Posts Tagged ‘Posttranslational modification’

Introduction to Protein Synthesis and Degradation

Curator: Larry H. Bernstein, MD, FCAP



Introduction to Protein Synthesis and Degradation

This chapter I made to follow signaling, rather than to precede it. I had already written much of the content before reorganizing the contents. The previous chapters on carbohydrate and on lipid metabolism have already provided much material on proteins and protein function, which was persuasive of the need to introduce signaling, which entails a substantial introduction to conformational changes in proteins that direct the trafficking of metabolic pathways, but more subtly uncovers an important role for microRNAs, not divorced from transcription, but involved in a non-transcriptional role.  This is where the classic model of molecular biology lacked any integration with emerging metabolic concepts concerning regulation. Consequently, the science was bereft of understanding the ties between the multiple convergence of transcripts, the selective inhibition of transcriptions, and the relative balance of aerobic and anaerobic metabolism, the weight of the pentose phosphate shunt, and the utilization of available energy source for synthetic and catabolic adaptive responses.

The first subchapter serves to introduce the importance of transcription in translational science.  The several subtitles that follow are intended to lay out the scope of the transcriptional activity, and also to direct attention toward the huge role of proteomics in the cell construct.  As we have already seen, proteins engage with carbohydrates and with lipids in important structural and signaling processes.  They are integrasl to the composition of the cytoskeleton, and also to the extracellular matrix.  Many proteins are actually enzymes, carrying out the transformation of some substrate, a derivative of the food we ingest.  They have a catalytic site, and they function with a cofactor – either a multivalent metal or a nucleotide.

The amino acids that go into protein synthesis include “indispensable” nutrients that are not made for use, but must be derived from animal protein, although the need is partially satisfied by plant sources. The essential amino acids are classified into well established groups. There are 20 amino acids commonly found in proteins.  They are classified into the following groups based on the chemical and/or structural properties of their side chains :

  1. Aliphatic Amino Acids
  2. Cyclic Amino Acid
  3. AAs with Hydroxyl or Sulfur-containing side chains
  4. Aromatic Amino Acids
  5. Basic Amino Acids
  6. Acidic Amino Acids and their Amides

Examples include:

Alanine                  aliphatic hydrophobic neutral
Arginine                 polar hydrophilic charged (+)
Cysteine                polar hydrophobic neutral
Glutamine             polar hydrophilic neutral
Histidine                aromatic polar hydrophilic charged (+)
Lysine                   polar hydrophilic charged (+)
Methionine            hydrophobic neutral
Serine                   polar hydrophilic neutral
Tyrosine                aromatic polar hydrophobic

Transcribe and Translate a Gene

  1. For each RNA base there is a corresponding DNA base
  2. Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein.
  3. mRNA is produced in the nucleus, and is transferred to the ribosome
  4. mRNA uses uracil instead of thymine
  5. the ribosome reads the RNA sequence and makes protein
  6. There is a sequence combination to fit each amino acid to a three letter RNA code
  7. The ribosome starts at AUG (start), and it reads each codon three letters at a time
  8. Stop codons are UAA, UAG and UGA


protein synthesis

protein synthesis






What about the purine inosine?

Inosine triphosphate pyrophosphatase – Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2′-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5′-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions.

Gastroenterology. 2011 Apr;140(4):1314-21. Epub 2011 Jan 1.

Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function.

Hitomi Y1, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB.

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown.

Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR).

ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

Ther Drug Monit. 2012 Aug;34(4):477-80.

Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.

Citterio-Quentin A1, Salvi JP, Boulieu R.

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.

The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.

The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.


System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3

PeerJ 2:e270;

Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher.We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression.We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 D 0.13). Based on this, our second strategy suggests that mRNA levels explain 81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimat that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the ab-sence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the 40% of genes that are non-expressed.


Related studies that reveal issues that are not part of this chapter:

  1. Ubiquitylation in relationship to tissue remodeling
  2. Post-translational modification of proteins
    1. Glycosylation
    2. Phosphorylation
    3. Methylation
    4. Nitrosylation
    5. Sulfation – sulfotransferases
      cell-matrix communication
    6. Acetylation and histone deacetylation (HDAC)
      Connecting Protein Phosphatase to 1α (PP1α)
      Acetylation complexes (such as CBP/p300 and PCAF)
      Rel/NF-kB Signal Transduction
      Homologous Recombination Pathway of Double-Strand DNA Repair
    7. Glycination
    8. cyclin dependent kinases (CDKs)
    9. lyase
    10. transferase


This year, the Lasker award for basic medical research went to Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) for their “discoveries concerning the unfolded protein response (UPR) — an intracellular quality control system that

detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.”

About UPR: Approximately a third of cellular proteins pass through the Endoplasmic Reticulum (ER) which performs stringent quality control of these proteins. All proteins need to assume the proper 3-dimensional shape in order to function properly in the harsh cellular environment. Related to this is the fact that cells are under constant stress and have to make rapid, real time decisions about survival or death.

A major indicator of stress is the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), which triggers a transcriptional cascade in order to increase the folding capacity of the ER. If the metabolic burden is too great and homeostasis cannot be achieved, the response shifts from

damage control to the induction of pro-apoptotic pathways that would ultimately cause cell death.

This response to unfolded proteins or the UPR is conserved among all eukaryotes, and dysfunction in this pathway underlies many human diseases, including Alzheimer’s, Parkinson’s, Diabetes and Cancer.


The discovery of a new class of human proteins with previously unidentified activities

In a landmark study conducted by scientists at the Scripps Research Institute, The Hong Kong University of Science and Technology, aTyr Pharma and their collaborators, a new class of human proteins has been discovered. These proteins [nearly 250], called Physiocrines belong to the aminoacyl tRNA synthetase gene family and carry out novel, diverse and distinct biological functions.

The aminoacyl tRNA synthetase gene family codes for a group of 20 ubiquitous enzymes almost all of which are part of the protein synthesis machinery. Using recombinant protein purification, deep sequencing technique, mass spectroscopy and cell based assays, the team made this discovery. The finding is significant, also because it highlights the alternate use of a gene family whose protein product normally performs catalytic activities for non-catalytic regulation of basic and complex physiological processes spanning metabolism, vascularization, stem cell biology and immunology


Muscle maintenance and regeneration – key player identified

Muscle tissue suffers from atrophy with age and its regenerative capacity also declines over time. Most molecules discovered thus far to boost tissue regeneration are also implicated in cancers.  During a quest to find safer alternatives that can regenerate tissue, scientists reported that the hormone Oxytocin is required for proper muscle tissue regeneration and homeostasis and that its levels decline with age.

Oxytocin could be an alternative to hormone replacement therapy as a way to combat aging and other organ related degeneration.

Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration (June 2014)


Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14289-94. Epub 2014 Sep 15.

Role of forkhead box protein A3 in age-associated metabolic decline.

Ma X1, Xu L1, Gavrilova O2, Mueller E3.

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter.


Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

RJ Weatheritt, TJ Gibson & MM Babu
Nature Structural & Molecular Biology 24 Aug, 2014; 21: 833–839

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.


An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.


An overview of the potential advantages conferred by distal-site protein synthesis

An overview of the potential advantages conferred by distal-site protein synthesis


Turquoise and red filled circle represents off-target and correct interaction partners, respectively. Wavy lines represent a disordered region within a distal site synthesis protein. Grey and red line in graphs represents profiles of t…


Tweaking transcriptional programming for high quality recombinant protein production

Since overexpression of recombinant proteins in E. coli often leads to the formation of inclusion bodies, producing properly folded, soluble proteins is undoubtedly the most important end goal in a protein expression campaign. Various approaches have been devised to bypass the insolubility issues during E. coli expression and in a recent report a group of researchers discuss reprogramming the E. coli proteostasis [protein homeostasis] network to achieve high yields of soluble, functional protein. The premise of their studies is that the basal E. coli proteostasis network is insufficient, and often unable, to fold overexpressed proteins, thus clogging the folding machinery.

By overexpressing a mutant, negative-feedback deficient heat shock transcription factor [σ32 I54N] before and during overexpression of the protein of interest, reprogramming can be achieved, resulting in high yields of soluble and functional recombinant target protein. The authors explain that this method is better than simply co-expressing/over-expressing chaperones, co-chaperones, foldases or other components of the proteostasis network because reprogramming readies the folding machinery and up regulates the essential folding components beforehand thus  maintaining system capability of the folding machinery.

The Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli (July 2014)


 Unfolded proteins collapse when exposed to heat and crowded environments

Proteins are important molecules in our body and they fulfil a broad range of functions. For instance as enzymes they help to release energy from food and as muscle proteins they assist with motion. As antibodies they are involved in immune defence and as hormone receptors in signal transduction in cells. Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions. Surprisingly, it has been shown that many important proteins occur as unfolded coils. Researchers seek to establish how these disordered proteins are capable at all of assuming highly complex functions.

Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect.

Measurements using the “molecular ruler”

“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.

With computer simulations the researchers have mimicked the behaviour of disordered proteins.
(Courtesy of Jose EDS Roselino, PhD.


MLKL compromises plasma membrane integrity

Necroptosis is implicated in many diseases and understanding this process is essential in the search for new therapies. While mixed lineage kinase domain-like (MLKL) protein has been known to be a critical component of necroptosis induction, how MLKL transduces the death signal was not clear. In a recent finding, scientists demonstrated that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death.

They also found a patch of positively charged amino acids on the surface of the 4HBD that bound to phosphatidylinositol phosphates (PIPs) and allowed the recruitment of MLKL to the plasma membrane that resulted in the formation of pores consisting of MLKL proteins, due to which cells absorbed excess water causing them to explode. Detailed knowledge about how MLKL proteins create pores offers possibilities for the development of new therapeutic interventions for tolerating or preventing cell death.

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates (May 2014)


Mitochondrial and ER proteins implicated in dementia

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations that facilitate a number of cellular functions. However, the molecular mechanisms of these interactions aren’t properly understood.

A group of researchers showed that the ER protein VAPB interacted with mitochondrial protein PTPIP51 to regulate ER-mitochondria associations and that TDP-43, a protein implicated in dementia, disturbs this interaction to regulate cellular Ca2+ homeostasis. These studies point to a new pathogenic mechanism for TDP-43 and may also provide a potential new target for the development of new treatments for devastating neurological conditions like dementia.

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nature (June 2014)


A novel strategy to improve membrane protein expression in Yeast

Membrane proteins play indispensable roles in the physiology of an organism. However, recombinant production of membrane proteins is one of the biggest hurdles facing protein biochemists today. A group of scientists in Belgium showed that,

by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis,

enhanced expression of recombinant membrane proteins in yeast is achieved.

Specifically, they engineered the oleotrophic yeast, Yarrowia lipolytica, by

deleting the phosphatidic acid phosphatase, PAH1 gene,

which led to massive proliferation of endoplasmic reticulum (ER) membranes.

For all 8 tested representatives of different integral membrane protein families, they obtained enhanced protein accumulation.


An unconventional method to boost recombinant protein levels

MazF is an mRNA interferase enzyme in E.coli that functions as and degrades cellular mRNA in a targeted fashion, at the “ACA” sequence. This degradation of cellular mRNA causes a precipitous drop in cellular protein synthesis. A group of scientists at the Robert Wood Johnson Medical School in New Jersey, exploited the degeneracy of the genetic code to modify all “ACA” triplets within their gene of interest in a way that the corresponding amino acid (Threonine) remained unchanged. Consequently, induction of MazF toxin caused degradation of E.coli cellular mRNA but the recombinant gene transcription and protein synthesis continued, causing significant accumulation of high quality target protein. This expression system enables unparalleled signal to noise ratios that could dramatically simplify structural and functional studies of difficult-to-purify, biologically important proteins.


Tandem fusions and bacterial strain evolution for enhanced functional membrane protein production

Membrane protein production remains a significant challenge in its characterization and structure determination. Despite the fact that there are a variety of host cell types, E.coli remains the popular choice for producing recombinant membrane proteins. A group of scientists in Netherlands devised a robust strategy to increase the probability of functional membrane protein overexpression in E.coli.

By fusing Green Fluorescent Protein (GFP) and the Erythromycin Resistance protein (ErmC) to the C-terminus of a target membrane protein they wer e able to track the folding state of their target protein while using Erythromycin to select for increased expression. By increasing erythromycin concentration in the growth media and testing different membrane targets, they were able to identify four evolved E.coli strains, all of which carried a mutation in the hns gene, whose product is implicated in genome organization and transcriptional silencing. Through their experiments the group showed that partial removal of the transcriptional silencing mechanism was related to production of proteins that were essential for functional overexpression of membrane proteins.


The role of an anti-apoptotic factor in recombinant protein production

In a recent study, scientists at the Johns Hopkins University and Frederick National Laboratory for Cancer Research examined an alternative method of utilizing the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, specifically, through the co-transfection of Bcl-xL along with the product-coding target gene.

Chinese Hamster Ovary(CHO) cells were co-transfected with the product-coding gene and a vector containing Bcl-xL, using Polyethylenimine (PEI) reagent. They found that the cells co-transfected with Bcl-xL demonstrated reduced apoptosis, increased specific productivity, and an overall increase in product yield.

B-cell lymphoma-extra-large (Bcl-xL) is a mitochondrial transmembrane protein and a member of the Bcl-2 family of proteins which are known to act as either pro- or anti-apoptotic proteins. Bcl-xL itself acts as an anti-apoptotic molecule by preventing the release of mitochondrial contents such as cytochrome c, which would lead to caspase activation. Higher levels of Bcl-xL push a cell toward survival mode by making the membranes pores less permeable and leaky.




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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

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified

Mitochondrial Damage and Repair under Oxidative Stress

Bzzz! Are fruitflies like us?

Discovery of Imigliptin, a Novel Selective DPP-4 Inhibitor for the Treatment of Type 2 Diabetes

Molecular biology mystery unravelled

Gene Switch Takes Blood Cells to Leukemia and Back Again

Wound-healing role for microRNAs in colon offer new insight to inflammatory bowel diseases

Targeting a key driver of cancer

Tang Prize for 2014: Immunity and Cancer

Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad                             Demet Sag, PhD

3:45 – 4:15, 2014, Scott Lowe “Tumor suppressor and tumor maintenance genes”

12:00 – 12:30, 6/13/2014, John Maraganore “Progress in advancement of RNAi therapeutics”

9:30 – 10:00, 6/13/2014, David Bartel “MicroRNAs, poly(A) tails and post-transcriptional gene regulation.”

10:00 – 10:30, 6/13/2014, Joshua Mendell “Novel microRNA functions in mammalian physiology and cancer”

Aviva Lev-Ari, PhD, RN

Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases          Aviva Lev-Ari, PhD, RN

Illana Gozes discovered Novel Protein Fragments that have proven Protective Properties for Cognitive Functioning

Aviva Lev-Ari, PhD, RN







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CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way – Part IIA

Curator: Larry H Bernstein, MD, FCAP

Introduction and purpose

This material goes beyond the Initiation Phase of Molecular Biology, Part I.
Part II reviews the Human Genome Project and the decade beyond.

In a three part series:
Part IIA.  CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way
Part IIB.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
Part IIC.  CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease

Part III will conclude with Ubiquitin, it’s Role in Signaling and Regulatory Control.
Part I reviewed the huge expansion of the biological research enterprise after the Second World War. It concentrated on the

  • discovery of cellular structures,
  • metabolic function, and
  • creation of a new science of Molecular Biology.

Part II follows the race to delineation of the Human Genome, discovery methods and fundamental genomic patterns that are ancient in both animal and plant speciation. But it explores both the complexity and the systems view of the architecture that underlies and understanding of the genome.

These articles review a web-like connectivity between inter-connected scientific discoveries, as significant findings have led to novel hypotheses and many expectations over the last 75 years. This largely post WWII revolution has driven our understanding of biological and medical processes at an exponential pace owing to successive discoveries of

  • chemical structure,
  • the basic building blocks of DNA  and proteins,
  • nucleotide and protein-protein interactions,
  • protein folding, allostericity,
  • genomic structure,
  • DNA replication,
  • nuclear polyribosome interaction, and
  • metabolic control.

In addition, the emergence of methods for

  • copying,
  • removal,
  • insertion,
  • improvements in structural analysis
  • developments in applied mathematics that have transformed the research framework.

Part IIA:


Milestones along the Way

A NOVA interview with Francis Collins (NHGRI) (FC), J. Craig Venter (CELERA)(JCV), and Eric Lander (EL).
RK: For the past ten years, scientists all over the world have been painstakingly trying to read the tiny instructions buried inside our DNA. And now, finally, the “Human Genome” has been decoded.
EL: The genome is a storybook that’s been edited for a couple billion years.
The following will address the odd similarity of genes between man and yeast

EL: In the nucleus of your cell the DNA molecule resides that is about 10 angstroms wide curled up, but the amount of curling is limited by the negative charges that repel one another, but there are folds upon folds. If the DNA is stretched the length of the DNA would be thousands of feet.
EL: We have known for 2000 years that your kids look a lot like you. Well it’s because you must pass them instructions that give them the eyes, the hair color, and the nose shape they have. RK: Cracking the code of those minuscule differences in DNA that influence health and illness is what the Human Genome Project is all about. Since 1990, scientists all over the world have been involved in the effort to read all three billion As, Ts, Gs, and Cs of human DNA.  It took 10 years to find the one genetic mistake that causes cystic fibrosis. Another 10 years to find the gene for Huntington’s disease. Fifteen years to find one of the genes that increase the risk for breast cancer. One letter at a time, painfully slowly…     And then came the revolution. In the last ten years the entire process has been computerized. The computations can do a thousand every second and that has made all the difference. EL: This is basically a parts list with a lot of parts. If you take an airplane, a Boeing 777, I think it has like 100,000 parts. If I gave you a parts list for the Boeing 777 in one sense you’d know 100,000 components, screws and wires and rudders and things like that.  But you wouldn’t know how to put it together, or why it flies. We now have a parts list, and that’s not enough to understand why it flies.

The Human Genome

The Human Genome (Photo credit: dullhunk)

A Quest For Clarity

Tracy Vence is a senior editor of Genome Technology
Tracy Vence @GenomeTechMag
Projects supported by the US National Institutes of Health will have produced 68,000 total human genomes — around 18,000 of those whole human genomes — through the end of this year, National Human Genome Research Institute estimates indicate. And in his book, The Creative Destruction of Medicine, the Scripps Research Institute’s Eric Topol projects that 1 million human genomes will have been sequenced by 2013 and 5 million by 2014.
Daniel MacArthur, a group leader in Massachusetts General Hospital’s Analytic and Translational Genetics Unit estimates that “From a capacity perspective … millions of genomes are not that far off. If you look at the rate that we’re scaling, we can certainly achieve that.”    The prospect of so many genomes has brought clinical interpretation into focus. But there is an important distinction to be made between the interpretation of an apparently healthy person’s genome and that of an individual who is already affected by a disease.
In an April Science Translational Medicine paper, Johns Hopkins University School of Medicine‘s Nicholas Roberts and his colleagues reported that personal genome sequences for healthy monozygotic twin pairs are not predictive of significant risk for 24 different diseases in those individuals. The researchers concluded that whole-genome sequencing was not likely to be clinically useful. Ambiguities have clouded even the most targeted interpretation efforts.

  • Technological challenges,
  • meager sample sizes,
  • a need for increased,
  • fail-safe automation and most important
  • a lack of community-wide standards for the task.

have hampered researchers’ attempts to reliably interpret the clinical significance of genomic variation.

How signals from the cell surface affect transcription of genes in the nucleus.

James Darnell, Jr., MD, Astor Professor, Rockefeller
After graduation from Washington University School of Medicine he worked with Francois Jacob at the Pasteur Institute in Paris and served as Vice President for Academic Affairs at Rockefeller in 1990-91. He is the coauthor with S.E. Luria of General Virology and the founding author with Harvey Lodish and David Baltimore of Molecular Cell Biology, now in its sixth edition. His book RNA, Life’s Indispensable Molecule was published in July 2011 by Cold Spring Harbor Laboratory Press. A member of the National Academy of Sciences since 1973, recipient of  numerous awards, including the 2003 National Medal of Science, the 2002 Albert Lasker Award.
Using interferon as a model cytokine, the Darnell group discovered that cell transcription was quickly changed by binding of cytokines to the cell surface. The bound interferon led to the tyrosine phosphorylation of latent cytoplasmic proteins now called STATs (signal transducers and activators of transcription) that dimerize by

  • reciprocal phosphotyrosine-SH2 interchange.
  • accumulate in the nucleus,
  • bind DNA and drive transcription.

This pathway has proved to be of wide importance with seven STATs now known in mammals that take part in a wide variety of developmental and homeostatic events in all multicellular animals. Crystallographic analysis defined functional domains in the STATs, and current attention is focused on two areas:

  • how the STATs complete their cycle of  activation and inactivation, which requires regulated tyrosine dephosphorylation; and how
  • persistent activation of STAT3 that occurs in a high proportion of many human cancers contributes to blocking apoptosis in cancer cells.

Current efforts are devoted to inhibiting STAT3 with modified peptides that can enter cells.

Cell cycle regulation and the cellular response to genotoxic stress

Stephen J Elledge, PhD, Gregor Mendel Professor of Genetics and Medicine, Investigator, Howard Hughes Medical Institute, Harvard Medical School
As a postdoctoral fellow at Stanford working on eukaryotic homologous recombination, he serendipitously found a family of genes known as ribonucleotide reductases. He subsequently showed that

  • these genes are activated by DNA damage and
  • could serve as tools to help scientists dissect the signaling pathways
  • through which cells sense and respond to DNA damage and replication stress.

At Baylor College of Medicine he made a second major breakthrough with the discovery of the cyclin-dependent kinase 2 gene (Cdk2), which

  • controls the G1-to-S cell cycle transition,
  • an entry checkpoint for the cell proliferation cycle and
  • a critical regulatory step in tumorigenesis.

From there, using a novel “two-hybrid” cloning method he developed, Elledge and Wade Harper, PhD, proceeded to

  • isolate several members of the Cdk2-inhibitory family.

Their discoveries included the p21 and p57 genes, mutations in the latter (responsible for Beckwith-Wiedemann syndrome), characterized by somatic overgrowth and increased cancer risk. Elledge is also recognized for his work in understanding

  • proteome remodeling through ubiquitin-mediated proteolysis.
  • they identified F-box proteins that regulate protein degradation in the cell by
  1. binding to specific target protein sequences and then
  2. marking them with ubiquitin for destruction by the cell’s proteasome machinery.

This breakthrough resulted in

  • the elucidation of the cullin ubiquitin ligase family,
  • which controls regulated protein stability in eukaryotes.

nature10774-f5.2  nature10774-f3.2   ubiquitin structures  Rn1  Rn2

Elledge’s recent research has focused on the cellular mechanisms underlying DNA damage detection and cancer using genetic technologies. In collaboration with Cold Spring Harbor Laboratory researcher Gregory Hannon, PhD, Elledge has generated complete human and mouse short hairpin RNA (shRNA) libraries for genome-wide loss-of-function studies. Their efforts have led to

  • the identification of a number of tumor suppressor proteins
  • genes upon which cancer cells uniquely depend for survival.

This work led to the development of the “non-oncogene addiction” concept. This is noted as follows:

  • proteome remodeling through ubiquitin-mediated proteolysis
  • F-box proteins regulate protein degradation in the cell by binding to specific target protein sequences
  • and then marking them with ubiquitin for destruction by the cell’s proteasome machinery
  • elucidation of the cullin ubiquitin ligase family, which controls regulated protein stability in eukaryotes

Playing the dual roles of inventor and investigator, Elledge developed original techniques to define

  • what drives the cell cycle and
  • how cells respond to DNA damage.

By using these tools, he and his colleagues have identified multiple genes involved in cell-cycle regulation.

Elledge’s work has earned him many awards, including a 2001 Paul Marks Prize for Cancer Research and a 2003 election to the National Academy of Sciences. In his Inaugural Article (1), published in this issue of PNAS, Elledge and his colleagues describe the function of Fbw7, a protein involved in controlling cell proliferation (see below). Elledge studied the error-prone DNA repair mechanism in E-Coli (Escherichia coli) called SOS mutagenesis for his PhD thesis at MIT. His work identified  and described

  • the regulation of a group of enzymes now known as error-prone polymerases,
  • the first members of which were the umuCD genes in E. coli.

It was then that he developed a new cloning tool. Elledge invented a technique that allowed him to approach future cloning problems of this type with great rapidity. With the new technique, “you could make large libraries in lambda that behave like plasmids. We called them `phasmid’ vectors, like plasmid and phage together”. The phasmid cloning method was an early cornerstone for molecular biology research.

Elledge began working on homologous recombination in postdoctoral fellowship at Stanford University, an important niche in the field of eukaryotic genetics. Working with the yeast genome, Elledge searched for rec A, a gene that allows DNA to recombine homologously. Although he never located rec A, he discovered a family of genes known as ribonucleotide reductases (RNRs), which are involved in DNA production. Rec A and RNRs share the same last 4 amino acids, which caused an antibody crossreaction in one of Elledge’s experiments. Initially disappointed with the false positives in his hunt for rec A, Elledge was later delighted with his luck. He found that

  • RNRs are turned  on by DNA damage, and
  • these genes are regulated by the cell cycle.

Prior to leaving Stanford, Elledge attended a talk at the University of California, San Francisco, by Paul Nurse, a leader in cell-cycle research who would later win the 2001 Nobel Prize in medicine. Nurse described his success in isolating the homolog of a key human cell-cycle kinase gene, Cdc2, by using a mutant strain of yeast (8). Although Nurse’s methods were primitive, Elledge was struck by the message he carried: that

  • cell-cycle regulation was functionally conserved, and
  • many human genes could be isolated by looking for complimentary genes in yeast.

Elledge then took advantage of his past successes in building phasmid vectors to build a versatile human cDNA library that could be expressed in yeast. After setting up a laboratory at Baylor, he introduced this library into yeast, screening for complimentary cell-cycle genes.  He quickly identified the same Cdc2 gene isolated by Nurse. However, Elledge also discovered a related gene known as Cdk2. Elledge subsequently found that

  • Cdk2 controlled the G1 to S cell-cycle transition, a step that often goes awry in cancer. These results were published in the EMBO Journal in 1991.

He then continued to use

  • RNRs to perform genetic screens to
  • identify genes involved in sensing and responding to DNA damage.

He subsequently worked out the

  • signal transduction pathways in both yeast and humans that recognize damaged DNA and replication problems.

These “checkpoint” pathways are central to the

  • prevention of genomic instability and a key to understanding tumorigenesis.

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 2003.

Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein.

Tetzlaff MT, Yu W, Li M, Zhang P, Finegold M, Mahon K , Harper JW, Schwartz RJ, and SJ Elledge. PNAS 2004; 101(10): 3338-3345. cgi doi 10.1073.  pnas.0307875101

The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in

  • the ubiquitin-mediated turnover of cyclin E
  • as well as the Notch Lin-12 family of transcriptional activators. Both unregulated
  1. Notch and cyclin E
  2. promote tumorigenesis, and
  3. inactivate mutations in human

Fbw7 studies suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7.  Fbw7-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber mutations. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.

Science as an Adventure


Prof. Avram Hershko – Science as an Adventure
Prof. Avram Hershko shared the 2004 Nobel Prize in Chemistry with Aaron Ciechanover and Irwin Rose for “for the discovery of ubiquitin-mediated protein degradation.”

Gene Switches

Nipam Patel is a professor in the Departments of Molecular and Cell Biology and Integrative Biology at UC Berkeley and runs a research laboratory that studies the role, during embryonic development, of homeotic genes (the genetic switches described in this feature). “Ghost in Your Genes” focuses on epigenetic “switches” that turn genes “on” or “off.” But not all switches are epigenetic; some are genetic. That is, other genes within the chromosome turn genes on or off. In an animal’s embryonic stage, these gene switches play a predominant role in laying out the animal’s basic body plan and perform other early functions;

  • the epigenome begins to take over during the later stages of embryogenesis.

Beginning as a fertilized single egg that egg becomes many different kinds of cells.  Altogether, multicellular organisms like humans have thousands of differentiated cells. Each is optimized for use in the brain, the liver, the skin, and so on. Remarkably, the DNA inside all these cells is exactly the same. What makes the cells differ from one another is that different genes in that DNA are either turned on or off in each type of cell.

Take a typical cell, such as a red blood cell. Each gene within that cell has a coding region that encodes the information used to make a particular protein. (Hemoglobin shuttles oxygen to the tissues and carbon dioxide back out to the lungs—or gills, if you’re a fish.) But another region of the gene, called “regulatory DNA,” determines whether and when the gene will be expressed, or turned on, in a particular kind of cell. This precise transcribing of genes is handled by proteins known as transcription factors, which bind to the regulatory DNA, thereby generating instructions for the coding region.

One important class of transcription factors is encoded by the so called homeotic, or Hox, genes. Found in all animals, Hox genes act to “regionalize” the body along the embryo’s anterior-to-posterior (head-to-tail) axis. In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Amazingly, all animals, from fruit flies to mice to people, rely on the same basic Hox-gene complex. Using different-colored antibody stains, we can see exactly where and to what degree Hox genes are expressed. Each Hox gene is expressed in a specific region along the anterior-to-posterior axis of the embryo.

A fly’s body has three main divisions: head, thorax, and abdomen. We’ll focus on the thorax, which itself has three main segments. In a normal adult fly, the second thoracic segment features a pair of wings, while the third thoracic segment has a pair of small, balloon-shaped structures called halteres. A modified second wing, the haltere serves as a flight stabilizer. In order for the pair of wings and the pair of halteres (as well as all other parts of the fly) to develop properly, the fly’s suite of

  • Hox genes must be expressed in a precise way and at precise times.

During development, the fly’s two wings grow from a structure in the larva known as the wing imaginal disk. (An imago is an insect in its final, adult state.) The haltere grows from the larval haltere imaginal disk. Remember the Ubx Hox gene? Using staining again, we can detect the gene product of Ubx. This reveals that

  • the Ubx gene is naturally “off” in the wing disk—
  • and is “on” in the haltere disk.
  • Now you’ll see what happens when the Ubx gene—just one of a large number of Hox genes—is turned off in the haltere disk. What if a genetic mutation caused the Ubx gene to be turned off, during the larval stage, in the third thoracic segment, the segment that normally produces the haltere? Instead of a pair of halteres, the fly has a second set of wings. With the switch of that single Hox gene, Ubx, from on to off, the third thoracic segment becomes an additional second thoracic segment and the pair of halteres became a second pair of wings. This illustrates the remarkable ability of transcription factors like Ubx to control patterning as well as cell type during development.


A. Data Suggests “Gene” Redefinition

As part of a huge collaborative effort called ENCODE (Encyclopedia of DNA Elements), a research team led by Cold Spring Harbor Laboratory (CSHL) Professor Thomas Gingeras, PhD, publishes a genome-wide analysis of RNA messages, called transcripts, produced within human cells.
Their analysis—one component of a massive release of research results by ENCODE teams from 32 institutes in 5 countries, with 30 papers appearing in 3 different high-level scientific journals—shows that three-quarters of the genome is capable of being transcribed.  This indicates that nearly all of our genome is dynamic and active.  It stands in marked contrast to consensus views prior to ENCODE’s comprehensive research efforts, which suggested that

  • only the small protein-encoding fraction of the genome was transcribed.

The vast amount of data generated with advanced technologies by Gingeras’ group and others in the ENCODE project changes the prevailing understanding of what defines a gene. The current outstanding question concerns

  • the nature and range of those functions.  It is thought that these
  • “non-coding” RNA transcripts act something like components of a giant, complex switchboard, controlling a network of  many events in the cell by
  1. regulating the processes of
  2. replication,
  3. transcription
  4. and translation

– that is, the copying of DNA and the making of proteins is based on information carried by messenger RNAs.  With the understanding that so much of our DNA can be transcribed into RNA comes the realization that there is much less space between what we previously thought of as genes, Gingeras points out.

The full ENCODE Consortium data sets can be freely accessed through

  • the ENCODE project portal as well as at the University of California at Santa Cruz genome browser,
  • the National Center for Biotechnology Information, and
  • the European Bioinformatics Institute.

Topic threads that run through several different papers can be explored via the ENCODE microsite page at    Date: September 5, 2012   Source: Cold Spring Harbor Laboratory

1000 Genomes Project Team Reports on Variation Patterns

(from Phase I Data) October 31, 2012 GenomeWeb

In a study appearing online today in Nature, members of the 1000 Genomes Project Consortium presented an integrated haplotype map representing the genomic variation present in more than 1,000 individuals from 14 human populations.  Using data on 1,092 individuals tested by

  • low-coverage whole-genome sequencing,
  • deep exome sequencing, and/or
  • dense genotyping,

the team looked at the nature and extent of the rare and common variation present in the genomes of individuals within these populations. In addition to population-specific differences in common variant profiles, for example, the researchers found distinct rare variant patterns within populations from different parts of the world — information that is expected to be important in interpreting future disease studies. They also encountered a surprising number of the variants that are expected to impact gene function, such as

  • non-synonymous changes,
  • loss-of-function variants, and, in some cases,
  • potentially damaging mutations.

ENCODE was designed to pick up where the Human Genome Project left off.
Although that massive effort revealed the blue­print of human biology, it quickly became clear that the instruction manual for reading the blueprint was sketchy at best. Researchers could identify in its 3 billion letters many of the regions that code for proteins, but they make up little more than 1% of the genome, contained in around 20,000 genes. ENCODE, which started in 2003, is a massive data-collection effort designed to catalogue the

  • ‘functional’ DNA sequences,
  • learn when and in which cells they are active and
  • trace their effects on how the genome is
  1. packaged,
  2. regulated and
  3. read.

After an initial pilot phase, ENCODE scientists started applying their methods to the entire genome in 2007. That phase came to a close with the publication of 30 papers, in Nature, Genome Research and Genome Biology. The consortium has assigned some sort of function to roughly 80% of the genome, including

  • more than 70,000 ‘promoter’ regions — the sites, just upstream of genes, where proteins bind to control gene expression —
  • and nearly 400,000 ‘enhancer’ regions that regulate expression of  distant genes (see page 57)1. But the job is far from done.

Junk DNA? What Junk DNA?

New data reveals that at least 80% of the human genome encodes elements that have some sort of biological function. [© Gernot Krautberger –] Far from containing vast amounts of junk DNA between its protein-coding genes, at least 80% of the human genome encodes elements that have some sort of biological function, according to newly released data from the Encyclopedia of DNA Elements (Encode) project, a five-year initiative that aims to delineate all functional elements within human DNA. The massive international project, data from which are published in 30 different papers in Nature, Genome Research, Genome Biology, the Journal of Biological Chemistry, Science, and Cell, has identified four million gene switches, effectively

  • regulatory regions in the genome where
  • proteins interact with the DNA to control gene expression.

Overall, the Encode data define regulatory switches that are scattered all over the three billion nucleotides of the genome. In fact, the data suggests,

  • the regions that lie between gene-coding sequences contain a wealth of previously unrecognized functional elements,Including
  • nonprotein-coding RNA transcribed sequences,
  • transcription factor binding sites,
  • chromatin structural elements, and
  • DNA methylation sites.

The combined results suggest that 95% of the genome lies within 8 kb of a DNA-protein interaction, and 99% lies within 1.7 kb of at least one of the biochemical events, the researchers say. Importantly, given the complex three-dimensional nature of DNA, it’s also apparent that

  • a regulatory element for one gene may be located quite some ‘linear’ distance from the gene itself.

“The information processing and the intelligence of the genome reside in the regulatory elements,” explains Jim Kent, director of the University of California, Santa Cruz Genome Browser project and head of the Encode Data Coordination Center. “With this project, we probably went from understanding less than 5% to now around 75% of them.”
The ENCODE results also identified SNPs within regulatory regions that are associated with a range of diseases, providing new insights into the roles that

  • noncoding DNA plays in disease development.

“As much as nine out of 10 times, disease-linked genetic variants are not in protein-coding regions,” comments Mike Pazin, Encode program director at the National Human Genome Research Institute.  “Far from being junk DNA, this regulatory DNA clearly makes important contributions to human disease.”

Other Related Articles on this Open Access Online Scientific Journal, include the following: 

Big Data in Genomic Medicine LHB

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair S Saha

Computational Genomics Center: New Unification of Computational Technologies at Stanford A Lev-Ari

Personalized medicine gearing up to tackle cancer ritu saxena

Differentiation Therapy – Epigenetics Tackles Solid Tumors sj Williams

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment A Lev-Ari

The Molecular pathology of Breast Cancer Progression tilde barliya`

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1 ( A Lev-Ari

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2 A Lev-Ari

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3 A Lev-Ari

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ ALA Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders/

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial” A Lev-Ari

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors S Saha

Personalized medicine-based cure for cancer might not be far away ritu saxena

Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence A Lev-Ari

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition sjwilliams

Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics A Lev-Ari

The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953 A Lev-Ari

Directions for genomics in personalized medicine lhb

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis. SJwilliams

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

Mitochondrial fission and fusion: potential therapeutic targets? Ritu saxena

Mitochondrial mutation analysis might be “1-step” away ritu saxena

mRNA interference with cancer expression lhb

Expanding the Genetic Alphabet and linking the genome to the metabolome LHB

Breast Cancer, drug resistance, and biopharmaceutical targets lhb

Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis A Lev-Ari

Gastric Cancer: Whole-genome reconstruction and mutational signatures A Lev-Ari

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

Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology A Lev-Ari

Reveals from ENCODE project will invite high synergistic collaborations to discover specific targets A. Sarkar

ENCODE: the key to unlocking the secrets of complex genetic diseases R. Saxena

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations s Saha

ENCODE Findings as Consortium A Lev-Ari

Genomics Orientations for Personalized Medicine SJH, ALA, LHB

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

 Related Articles

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Author: Stephen J. Williams, PhD

     The finding that a substance, derived from vascular endothelium, that could control vascular tone and induce smooth muscle relaxation, led to the discovery of nitric oxide (NO) as a major physiological mediator (1) in many cell types and processes.  Other investigators, working with platelets, determined that nitric oxide is a potent inhibitor, via an autocrine pathway, of platelet aggregation and adhesion to the vessel wall (2).  Nitric oxide is also an important regulator of neurotransmission in the nonadrenergic-noncholinergic system in gastric tissue (3,4).   In addition nitric oxide is involved in macrophage-mediated cytotoxicity, (5)based on the observation the cytotoxic action of macrophages required external arginine, which summarily was converted to citrulline, releasing  the nitric oxide involved in the cell-killing process.  The above physiological responses represent highly regulated, short-term responses that, as seen with classical receptor-based agonists such as epinephrine, terminate once the agonist (NO) is removed.   Given the short half-life of nitric oxide and these rapid physiologic responses, nitric oxide has been given the role of a second messenger within the cell.

However nitric oxide also produces some physiologically, pharmacologically, and pathologically relevant changes, lasting longer time periods, which is the main focus of this article.  For example, nitric oxide is important in the development of long term potentiation (a model of learning and memory), neural plasticity, and neurite outgrowth, revealing nitric oxide can induce more permanent changes in cellular and tissue reorganization (6-9).  Other pathologic and toxicological responses to nitric oxide include cell death from excitotoxic amino acids (glutamate, kainite), oxidative stress, DNA and protein damage, and disease progression in Alzheimer’s disease, epilepsy, aging, apoptosis and Huntington’s chorea (10-12).  These effects persist over longer time frames than the effects which most second messenger systems occur.  These cellular changes can be described by biochemical changes on protein and nucleic acid modification, metabolism (13-15), DNA synthesis and replication, and molecular and organelle reorganization.  The pharmacological and toxicological implications of such cellular changes are inherent in the persistent effects of nitric oxide on biological systems.  The mechanism of nitric oxide-induced physiology and toxicology had been assumed to involve the stimulation of soluble guanylate cyclase, raising intracellular cGMP levels.  As discussed further, this mechanism of action does not account for all the actions of nitric oxide, especially in nitric oxide-induced pathologies.  Other mechanisms of action include post-translational modifications of proteins such as S-nitrosylations, ADP-ribosylations, and a unique nonenzymatic covalent attachment of NAD+ to the regulatory site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a reaction specific to this dehydrogenase.  GAPDH is a true multifunctional protein involved in diverse cellular functions such as glycolysis, endocytosis, RNA processing and stability, DNA replication and repair, and involved in apoptosis.  GAPDH has been implicated in trinucleotide repeat neurodegenerative disorders such as Huntington’s disease, spinocerebellar ataxia, via binding to the polyglutamated forms of huntingtin and ataxin, protein modifications only seen in these respective diseases. GAPDH has also been implicated in Alzheimer’s disease as well, in genetic linkage studies as well as a β-amyloid precursor protein binding partner (for reviews see (16-20)).

Next to phosphorylation, ADP ribosylation and NAD+ modifications are the second most  common enzymatic  protein modifications in nature and regulates many cellular processes in nervous tissue, tumoral cell growth, cytoskeletal function, cell death and apoptosis, immune function, and bacterial cytotoxicity(21,22). These include poly ADP-ribosylations such as histones in the apoptotic process, and ADP-ribosylation of G-proteins by pertussis and cholera toxin. Interestingly, nitric oxide and other oxidants promote nonenzymatic ribosylation of proteins such as GAPDH.  Unlike the enzymatic reactions, this modification is covalent and generally considered irreversible and either involves nitrosylation of critical reactive cysteine residues or nitric oxide-mediated attachment of the whole NAD+ moiety, a reaction akin to aging of enzymes by reactive oxygen species.  There have been multiple intracellular targets of nitric oxide, with the result of inhibiting activity and/or protein interactions.  These include mitochondrial enzymes such as aconitase (23) and cytochrome oxidase (24), cytosolic enzymes such as cyclooxygenase and affect heme-containing proteins hemoglobin and myoglobin.  Such nitric oxide mediated effects on these systems were cGMP-independent, therefore independent of nitric oxide synthase.  The inhibition of GAPDH glycolytic activity by nitric oxide and NO-mediated NAD+ modification has been widely studied (21,25) and widely accepting to be important in nitric oxide mediated pathology (16,26-33).

So can this NO-NAD+ modification of GAPDH be useful as a therapeutic target for diseases such as Huntington’s, Alzheimer’s or other nitric oxide associated pathologies?   This is as much an intriguing idea as one fraught with caveats and technical issues.   First there is ample evidence that alterations of GAPDH structure/function exist in these neurodegenerative diseases and evidence that this type of modification may be important in the etiology of such diseases(34-41).

Second, as mentioned before, this modification is unique for GAPDH and would offer a disease-specific target(42).  Third, and most interesting, is the multifunctionality of GAPDH, therefore such modification has the possibility for affecting many processes involved in the disease progression.    However there is the big caveat and problem.  Such NO-NAD+ modifications are a covalent reaction, thought to be irreversible.  Studies on purified GAPDH reveal such modification is released by chemicals that can reduce the cysteine covalent bond such as HgCl2 or NaOH treatment(17).  However such treatment would be impractical for in-vivo use.  The ideal situation would be the discovery of an enzyme activity comparable to phosphatases which could enzymatically release the NO-NAD+ modification from GAPDH. A proof of concept experiment could involve creation of a genetically engineered enzyme capable of this reaction.  Therapeutic use of such an enzyme would depend of course on bioavailability.  Interestingly there has been evidence of cellular NO reductase activities, capable of removing the S-nitrosylation on reactive thiols.  Enzymes with denitrosylation activities include the thioredoxin system, superoxide dismutase, and xanthine oxidoreductase (34-40).  Possible therapeutic strategies may include regulation of these intracellular reductase activities.

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Other research paper on Nitric Oxide were published on this Scientific Web site as follows:

Discovery of nitric oxide and its role in vascular biology

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

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