORC1 via Akt, which inactivates TSC2 to prevent inhibition of mTORC1. Alternatively, low ATP levels lead to the AMPK-dependent activation of TSC2 and phosphorylation of raptor to reduce mTORC1 signaling. Amino acid availability is signaled to mTORC1 via a pathway involving the Rag and Ragulator (LAMTOR1-3) proteins. Active mTORC1 has a number of downstream biological effects including translation of mRNA via the phosphorylation of downstream targets (4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1), ribosome biogenesis, and activation of transcription leading to mitochondrial metabolism or adipogenesis. The mTOR complex 2 (mTORC2) is composed of mTOR, Rictor, GβL, Sin1, PRR5/Protor-1, and Deptor and promotes cellular survival by activating Akt. mTORC2 also regulates cytoskeletal dynamics by activating PKCα and regulates ion transport and growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states including cancer, cardiovascular disease, and metabolic disorders.

Selected Reviews:

We would like to thank Carson Thoreen and Prof. David Sabatini, Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, for reviewing this diagram. revised November 2012

Protein Folding




Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.


Heat Shock Proteins (HSPs) form seven families (small HSPs (sHSPs), HSP10, 40, 60, 70, 90, and 100) of molecular chaperone proteins that play a central role in the cellular resistance to stress and actin organization. They are involved in the proper folding of proteins and the recognition and refolding of misfolded proteins. HSP expression is induced by a variety of environmental stresses, including heat, hypoxia, nutrient deficiency, free radicals, toxins, ischemia, and UV radiation. HSP27 is a member of the sHSP family. It is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the activation of the p38 MAP kinase pathway. Phosphorylation and increased concentration of HSP27 has been implicated in actin polymerization and reorganization. HSP70 and HSP90 interact with unfolded proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP- and co-chaperone-dependent manner. HSP70 has a broad range of substrates including newly synthesized and denatured proteins, while HSP90 tends to have a more limited subset of substrates, most of which are signaling molecules. HSP70 and HSP90 are also essential for the maturation and inactivation of nuclear hormones and other signaling molecules.


  1. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Rev. Mol. Cell Biol. 10(8), 513–25.
  2. Horgan CP, McCaffrey MW (2009) The dynamic Rab11-FIPs. Soc. Trans. 37(Pt 5), 1032–6.
  3. Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. Med. Chem. 53(12), 4585–602.
  4. Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. ScientificWorldJournal 10, 1543–52.
  5. Ghayour-Mobarhan M, Saber H, Ferns GA (2012) The potential role of heat shock protein 27 in cardiovascular disease. Chim. Acta 413(1-2), 15–24.
  6. Horgan CP, McCaffrey MW (2011) Rab GTPases and microtubule motors. Soc. Trans. 39(5), 1202–6.
  7. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Rev. Mol. Cell Biol. 10(8), 513–25.
  8. Horgan CP, McCaffrey MW (2009) The dynamic Rab11-FIPs. Soc. Trans. 37(Pt 5), 1032–6.
  9. Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. Med. Chem. 53(12), 4585–602.
  10. Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. ScientificWorldJournal 10, 1543–52.
  11. Ghayour-Mobarhan M, Saber H, Ferns GA (2012) The potential role of heat shock protein 27 in cardiovascular disease. Chim. Acta 413(1-2), 15–24.
  12. Horgan CP, McCaffrey MW (2011) Rab GTPases and microtubule motors. Soc. Trans. 39(5), 1202–6

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Overview of Posttranslational Modification (PTM)

Curator:  Larry H. Bernstein, MD, FCAP


This is the second discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.

1.  A Primer on DNAand DNA Replication

2. Overview of translational medicine

3. Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation


Posttranslational modification is a step in protein biosynthesis. Proteins are created by ribosomes translating mRNA into polypeptide chains. These polypeptide chains undergo
PTM before becoming the mature protein product.

Protein phosphorylation is one type of post-translational modification. Wikipedia

Explore: Phosphorylation

Glycosylation is a form of co-translational and post-translational modification. Wikipedia

Explore: Glycosylation

Acetylation occurs as a co-translational and post-translational modification of proteins, for example, histones, p53, and tubulins.


Post-Translational Modifications
As noted above, the large number of different PTMs precludes a thorough review of all possible protein modifications. Therefore, this overview only touches on a small number of the most common types of PTMs studied in protein research today. Furthermore, greater focus is placed on phosphorylation, glycosylation and ubiquitination, and therefore these PTMs are described in greater detail on pages dedicated to the respective PTM.
PhosphorylationReversible protein phosphorylation, principally on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications. Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.
GlycosylationProtein glycosylation is acknowledged as one of the major post-translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity. Glycosylation encompasses a diverse selection of sugar-moiety additions to proteins that ranges from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide changes of cell surface receptors. Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins.
UbiquitinationUbiquitin is an 8-kDa polypeptide consisting of 76 amino acids that is appended to lysine in target proteins via the C-terminal glycine of ubiquitin. A ubiquitin polymer is formed after  initial monoubiquitination. Polyubiquitinated proteins are degraded recycling the ubiquitin.
S-NitrosylationNitric oxide (NO) is produced by three isoforms of nitric oxide synthase (NOS) and is a chemical messenger that reacts with free cysteine residues to form S-nitrothiols (SNOs). S-nitrosylation is a critical PTM used by cells to stabilize proteins, regulate gene expression and provide NO donors, and the generation, localization, activation and catabolism of SNOs are tightly regulated.S-nitrosylation is a reversible reaction, and SNOs have a short half life in the cytoplasm because of the host of reducing enzymes, including glutathione (GSH) and thioredoxin, that denitrosylate proteins. Therefore, SNOs are often stored in membranes, vesicles, the interstitial space and lipophilic protein folds to protect them from denitrosylation (5). For example, caspases, which mediate apoptosis, are stored in the mitochondrial intermembrane space as SNOs. In response to extra- or intracellular cues, the caspases are released into the cytoplasm, and the highly reducing environment rapidly denitrosylates the proteins, resulting in caspase activation and the induction of apoptosis.Only specific cysteine residues are S-nitrosylated. Proteins may contain multiple cysteines and due to the labile nature of SNOs, S-nitrosylated cysteines can be difficult to detect and distinguish from non-S-nitrosylated amino acids. The biotin switch assay, developed by Jaffrey et al., is a common method of detecting SNOs, and the steps of the assay are listed below (6):

  • All free cysteines are blocked.
  • All remaining cysteines (presumably only those that are denitrosylated) are denitrosylated.
  • The now-free thiol groups are then biotinylated.
  • Biotinylated proteins are detected by SDS-PAGE and Western blot analysis or mass spectrometry (7).
MethylationThe transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine (SAM) is the primary methyl group donor.Methylation occurs so often that SAM has been suggested to be the most-used substrate in enzymatic reactions after ATP (4). Additionally, while N-methylation is irreversible, O-methylation is potentially reversible. Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and demethylation influences the availability of DNA for transcription.
N-AcetylationN-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost all eukaryotic proteins through both irreversible and reversible mechanisms. N-terminal acetylation requires the cleavage of the N-terminal methionine by methionine aminopeptidase (MAP) before replacing the amino acid with an acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes. This type of acetylation is co-translational, in that N-terminus is acetylated on growing polypeptide chains that are still attached to the ribosome.Acetylation at the ε-NH2 of lysine (termed lysine acetylation) on histone N-termini is a common method of regulating gene transcription. Histone acetylation is a reversible event that reduces chromosomal condensation to promote transcription, and the acetylation of these lysine residues is regulated by transcription factors that contain histone acetyletransferase (HAT) activity. While transcription factors with HAT activity act as transcription co-activators, histone deacetylase (HDAC) enzymes are co-repressors that reverse the effects of acetylation by reducing the level of lysine acetylation and increasing chromosomal condensation.Sirtuins (silent information regulator) are a group of NAD-dependent deacetylases that target histones. As their name implies, they maintain gene silencing by hypoacetylating histones and have been reported to aid in maintaining genomic stability (8).Cytoplasmic proteins may also be acetylated, and therefore acetylation seems to play a greater role in cell biology than simply transcriptional regulation (9). Furthermore, crosstalk between acetylation and other post-translational modifications, including phosphorylation, ubiquitination and methylation, can modify the biological function of the acetylated protein (10).
LipidationLipidation is a method to target proteins to membranes in organelles (endoplasmic reticulum [ER], Golgi apparatus, mitochondria), vesicles (endosomes, lysosomes) and the plasma membrane. The four types of lipidation are:

  • C-terminal glycosyl phosphatidylinositol (GPI) anchor
  • N-terminal myristoylation
  • S-myristoylation
  • S-prenylation

Each type of modification gives proteins distinct membrane affinities, although all types of lipidation increase the hydrophobicity of a protein and thus its affinity for membranes. The different types of lipidation are not mutually exclusive, in that two or more lipids can be attached to a given protein.

GPI anchors tether cell surface proteins to the plasma membrane. These hydrophobic moieties are prepared in the ER, where they are then added to the nascent protein en bloc. GPI-anchored proteins are often localized to cholesterol- and sphingolipid-rich lipid rafts, which act as signaling platforms on the plasma membrane.

is a method to give proteins a hydrophobic handle for membrane localization. The myristoyl group is a 14-carbon saturated fatty acid (C14), which gives the protein sufficient hydrophobicity and affinity for membranes, but not enough to permanently anchor the protein in the membrane. N-myristoylation can therefore act as a conformational localization switch, in which protein conformational changes influence the availability of the handle for membrane attachment.

N-myristoylation, facilitated specifically by N-myristoyltransferase (NMT), uses myristoyl-CoA to attach the myristoyl group to the N-terminal glycine. This PTM requires methionine cleavage prior to addition of the myristoyl group because methionine is the N-terminal amino acid of all eukaryotic proteins.

 S-palmitoylation adds a C16 palmitoyl group from palmitoyl-CoA to the thiolate side chain of cysteine residues via palmitoyl acyl transferases (PATs). Because of the longer hydrophobic group, this anchor can permanently anchor the protein to the membrane. S-palmitoylation is used as an on/off switch to regulate membrane localization.

S-prenylation covalently adds a farnesyl (C15) or geranylgeranyl (C20) group to specific cysteine residues within 5 amino acids from the C-terminus via farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II). All members of the Ras superfamily are prenylated. These proteins have specific 4-amino acid motifs at the C-terminus that determine the type of prenylation at single or dual cysteines. Prenylation occurs in the ER and is often part of a stepwise process of PTMs that is followed by proteolytic cleavage by Rce1 and methylation by isoprenyl cysteine methyltransferase (ICMT).

ProteolysisPeptide bonds are indefinitely stable under physiological conditions, and therefore cells require some mechanism to break these bonds. Proteases comprise a family of enzymes that cleave the peptide bonds of proteins and are critical in antigen processing, apoptosis, surface protein shedding and cell signaling.Degradative proteolysis is critical to remove unassembled protein subunits and misfolded proteins and to maintain protein concentrations at homeostatic concentrations.Proteolysis is a thermodynamically favorable and irreversible reaction. Therefore, protease activity is tightly regulated to avoid uncontrolled proteolysis through temporal and/or spatial control mechanisms including regulation by cleavage in cis or trans and compartmentalization (e.g., proteasomes, lysosomes).


The diverse family of proteases can be classified by the site of action, such as aminopeptidases and carboxypeptidase, which cleave at the amino or carboxy terminus of a protein, respectively. Another type of classification is based on the active site groups of a given protease that are involved in proteolysis. Based on this classification strategy, greater than 90% of known proteases fall into one of four categories as follows:

  • Serine proteases
  • Cysteine proteases
  • Aspartic acid proteases
  • Zinc metalloproteases
  1. International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature. 431, 931-45.
  2. Jensen O. N. (2004) Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Curr Opin Chem Biol. 8, 33-41.
  3. Ayoubi T. A. and Van De Ven W. J. (1996) Regulation of gene expression by alternative promoters. FASEB J. 10, 453-60.
  4. Walsh C. (2006) Posttranslational modification of proteins : Expanding nature’s inventory. Englewood, Colo.: Roberts and Co. Publishers. xxi, 490 p. p.
  5. Gaston B. M. et al. (2003) S-nitrosylation signaling in cell biology. Mol Interv. 3, 253-63.
  6. Jaffrey S. R. and Snyder S. H. (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE. 2001, pl1.
  7. Han P. and Chen C. (2008) Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins. Rapid Commun Mass Spectrom. 22, 1137-45.
  8. Imai S. et al. (2000) Transcriptional silencing and longevity protein SIR2 is an NAD-dependent histone deacetylase. Nature. 403, 795-800.
  9. Glozak M. A. et al. (2005) Acetylation and deacetylation of non-histone proteins. Gene. 363, 15-23.
  10. Yang X. J. and Seto E. (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell. 31, 449-61


Protein phosphorylation

From Wikipedia, the free encyclopedia

Protein phosphorylation is a post-translational modification of proteins in which a serine, a threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Regulation of proteins by phosphorylation is one of the most common modes of regulation of protein function, and is often termed “phosphoregulation”. In almost all cases of phosphoregulation, the protein switches between a phosphorylated and an unphosphorylated form, and one of these two is an active form, while the other one is an inactive form.

Functions of phosphorylation[edit]

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

In other reactions, phosphorylation of a protein substrate can inhibit its activity (as when AKT phosphorylates the enzyme GSK-3). One common mechanism for phosphorylation-mediated enzyme inhibition was demonstrated in the tyrosine kinase called “src” (pronounced “sarc”, see: Src (gene)). When src is phosphorylated on a particular tyrosine, it folds on itself, and thus masks its own kinase domain, and is thus turned “off”.

In still other reactions, phosphorylation of a protein causes it to be bound to other proteins which have “recognition domains” for a phosphorylated tyrosineserine, or threoninemotif. As a result of binding a particular protein, a distinct signaling system may be activated or inhibited.

In the late 1990s it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. These target proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated.


Oxidative phosphorylation

From Wikipedia, the free encyclopedia

Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which the mitochondria in cellsuse their structure, enzymes, and energy released by the oxidation of nutrients to reform ATP. Although the many forms of life on earth use a range of different nutrients, ATP is the molecule that supplies energy tometabolism. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentationprocesses such as anaerobic glycolysis.

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

The electron transport chain in the mitochondrion is the site of oxidative phosphorylation in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized, releasing energy to power the ATP synthase.

These linked sets of proteins are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors.

electron transport chain in the mitochondrion

The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, in a process called electron transport. This generates potential energy in the form of a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to flow back across the membrane and down this gradient, through a large enzymecalled ATP synthase; this process is known as chemiosmosis. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a phosphorylation reaction. This reaction is driven by the proton flow, which forces the rotation of a part of the enzyme; the ATP synthase is a rotary mechanical motor.

Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such assuperoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease and, possibly, aging (senescence). The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons that inhibit their activities.

Additional References in Leaders in Pharmaceutical Intelligence

Proteomics and Biomarker Discovery


Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets


Immune activation, immunity, antibacterial activity


Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III


Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis


Research on inflammasomes opens therapeutic ways for treatment of rheumatoid arthritis


Update on mitochondrial function, respiration, and associated disorders


Insert – on ETC

Overview of energy transfer by chemiosmosis[edit]

Further information: Chemiosmosis and Bioenergetics

Oxidative phosphorylation works by using energy-releasing chemical reactions to drive energy-requiring reactions: The two sets of reactions are said to be coupled. This means one cannot occur without the other. The flow of electrons through the electron transport chain, from electron donors such as NADH to electron acceptors such as oxygen, is anexergonic process – it releases energy, whereas the synthesis of ATP is an endergonic process, which requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane, and energy is transferred from electron transport chain to the ATP synthase by movements of protons across this membrane, in a process called chemiosmosis.[1] In practice, this is like a simple electric circuit, with a current of protons being driven from the negative N-side of the membrane to the positive P-side by the proton-pumping enzymes of the electron transport chain. These enzymes are like a battery, as they perform work to drive current through the circuit. The movement of protons creates an electrochemical gradient across the membrane, which is often called the proton-motive force. It has two components: a difference in proton concentration (a H+gradient, ΔpH) and a difference in electric potential, with the N-side having a negative charge.[2]

ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the electrochemical gradient, back to the N-side of the membrane.[3] This kinetic energy drives the rotation of part of the enzymes structure and couples this motion to the synthesis of ATP.

The two components of the proton-motive force are thermodynamically equivalent: In mitochondria, the largest part of energy is provided by the potential; in alkaliphile bacteria the electrical energy even has to compensate for a counteracting inverse pH difference. Inversely, chloroplasts operate mainly on ΔpH. However, they also require a small membrane potential for the kinetics of ATP synthesis. At least in the case of the fusobacterium P. modestum it drives the counter-rotation of subunits a and c of the FO motor of ATP synthase.[2]

The amount of energy released by oxidative phosphorylation is high, compared with the amount produced by anaerobic fermentationGlycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one molecule of glucoseto carbon dioxide and water,[4] while each cycle of beta oxidation of a fatty acid yields about 14 ATPs. These ATP yields are theoretical maximum values; in practice, some protons leak across the membrane, lowering the yield of ATP.[5]

Electron and proton transfer molecules[edit]

Further information: Coenzyme and Cofactor

The electron transport chain carries both protons and electrons, passing electrons from donors to acceptors, and transporting protons across a membrane. These processes use both soluble and protein-bound transfer molecules. In mitochondria, electrons are transferred within the intermembrane space by the water-soluble electron transfer protein cytochrome c.[6] This carries only electrons, and these are transferred by the reduction and oxidation of an iron atom that the protein holds within a heme group in its structure. Cytochrome c is also found in some bacteria, where it is located within the periplasmic space.[7]

Krebs_Cycler_1402785124Overview of The Electron Transport Chain






Reduction of coenzyme Q from itsubiquinone form (Q) to the reduced ubiquinol form (QH2).


Within the inner mitochondrial membrane, the lipid-soluble electron carrier coenzyme Q10 (Q) carries both electrons and protons by a redox cycle.[8] This small benzoquinone molecule is very hydrophobic, so it diffuses freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to the ubiquinol form (QH2); when QH2 releases two electrons and two protons, it becomes oxidized back to the ubiquinone (Q) form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH2 oxidized on the other, ubiquinone will couple these reactions and shuttle protons across the membrane.[9] Some bacterial electron transport chains use different quinones, such as menaquinone, in addition to ubiquinone.[10]

Within proteins, electrons are transferred between flavin cofactors,[3][11] iron–sulfur clusters, and cytochromes. There are several types of iron–sulfur cluster. The simplest kind found in the electron transfer chain consists of two iron atoms joined by two atoms of inorganic sulfur; these are called [2Fe–2S] clusters. The second kind, called [4Fe–4S], contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an additional amino acid, usually by the sulfur atom of cysteine. Metal ion cofactors undergo redox reactions without binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors.[12] This occurs by quantum tunnelling, which is rapid over distances of less than 1.4×10−9 m.[13]

Eukaryotic electron transport chains[edit]

Further information: Electron transport chain and Chemiosmosis

Many catabolic biochemical processes, such as glycolysis, the citric acid cycle, and beta oxidation, produce the reduced coenzyme NADH. This coenzyme contains electrons that have a high transfer potential; in other words, they will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of complexes I through IV, is called the electron transport chain and is found in the inner membrane of the mitochondrion. Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different point.

In eukaryotes, the enzymes in this electron transport system use the energy released from the oxidation of NADH to pump protons across the inner membrane of the mitochondrion. This causes protons to build up in the intermembrane space, and generates an electrochemical gradient across the membrane. The energy stored in this potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the exception of anaerobic protozoa such as Trichomonas vaginalis that instead reduce protons to hydrogen in a remnant mitochondrion called a hydrogenosome.[14]


Typical respiratory enzymes and substrates in eukaryotes.
Respiratory enzyme Redox pair Midpoint potential (Volts)
NADH dehydrogenase NAD+ / NADH −0.32[15]
Succinate dehydrogenase FMN or FAD / FMNH2 or FADH2 −0.20[15]
Cytochrome bc1 complex Coenzyme Q10ox / Coenzyme Q10red +0.06[15]
Cytochrome bc1 complex Cytochrome box / Cytochrome bred +0.12[15]
Complex IV Cytochrome cox / Cytochrome cred +0.22[15]
Complex IV Cytochrome aox / Cytochrome ared +0.29[15]
Complex IV O2 / HO +0.82[15]
Conditions: pH = 7[15]


NADH-coenzyme Q oxidoreductase (complex I)[edit]

NADH-coenzyme Q oxidoreductase, also known as NADH dehydrogenase or complex I, is the first protein in the electron transport chain.[16] Complex I is a giant enzyme with the mammalian complex I having 46 subunits and a molecular mass of about 1,000 kilodaltons (kDa).[17] The structure is known in detail only from a bacterium;[18][19]  in most organisms the complex resembles a boot with a large “ball” poking out from the membrane into the mitochondrion.[20][21]

Complex I or NADH-Q oxidoreductase



Complex I or NADH-Q oxidoreductase. The abbreviations are discussed in the text. In all diagrams of respiratory complexes in this article, the matrix is at the bottom, with the intermembrane space above.

The genes that encode the individual proteins are contained in both the cell nucleus and themitochondrial genome, as is the case for many enzymes present in the mitochondrion.

The reaction that is catalyzed by this enzyme is the two electron oxidation of NADH by coenzyme Q10 or ubiquinone(represented as Q in the equation below), a lipid-soluble quinone that is found in the mitochondrion membrane:

The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I and the donation of two electrons. The electrons enter complex I via a prosthetic group attached to the complex, flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH2. The electrons are then transferred through a series of iron–sulfur clusters: the second kind of prosthetic group present in the complex.[18] There are both [2Fe–2S] and [4Fe–4S] iron–sulfur clusters in complex I.

As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. Exactly how this occurs is unclear, but it seems to involve conformational changes in complex I that cause the protein to bind protons on the N-side of the membrane and release them on the P-side of the membrane.[22] Finally, the electrons are transferred from the chain of iron–sulfur clusters to a ubiquinone molecule in the membrane.[16] Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the matrix as it is reduced to ubiquinol (QH2).

Succinate-Q oxidoreductase (complex II)[edit]

Succinate-Q oxidoreductase, also known as complex II or succinate dehydrogenase, is a second entry point to the electron transport chain.[23] It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of four protein subunits and contains a bound flavin adenine dinucleotide (FAD) cofactor, iron–sulfur clusters, and a hemegroup that does not participate in electron transfer to coenzyme Q, but is believed to be important in decreasing production of reactive oxygen species.[24][25]

Complex II



Complex II: Succinate-Q oxidoreductase.

It oxidizes succinate to fumarate and reduces ubiquinone.As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.

In some eukaryotes, such as the parasitic worm Ascaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment of the large intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor.[26] Another unconventional function of complex II is seen in the malaria parasite Plasmodium falciparum. Here, the reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an unusual form ofpyrimidine biosynthesis.[27]

Electron transfer flavoprotein-Q oxidoreductase[edit]

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone.[28] This enzyme contains a flavin and a [4Fe–4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.[29]

In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and choline, as it accepts electrons from multiple acetyl-CoAdehydrogenases.[30][31] In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.[32]


Q-cytochrome c oxidoreductase (complex III)[edit]

Q-cytochrome c oxidoreductase is also known as cytochrome c reductasecytochrome bc1 complex, or simply complex III.[33][34] In mammals, this enzyme is a dimer, with each subunit complex containing 11 protein subunits, an [2Fe-2S] iron–sulfur cluster and three cytochromes: one cytochrome c1 and two bcytochromes.[35] A cytochrome is a kind of electron-transferring protein that contains at least one hemegroup. The iron atoms inside complex III’s heme groups alternate between a reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein.

complex III



The two electron transfer steps in complex III: Q-cytochrome c oxidoreductase. After each step, Q (in the upper part of the figure) leaves the enzyme.

The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two molecules of cytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron.

As only one of the electrons can be transferred from the QH2 donor to a cytochrome c acceptor at a time, the reaction mechanism of complex III is more elaborate than those of the other respiratory complexes, and occurs in two steps called the Q cycle.[36] In the first step, the enzyme binds three substrates, first, QH2, which is then oxidized, with one electron being passed to the second substrate, cytochrome c. The two protons released from QH2 pass into the intermembrane space. The third substrate is Q, which accepts the second electron from the QH2 and is reduced to Q.-, which is the ubisemiquinone free radical. The first two substrates are released, but this ubisemiquinone intermediate remains bound. In the second step, a second molecule of QH2 is bound and again passes its first electron to a cytochrome c acceptor. The second electron is passed to the bound ubisemiquinone, reducing it to QH2 as it gains two protons from the mitochondrial matrix. This QH2 is then released from the enzyme.[37]

As coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidized to ubiquinone on the other, a net transfer of protons across the membrane occurs, adding to the proton gradient.[3] The rather complex two-step mechanism by which this occurs is important, as it increases the efficiency of proton transfer. If, instead of the Q cycle, one molecule of QH2 were used to directly reduce two molecules of cytochrome c, the efficiency would be halved, with only one proton transferred per cytochrome c reduced.[3]


Cytochrome c oxidase (complex IV)[edit]

For more details on this topic, see cytochrome c oxidase.

Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron transport chain.[38] The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors – in all, three atoms of copper, one of magnesium and one of zinc.[39]

This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane.[40] The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:

Complex IV



Complex IV: cytochrome c oxidase.

Organization of complexes[edit]

The original model for how the respiratory chain complexes are organized was that they diffuse freely and independently in the mitochondrial membrane.[17] However, recent data suggest that the complexes might form higher-order structures called supercomplexes or “respirasomes.”[49] In this model, the various complexes exist as organized sets of interacting enzymes.[50] These associations might allow channeling of substrates between the various enzyme complexes, increasing the rate and efficiency of electron transfer.[51] Within such mammalian supercomplexes, some components would be present in higher amounts than others, with some data suggesting a ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4.[52] However, the debate over this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.[17][53]


Reversible protein phosphorylation, principally on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications. Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.

Phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell. While phosphorylation has been observed in bacterial proteins, it is considerably more pervasive in eukaryotic cells. It is estimated that one-third of the proteins in the human proteome are substrates for phosphorylation at some point (1). Indeed, phosphoproteomics has been established as a branch of proteomics that focuses solely on the identification and characterization of phosphorylated proteins.

Mechanism of Phosphorylation
While phosphorylation is a prevalent post-translational modification (PTM) for regulating protein function, it only occurs at the side chains of three amino acids, serine, threonine and tyrosine, in eukaryotic cells. These amino acids have a nucleophilic (–OH) group that attacks the terminal phosphate group (γ-PO32-) on the universal phosphoryl donor adenosine triphosphate (ATP), resulting in the transfer of the phosphate group to the amino acid side chain. This transfer is facilitated by magnesium (Mg2+), which chelates the γ- and β-phosphate groups to lower the threshold for phosphoryl transfer to the nucleophilic (–OH) group. This reaction is unidirectional because of the large amount of free energy that is released when the phosphate-phosphate bond in ATP is broken to form adenosine diphosphate (ADP).

Serine Phosphorylation





Diagram of serine phosphorylation. Enzyme-catalyzed proton transfer from the (–OH) group on serine stimulates the nucleophilic attack of the γ-phosphate group on ATP, resulting in transfer of the phosphate group to serine to form phosphoserine and ADP. (—B:) indicates the enzyme base that initiates proton transfer.

For a large subset of proteins, phosphorylation is tightly associated with protein activity and is a key point of protein function regulation. Phosphorylation regulates protein function and cell signaling by causing conformational changes in the phosphorylated protein. These changes can affect the protein in two ways. First, conformational changes regulate the catalytic activity of the protein. Thus, a protein can be either activated or inactivated by phosphorylation. Second, phosphorylated proteins recruit neighboring proteins that have structurally conserved domains that recognize and bind to phosphomotifs. These domains show specificity for distinct amino acids. For example, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains show specificity for phosphotyrosine (pY), although distinctions in these two structures give each domain specificity for distinct phosphotyrosine motifs (2). Phosphoserine (pS) recognition domains include MH2 and the WW domain, while phosphothreonine (pT) is recognized by forkhead-associated (FHA) domains. The ability of phosphoproteins to recruit other proteins is critical for signal transduction, in which downstream effector proteins are recruited to phosphorylated signaling proteins.

Protein phosphorylation is a reversible PTM that is mediated by kinases and phosphatases, which phosphorylate and dephosphorylate substrates, respectively. These two families of enzymes facilitate the dynamic nature of phosphorylated proteins in a cell. Indeed, the size of the phosphoproteome in a given cell is dependent upon the temporal and spatial balance of kinase and phosphatase concentrations in the cell and the catalytic efficiency of a particular phosphorylation site.

Phosphorylation is a reversible PTM that regulates protein function





Phosphorylation is a reversible PTM that regulates protein function. Left panel: Protein kinases mediate phosphorylation at serine, threonine and tyrosine side chains, and phosphatases reverse protein phosphorylation by hydrolyzing the phosphate group. Right panel: Phosphorylation causes conformational changes in proteins that either activate (top) or inactivate (bottom) protein function.

Protein Kinases
Kinases are enzymes that facilitate phosphate group transfer to substrates. Greater than 500 kinases have been predicted in the human proteome; this subset of proteins comprises the human kinome (3). Substrates for kinase activity are diverse and include lipids, carbohydrates, nucleotides and proteins.ATP is the cosubstrate for almost all protein kinases, although guanosine triphosphate is used by a small number of kinases. ATP is the ideal structure for the transfer of α-, β- or γ-phosphate groups for nucleotidyl-, pyrophosphoryl- or phosphoryltransfer, respectively (4). While the substrate specificity of kinases varies, the ATP-binding site is generally conserved (5).Protein kinases are categorized into subfamilies that show specificity for distinct catalytic domains and include tyrosine kinases or serine/threonine kinases. Approximately 80% of the mammalian kinome comprises serine/threonine kinases, and >90% of the phosphoproteome consists of pS and pT. Indeed, studies have shown that the relative abundance ratio of pS:pT:pY in a cell is 1800:200:1 (6). Although pY is not as prevalent as pS and pT, global tyrosine phosphorylation is at the forefront of biomedical research because of its relation to human disease via the dysregulation of receptor tyrosine kinases (RTKs).Protein kinase substrate specificity is based not only on the target amino acid but also on consensus sequences that flank it (7). These consensus sequences allow some kinases to phosphorylate single proteins and others to phosphorylate multiple substrates (>300) (5). Additionally, kinases can phosphorylate single or multiple amino acids on an individual protein if the kinase-specific consensus sequences are available.

Kinases have regulatory subunits that function as activating or autoinhibitory domains and have various regulatory substrates. Phosphorylation of these subunits is a common approach to regulating kinase activity (8). Most protein kinases are dephosphorylated and inactive in the basal state and are activated by phosphorylation. A small number of kinases are constitutively active and are made intrinsically inefficient, or inactive, when phosphorylated. Some kinases, such as Src, require a combination of phosphorylation and dephosphorylation to become active, indicating the high regulation of this proto-oncogene. Scaffolding and adaptor proteins can also influence kinase activity by regulating the spatial relationship between kinases and upstream regulators and downstream substrates.

Signal Transduction Cascades
The reversibility of protein phosphorylation makes this type of PTM ideal for signal transduction, which allows cells to rapidly respond to intracellular or extracellular stimuli. Signal transduction cascades are characterized by one or more proteins physically sensing cues, either through ligand binding, cleavage or some other response, that then relay the signal to second messengers and signaling enzymes. In the case of phosphorylation, these receptors activate downstream kinases, which then phosphorylate and activate their cognate downstream substrates, including additional kinases, until the specific response is achieved. Signal transduction cascades can be linear, in which kinase A activates kinase B, which activates kinase C and so forth. Signaling pathways have also been discovered that amplify the initial signal; kinase A activates multiple kinases, which in turn activate additional kinases. With this type of signaling, a single molecule, such as a growth factor, can activate global cellular programs such as proliferation (9).


Signal Transduction Pathways




Signal transduction cascades amplify the signal output. External and internal stimuli induce a wide range of cellular responses through a series of second messengers and enzymes. Linear signal transduction pathways yield the sequential activation of a discrete number of downstream effectors, while other stimuli elicit signal cascades that amplify the initial stimulus for large-scale or global cellular responses.

Protein Phosphatases
The intensity and duration of phosphorylation-dependent signaling is regulated by three mechanisms (5):

  • Removal of the activating ligand
  • Kinase or substrate proteolysis
  • Phosphatase-dependent dephosphorylation

The human proteome is estimated to contain approximately 150 protein phosphatases, which show specificity for pS/pT and pY residues (10,11). While dephosphorylation is the end goal of these two groups of phosphatases, they do it through separate mechanisms. Serine/threonine phosphatases mediate the direct hydrolysis of the phosphorus atom of the phosphate group using a bimetallic (Fe/Zn) center, while tyrosine phosphatases form a covalent thiophosphoryl intermediate that facilitates removal of the tyrosine residue.


Phosphorylation and Ubiquitylation

Almost all aspects of biology are regulated by reversible protein phosphorylation and ubiquitylation. Abnormalities in these pathways cause numerous diseases including cancer, neurodegeneration and inflammation – all conditions under intense scrutiny in our Unit. Deciphering how disruptions in phosphorylation and ubiquitin networks lead to disease will reveal novel drug targets and improved strategies to treat these maladies in the future.

Protein ubiquitylation is analogous to protein phosphorylation except that ubiquitin molecules are attached covalently to Lys residues, as opposed to phosphate groups becoming covalently attached to one or more Ser, Thr or Tyr residues. Like phosphorylation, ubiquitylation can alter protein properties and functions in every conceivable way. Ubiquitylation is likely to be a more versatile control mechanism than phosphorylation, as ubiquitin molecules can not only be linked to one or more amino acid residues on the same protein, but can also form ubiquitin chains.

Moreover, there are also several ubiquitin-like modifiers (ULMs), such as Nedd8, SUMO1, SUMO2, SUMO3, FAT10 and ISG15, which can become attached to proteins in reactions termed Neddylation, SUMOylation, Tenylation and ISGylation, while poly-SUMO chains (involving SUMO2 and SUMO3) are also formed in cells. Recent research has highlighted an exquisite interplay between phosphorylation and ubiquitin pathways that regulate many physiological systems.






Protein ubiquitylation is an even more versatile control mechanism
than protein phosphorylation

This includes pathways of relevance to understanding innate immunity, Parkinson’s disease and cancer, emphasising the importance of integrating phosphorylation and ubiquitylation research, and not considering these separate areas to be studied in isolation.

Phosphorylation Ubiquitylation
Discovered 1955 Discovered 1978
>500 protein kinases ~10 E1s, ~40 E2s
>600 E3 ligases
140 protein phosphatases ~100 deubiquitylases
Nobel Prize 1992 Nobel Prize 2004
First drug approval
2001 (Gleevec)
First drug approval
2003 (Bortezomib)
16 drugs approved,
>150 in clinical trials
15 drugs in Phase I/II
Current sales of
USS$15 billion p.a.
Current sales of
USS$1.5 billion p.a.
30% of Pharma R&D <<1% of Pharma R&D


History of the development of protein phoshorylation and ubiquitylation

The MRC-PPU research focuses on unravelling the roles of protein phosphorylation and ubiquitylation pathways that have strong links to understanding human disease. This is where we can make the best use of our expertise, grasp opportunities emerging from the golden era of genetic analysis of human disease, and make a significant contribution to medical research.

Our Principal Investigators (PIs) deploy a blend of creativity, curiosity, expertise and state-of-the-art technology to tackle their selected projects. Their aim is to uncover fundamentally new knowledge on how biological systems are controlled, hopefully shedding novel insights into the understanding and treatment of disease. Effective translation of our research will also be impossible without robust interactions with drug discovery units such as the MRC Technology Centre for Therapeutics Discovery, the University of Dundee’s Drug Discovery Unit and close collaboration with pharmaceutical companies.

The latter will be greatly enhanced by major collaborations with the six pharmaceutical companies that support the Division of Signal Transduction Therapy. Access to the exceptional support services available within the MRC-PPU and DSTT also helps to maximise the competitiveness of our research groups and reinforce collaborations with our external partners.

Central questions being addressed by our PIs include understanding how ubiquitin and phosphorylation pathways are organised, characterising the interplay between these pathways, determining how they recognise and respond to signals, and uncovering how disruption of these networks causes disease. The expectation is that the data, reagents and expertise emerging from our research and working effectively with clinicians and pharmaceutical industry will enable us to devise new

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

https://pharmaceuticalintelligence.com/7/17/2014/Genes, proteomes, and their interaction


This is the third discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.


1.  A Primer on DNAand DNA Replication


Dna triplex pic





2. Overview of translational medicine

3. Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation


This discussion is the beginning of a diversion away from the routine discussion of a specific sequence and pairing of nucleotides in the classic model, to explore the interaction between proteins, or folded proteins and RNA or hidtones that reside in the nucleus and contribute to induction or inactivation of gene expression.  The basic text document is rigid, inflexible, and resides in all cells.  Yet, in bacteria, yeast, and eukaryotic cells, there are models of gene expression, and in eukaryotes, there is the development of expressed organ systems.  These systems have similar proteins or enzymes that are functionally identical, but they have isoforms that bind with proteins, membranes, lipopolysaccharides, and lipoproteins – which has an impact on the catabolic and anabolic activity of the cells, and they are affected by oxidative stress, and they are often dependent on the energy of binding with metal ions,i.e., Mn, Cu, Cd, Zn,..,Fe, and in other cases anionic ligands, such as I, and they may transiently act through a nucleotide or influenced by a hormone.


This will be presented as a group of predetermined articles to follow:

1.   Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.  

2.  Thyroid Hormone Key to Lipid Kinase Regulation

3.  Mammalian Target of Rapamycin Complex 1 Orchestrates Invariant NKT Cell Differentiation and Effector Function

4   The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells

5.  Nf k-beta signaling pathway

6.  P181 cAMP-mediated Rac1 activation regulates the re-establishment of endothelial adherens junctions and barrier restoration during inflammation.

7.  Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

8.  Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia

9.  Removing parts of shape-shifting protein explains how blood clots



1.  Added Layers of Proteome Complexity

Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.  

By Anna Azvolinsky | July 17, 2014

added layers of proteome

added layers of proteome


There may be more to the human proteome than previously thought. Some genes are known to have several different alternatively spliced protein variants, but the Scripps Research Institute’s Paul Schimmel and his colleagues have now uncovered almost 250 protein splice variants of an essential, evolutionarily conserved family of human genes. The results were published today (July 17) in Science.

Focusing on the 20-gene family of aminoacyl tRNA synthetases (AARSs), the team captured AARS transcripts from human tissues—some fetal, some adult—and showed that many of these messenger RNAs (mRNAs) were translated into proteins. Previous studies have identified several splice variants of these enzymes that have novel functions, but uncovering so many more variants was unexpected, Schimmel said. Most of these new protein products lack the catalytic domain but retain other AARS non-catalytic functional domains.

“The main point is that a vast new area of biology, previously missed, has been uncovered,” said Schimmel.

“This is an incredible study that fundamentally changes how we look at the protein-synthesis machinery,” Michael Ibba, a protein translation researcher at Ohio State University who was not involved in the work, told The Scientist in an e-mail. “The unexpected and potentially vast expanded functional networks that emerge from this study have the potential to influence virtually any aspect of cell growth.”

The team—including researchers at the Hong Kong University of Science and Technology, Stanford University, and aTyr Pharma, a San Diego-based biotech company that Schimmel co-founded—comprehensively captured and sequenced the AARS mRNAs from six human tissue types using high-throughput deep sequencing. While many of the transcripts were expressed in each of the tissues, there was also some tissue specificity.

Next, the team showed that a proportion of these transcripts, including those missing the catalytic domain, indeed resulted in stable protein products: 48 of these splice variants associated with polysomes. In vitro translation assays and the expression of more than 100 of these variants in cells confirmed that many of these variants could be made into stable protein products.

The AARS enzymes—of which there’s one for each of the 20 amino acids—bring together an amino acid with its appropriate transfer RNA (tRNA) molecule. This reaction allows a ribosome to add the amino acid to a growing peptide chain during protein translation. AARS enzymes can be found in all living organisms and are thought to be among the first proteins to have originated on Earth.

To understand whether these non-catalytic proteins had unique biological activities, the researchers expressed and purified recombinant AARS fragments, testing them in cell-based assays for proliferation, cell differentiation, and transcriptional regulation, among other phenotypes. “We screened through dozens of biological assays and found that these variants operate in many signaling pathways,” said Schimmel.

“This is an interesting finding and fits into the existing paradigm that, in many cases, a single gene is processed in various ways [in the cell] to have alternative functions,” said­ Steven Brenner, a computational genomics researcher at the University of California, Berkeley.

The team is now investigating the potentially unique roles of these protein splice variants in greater detail—in both human tissue as well as in model organisms. For example, it is not yet clear whether any of these variants directly bind tRNAs.

“I do think [these proteins] will play some biological roles,” said Tao Pan, who studies the functional roles of tRNAs at the University of Chicago. “I am very optimistic that interesting biological functions will come out of future studies on these variants.”

Brenner agreed. “There could be very different biological roles [for some of these proteins]. Biology is very creative that way, [it’s] able to generate highly diverse new functions using combinations of existing protein domains.” However, the low abundance of these variants is likely to constrain their potential cellular functions, he noted.

Because AARSs are among the oldest proteins, these ancient enzymes were likely subject to plenty of change over time, said Karin Musier-Forsyth, who studies protein translational at the Ohio State University. According to Musier-Forsyth, synthetases are already known to have non-translational functions and differential localizations. “Like the addition of post-translational modifications, splicing variation has evolved as another way to repurpose protein function,” she said.

One of the protein variants was able to stimulate skeletal muscle fiber formation ex vivo and upregulate genes involved in muscle cell differentiation and metabolism in primary human skeletal myoblasts. “This was really striking,” said Musier-Forsyth. “This suggests that, perhaps, peptides derived from these splice variants could be used as protein-based therapeutics for a variety of diseases.”

W.S. Lo et al., “Human tRNA synthetase catalytic nulls with diverse functions,” Science,  http://dx.doi.org:/10.1126/science.1252943, 2014.

Tags  tRNAproteomicsprotein synthesis and human proteome project

2. Thyroid Hormone Key to Lipid Kinase Regulation

Published: Jul 16, 2014 | Updated: Jul 17, 2014
By Salynn Boyles, Contributing Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and
Dorothy Caputo, MA, BSN, RN, Nurse Planner

Action Points

  • Thyroid hormone is an essential regulator of human growth, brain maturation, and adult cognition and metabolism.
  • This study provides evidence that cytoplasmic thyroid hormone signaling through phosphatidylinositol 3-kinase appears to be an essential mechanism underlying normal synaptic maturation and plasticity in the postnatal mouse hippocampus

Thyroid hormones are key for brain development and synaptic maturation, and researchers have identified a specific molecular mechanism for rapid lipid kinase activation by the thyroid hormone receptor beta (TR-beta) that involves a cytoplasmic complex of the gene.

Many effects of the thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation and its relevance to brain development have not been clear, according to David L. Armstrong, PhD, of the National Institute of Environmental Health and Development in Research Triangle, N.C., and colleagues.

They identified a specific molecular mechanism for rapid PI3 kinase activation by TR-beta which involves a cytoplasmic complex of TR-beta, the p85 regulatory subunit of PI3 kinase and the Src family kinase, Lyn, they wrote in Endocrinology.Armstrong’s co-authors are from Duke University and Loyola University in Chicago.

This complex provides a unique mechanism for integrating growth signals through thyroid hormone and receptor tyrosine kinases, they explained.

“Most everyone agrees that thyroid hormones are essential for brain development and synaptic maturation, but we didn’t know how exactly,” Armstrong told MedPage Today. “We show that nongenomic signaling in TR-beta through PI3 kinase is essential for one of its physiological actions.”

The Role of T3 Hormone

The recognition that many hormones regulate gene expression through receptor proteins that bind to DNA is a major biological discovery over the past 50 years, the researchers noted.

“More recently, it has become clear that in many cases the same hormones produce rapid effects on cell physiology though the same receptors signaling in the cytoplasm,” they wrote. “However, testing the relative importance of the genomic and nongenomic mechanisms in vivo has been prevented by the absence of specific molecular mechanisms for the nongenomic effects that could be blocked by mutation of the receptor without disrupting its direct effects on gene expression.”

The thyroid hormone T3 has been shown to be a regulator of many physiological effects, including human growth, brain maturation, and adult cognition and metabolism.

Many of these effects have been found to be mediated through the regulation of gene expression by zinc-finger nuclear receptor proteins that are encoded by the THRA and THRB genes. But many in vitro effects of T3 are too rapid to be explained by transcriptional regulation, Armstrong and colleagues noted.

In earlier work, they identified PI3 kinase as a key player in these rapid effects. Like thyroid hormone, PI3 kinase activity has been identified as essential for growth, metabolism, and brain development.

PI3 kinase is regulated primarily by receptor tyrosine kinases, and an integrin receptor has been identified that mediates some of the PI3 kinase-dependent effects of thyroxine (T4), the widely circulated precursor of T3.

Both TR-alpha and TR-beta have also been reported to associate with PI3 kinase and stimulate its activity in many cell types. In a 2006 study in the Proceedings of the National Academy of Sciences, Armstrong and colleagues demonstrated that TRis required to reconstitute T3 and PI3 kinase-dependent regulation of Kv11.1 channels in cell-free membrane patches from Chinese hamster ovary (CHO) cells.

Based on that research, they concluded that TR-beta signaling through PI3K “provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.”

Measuring PIP3 Production

In the newly reported series of experiments, the researchers used fluorescent PIP3 indicator to directly measure PIP3 production in response to thyroid hormone on the same time scale as the electrophysiological measurements in the CHO cells expressing recombinant human thyroid hormone receptors.

The research revealed that, in the absence of hormone, the nuclear receptor TR-beta forms a cytoplasmic complex with the p85 subunit of PI3 kinase and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TR that are not conserved in  TR-beta

“When hormone is added, [TR-beta] dissociates and moves to the nucleus, and PIP3production goes up rapidly,” the researchers wrote. “Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3 kinase but does not prevent hormone-dependent transcription of genes with a thyroid hormone response element.”

“It is only when you have both thyroid hormone and phosphotyrosine signaling that you get maximal stimulation of PI3 kinase,” Armstrong said, adding that the novel methodology of the study, which involved serum from thyroidectomized animals, led to the finding.

These experiments led to in vivo research to test the physiological relevance of thyroid hormone signaling through PI3 kinase for brain development in a novel mouse line created by the researchers.

“We reasoned that blocking binding of TR-beta to p85 by mutating Y171 might eliminate any dominant negative effect of the mutant, in much the same way that receptor knockdown proved much less deleterious to the organism than hormone withdrawal, presumably because many of the effects of the receptor on gene expression are mediated by binding of the unliganded receptor,” they wrote.

They created a novel mouse line with a targeted mutation knocked into the THRB gene to substitute phenylalanine for tyrosine at residue 147 of TR-beta-1, which prevents Lyn binding to the mutant receptor.

They confirmed that the mutation did not alter total circulating levels of thyroxine (T4) or T3 by mass spectrometry of serum samples from 4-month-old mice.

“When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of THRB, circulating hormone levels, TR-betaexpression, and direct gene regulation by TR-beta in the brain and liver were all unaffected,” the researchers wrote. “The mutation did significantly impair maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TR-betawith PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases.”

A Novel Finding

The finding that thyroid hormone signaling through PI3 kinase appears to be an essential mechanism underlying normal synaptic maturation and plasticity in the postnatal mouse hippocampus is novel.

The researchers noted that they could not formally exclude some more subtle effects of the mutation on the regulation of an unknown gene that plays as central a role in synaptic development as PI3K, but the added that “our results do categorically rule out a role for other thyroid hormone receptors in this particular aspect of synaptic maturation in the mouse hippocampus.

“In either case, given the importance of thyroid hormone signaling for human brain development and adult metabolism, future studies will need to investigate whether PI3 kinase stimulation by thyroid hormone is also susceptible to disruption by environmental toxicants,” they wrote.

Armstrong also pointed out that the tyrosine motifs in TR-beta, which were shown to be essential for signaling through PI3 kinase, are present in all mammals, but not in other species with known genome data, with the exception of the gecko and the axolotl (Mexican salamander).

“Mammals evolved from reptiles, and the thinking is that they survived by adopting a nocturnal niche,” he said. “This is exactly what thyroid hormone does, so it may be that this mutation contributed to the (evolutionary) success of mammals.”

Primary source: Endocrinology
Source reference: Martin NP, et al “A rapid cytoplasmic mechanism for PI3 kinase regulation by the nuclear thyroid hormone receptor, TR beta, and genetic evidence for its role in the maturation of mouse hippocampal synapses in vivo”

Endocrinology 2014;         http://dx.doi.org:/10.1210/en.2013-2058.


3.  Mammalian Target of Rapamycin Complex 1 Orchestrates Invariant NKT Cell Differentiation and Effector Function.

Lianjun ZhangBenjamin O TschumiStéphanie CorgnacMarkus A Rüegg,Michael N HallJean-Pierre MachPedro RomeroAlena Donda

Journal of immunology (Baltimore, Md. : 1950) 07/2014;     http://dx.doi.org:/10.4049/jimmunol.1400769

Source: PubMed

ABSTRACT Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.


4.  PARC

The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells

Vivian Gama1,2, Vijay Swahari1,2, Johanna Schafer1*, Adam J. Kole2, Allyson Evans2, Yolanda Huang2, Anna Cliffe1,2, Brian Golitz3,4, Noah Sciaky3,4, Xin-Hai Pei5,6, Yue Xiong5,6, and Mohanish Deshmukh1,2,5

1 Neuroscience Center, 2 Department of Cell Biology and Physiology, 3 UNC RNAi Screening Facility,4 Department of Pharmacology, 5 Lineberger Comprehensive Cancer Center, 6 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.* Present address: Vanderbilt University, Nashville, TN 37232, USA.  Present address: Cell Press, Cambridge, MA 02139, USA.  Present address: Department of Anesthesiology, Columbia University Medical Center, New York, NY 10032, USA.

Abstract: The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.
Sci. Signal., 15 July 2014   Vol. 7, Issue 334, p. ra67

Citation: V. Gama, V. Swahari, J. Schafer, A. J. Kole, A. Evans, Y. Huang, A. Cliffe, B. Golitz, N. Sciaky, X.-H. Pei, Y. Xiong, M. Deshmukh, The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells. Sci. Signal. 7, ra67 (2014).

Killing the Killer: PARC/CUL9 Promotes Cell Survival by Destroying Cytochrome c

Jonathan Lopez and Stephen W. G. Tait*
Cancer Research UK Beatson Institute, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.

Abstract: Balanced amounts of apoptotic cell death are essential for health; its deregulation plays key roles in neurodegeneration, autoimmunity, and cancer. Mitochondria orchestrate apoptosis through a process called mitochondrial outer-membrane permeabilization (MOMP). After MOMP, mitochondrial cytochrome c is released into the cytoplasm, where it binds the adaptor molecule APAF1, triggering caspase protease activation and cell death. In this issue of Science Signaling, Deshmukh and colleagues define a new survival mechanism downstream of mitochondrial permeabilization. Specifically, they identify proteasomal degradation of cytochrome c as a major determinant of cell survival. In an unbiased approach, PARC (also known as CUL9) was found to be the ubiquitin ligase responsible for the ubiquitination and proteasomal degradation of cytochrome c. The consequences of this survival process may be double-edged because both cancer cells and postmitotic cells use PARC/CUL9–mediated cytochrome c degradation to ensure cell survival. Ultimately, differential targeting of this process may promote survival of postmitotic tissue or enhance tumor-specific killing.

Citation: J. Lopez, S. W. G. Tait, Killing the Killer: PARC/CUL9 Promotes Cell Survival by Destroying Cytochrome c. Sci. Signal. 7, pe17 (2014).

Sci. Signal., 15 July 2014  Vol. 7, Issue 334, p. pe17


4. The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

Dario R. Alessi1, Jinwei Zhang1, Arjun Khanna2, Thomas Hochdörfer1, Yuze Shang3, and Kristopher T. Kahle2,3*
1 MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
2 Department of Neurosurgery, Massachusetts General Hospital, and Harvard Medical School, 3 Manton Center for Orphan Disease Research, Boston Children’s Hospital, Boston, MA 02115, USA.

Abstract: The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress–responsive kinase 1). The WNK family senses changes in intracellular Cl concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na+), potassium (K+), and chloride (Cl) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl-importing, Na+-driven CCCs or inhibit the Cl-extruding, K+-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K+ secretion to lower blood pressure while maintaining serum K+. In neurons, WNK-SPAK/OSR1 inhibition could facilitate Clextrusion and promote -aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K+-sparing blood pressure–lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.
Citation: D. R. Alessi, J. Zhang, A. Khanna, T. Hochdörfer, Y. Shang, K. T. Kahle, The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters. Sci. Signal. 7, re3 (2014).

Sci. Signal., 15 July 2014  Vol. 7, Issue 334, p. re3


5. Nf k-beta signaling pathway

Cracking the NF-B Code

Karen E. Tkach, Jennifer E. Oyler, and Grégoire Altan-Bonnet*
ImmunoDynamics Group, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Abstract: The discovery of feedback loops between signaling and gene expression is ushering in new quantitative models of cellular regulation. In a recent issue of Science Signaling, Sung et al. showed how positive feedback downstream of nuclear factor B (NF-B) signaling enhances the capacity of macrophages to scale their antimicrobial responses to the dose of pathogen-associated molecular cues. This finding stemmed from analysis of cell-to-cell variability and computational modeling of time integration between signaling and transcriptional responses. Ultimately, such quantitative approaches challenge the oft-assumed time separation of “fast” signal transduction followed by “slow” gene expression, and they provide a better understanding of complex biological regulation over long time scales.

Citation: K. E. Tkach, J. E. Oyler, G. Altan-Bonnet, Cracking the NF-B Code. Sci. Signal. 7, pe5 (2014).

Sci. Signal., 18 February 2014  Vol. 7, Issue 313, p. pe5


Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced Nfk-B Signaling

Myong-Hee Sung1*, Ning Li2, Qizong Lao1, Rachel A. Gottschalk2, Gordon L. Hager1*, and Iain D. C. Fraser2*
1 Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, 2 Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract: A fundamental goal in biology is to gain a quantitative understanding of how appropriate cell responses are achieved amid conflicting signals that work in parallel. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor B (NF-B) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela, which encodes the RelA (p65) NF-B subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-B signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We suggest that this switching in the relative dominance of feedback loops (“feedback dominance switching”) may be a general mechanism in immune cells to integrate opposing feedback on a key transcriptional regulator and to set a response threshold for the host.

Citation: M.-H. Sung, N. Li, Q. Lao, R. A. Gottschalk, G. L. Hager, I. D. C. Fraser, Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced NF-B Signaling. Sci. Signal. 7, ra6 (2014).

Sci. Signal., 14 January 2014  Vol. 7, Issue 308, p. ra6

Drug development in the Alzheimer’s field has been riddled with failures, and most research efforts have focused on pinpointing genetic and environmental factors responsible for causing or accelerating the progression of the disease.

Now, researchers from Montreal’s Douglas Mental Health Institute and McGill University have identified a relatively frequent genetic variant that may provide protection against the devastating neurodegenerative disease.

“We found that specific genetic variants in a gene called HMG CoA reductase which normally regulates cholesterol production and mobilization in the brain can interfere with, and delay the onset of Alzheimer’s disease by nearly four years. This is an exciting breakthrough in a field where successes have been scarce these past few years,” said Dr. Judes Poirier, whose previous research led to the discovery that a genetic variant was formally associated with the common form of Alzheimer’s disease.

This variant may explain why some people who are carriers of predisposing genetic factors for the common form of Alzheimer’s do not develop the disease, living long lives without memory problems until their nineties.


6.  P181 cAMP-mediated Rac1 activation regulates the re-establishment of endothelial adherens junctions and barrier restoration during inflammation.

M AslamH NefC TroidlR SchulzT NollC HammD Guenduez

Cardiovascular research 07/2014; 103(suppl 1):S32.
Source: PubMed

ABSTRACT Inflammatory mediators like thrombin and TNFα disrupt endothelial junctions and barrier integrity, leading to edema formation. This increase in endothelial permeability is followed by slow restoration of the endothelial barrier, which is critical for the maintenance of basal endothelial permeability. However, the molecular mechanism of recovery of the endothelial barrier in response to inflammatory mediators has not yet been well delineated. The aim of the present study was to explore the mechanism of this barrier restoration. Specific emphasis was given to the role of Rac1 GTPase activation, which is an important regulator of endothelial adherens junction (AJ) integrity.


7.  Thalidomide

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Eric S. Fischer, Kerstin Böhm, John R. Lydeard, Haidi Yang, Michael B. Stadler, et al.
Nature (2014)     http://dx.doi.org:/10.1038/nature13527

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Figure 1: The overall structure of the DDB1–CRBN complex.


a, Cartoon representation of the structure of the complex of human DDB1, G. gallus CRBN and thalidomide: DDB1, highlighting the domains BPA (red), BPB (magenta), BPC (orange) and DDB1-CTD (grey); G. gallus CRBN, highlighting the domain…

Figure 2: IMiD binding to CRBN.


a, Chemical structure of lenalidomide. b, Chemical structure of pomalidomide. c, Sketch of thalidomide and its interactions with G. gallus CRBN. Hydrogen bonds are shown as dashed lines, and hydrophobic interactions are indicated as gr

Figure 3: CRBN is a substrate receptor in the ligase CRL4CRBN.


a, Architecture of the CRL4DDB2 complex bound to DNA (PDB ID 4A0K). b, Model of CRL4CRBN bound to thalidomide. c, Firefly luciferase (Fluc) to Renillaluciferase (Rluc) ratios (Fluc:Rluc) of IKZF1-reporter-plasmid-transfected HEK 293T…


Figure 5: Molecular model of IMiD function.


a, Thalidomide binds to CRBN at the canonical substrate-binding site. b, The potent anti-myeloma drug thalidomide and its derivatives lenalidomide and pomalidomide occupy the same site but with different solvent-exposed moieties. c, Bi…


8. Preeclampsia of pregnancyand protein misfolding

Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia

Irina A. Buhimschi1,2,*Unzila A. Nayeri2Guomao Zhao1Lydia L. Shook2Anna Pensalfini3, et al.
1Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, 4Depart of ObGyn, The Ohio State University College of Medicine, Columbus, OH
2Depart of ObGyn and Reproductive Sciences, Yale University School of Medicine, New Haven, CT

3Center for Dementia Research, Nathan Kline Institute for Psychiatric Research and Department of Psychiatry, New York University School of Medicine, New York, NY
5Depart of ObGyn and Reproductive Sciences, University of Vermont College of Medicine, Burlington, VT .
6Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92617, USA.
7Department of Biochemistry and Experimental Biochemistry Unit, King Abdulaziz Univ, Jeddah , Saudi Arabia.

Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state–dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state–dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer’s β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.

Citation: I. A. Buhimschi, U. A. Nayeri, G. Zhao, L. L. Shook, A. Pensalfini, E. F. Funai, I. M. Bernstein, C. G. Glabe, C. S. Buhimschi,Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia. Sci. Transl. Med. 6, 245ra92 (2014).


9. Blood Clotting

Removing parts of shape-shifting protein explains how blood clots

prothrombin (FII)

prothrombin (FII)




Using x-ray crystallography, SLU researchers published the first image of the important blood-clotting protein prothrombin (coagulation factor II). The protein’s flexible structure is key to the development of blood-clotting.In results recently published in Proceedings of the National Academy of Sciences (PNAS), Saint Louis University scientists have discovered that removal of disordered sections of a protein’s structure reveals the molecular mechanism of a key reaction that initiates blood clotting.

Enrico Di Cera, M.D., chair of the Edward A. Doisy department of biochemistry and molecular biology at Saint Louis University, studies thrombin, a key vitamin K-dependent blood-clotting protein, and its inactive precursor prothrombin (or coagulation factor II).

“Prothrombin is essential for life and is the most important clotting factor,” Di Cera said. “We are proud to report that our lab here at SLU has finally succeeded in crystallizing prothrombin for the first time.”

Blood-clotting has long ensured our survival, stopping blood loss after an injury. However, when triggered in the wrong circumstances, clotting can lead to debilitating or fatal conditions such as a heart attack, stroke or deep vein thrombosis.

Before thrombin becomes active, it circulates throughout the blood in the inactive (zymogen) form called prothrombin. When the active enzyme is needed (after a vascular injury, for example), the coagulation cascade is initiated and prothrombin is converted into the active enzyme thrombin that causes blood to clot.

X-ray crystallography is one tool in scientists’ toolbox for understanding processes at the molecular level. It offers a way to obtain a “snap shot” of a protein’s structure.

In this technique, scientists grow crystals of the protein they want to study, shoot x-rays at them and record data about the way the rays are scattered by crystals. Then they use computer programs to create an image of the protein based on that data.

Once scientists can visualize the three dimensional structure of a molecule, they can begin to piece together the way in which the protein functions and interacts with other molecules in the body, or with drugs.

Last year, Di Cera and colleagues published the first structure of prothrombin. This first structure lacked a domain responsible for interaction with membranes and certain other sections were not detected by x-ray analysis. Though the scientists were able to crystallize the protein, there were disordered regions in the structure that they could not see.

Within prothrombin there are two kringle domains (looped sections of a protein named after the Scandinavian pastry) connected by a “linker” region that intrigued the SLU investigators because of its intrinsic disorder.

“We deleted this linker and crystals grew in a few days instead of months, revealing for the first time the full architecture of prothrombin,” Di Cera said.

In addition to this remarkable discovery, Di Cera and colleagues found that the deleted version of prothrombin is activated to thrombin much faster than the intact prothrombin. The structure without the disordered linker is in fact optimized for conversion to thrombin and reveals key information on the mechanism of prothrombin activation.

For over four decades, scientists have tried to crystallize prothrombin but without success.

“It took us almost two years to discover that the disordered linker was the key,” Di Cera said.  “Finally, prothrombin revealed its secrets and with that the molecular mechanism of a key reaction of blood clotting finally becomes amenable to rational drug design for therapeutic intervention.”

SLU researchers Nicola Pozzi, Ph.D., Zhiwei Chen, Leslie Pelc and Daniel Shropshire also are authors on the paper.

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