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DNA and protein synthesis: Post-translation

Introduction

Proteins are synthesized through a process called translation and nearly all of them are formed by mRNA-directed mechanisms. This process involves a lot of enzymatic mechanisms to ensure that the proteins are correctly synthesized, folded and assigned various functions including enzymes, hormones, transcription factors, antibodies, and membrane proteins.

It is estimated that the human genome comprises between 20,000 and 25,000 genes. However, the total number of proteins present in the human proteosome is estimated at over 1 million. This means that multiple proteins are encoded by a single gene. This is in part due to splice variants where different processing of mRNA leads to transcription of similar proteins with different features and lengths. The organizational complexity from the level of the genome to that of the proteosome is further explained by the process of post-translational modification.

Post-Translational Modifications

Post-translational modifications are series of covalent processing events that change the properties of a protein by either proteolytic cleavage or the addition of a modifying group to one or more amino acids. This process has a great influence on the nature of a protein as it can regulate its activity, localization, turn-over and interaction with other proteins and molecules like nucleic acids, lipids and cofactors.

As the human proteosome is highly dynamic and changes in response to various forms of stimuli, post-translational modifications serve to regulate the cellular activities. PTMs are often mediated by enzymes and occur at distinct amino acid side chains and peptide linkages. These enzymes include the kinases, transferases, phosphatases and ligases which add or remove functional groups, proteins, lipids or sugars to or from amino acid side chains. Conversely, proteases are enzymes that cleave peptide bonds to remove specific sequences or regulatory subunits. Other proteins have the ability to modify themselves by using autokinases or autoproteolytic domains – these are examples of autocatalytic domains.

Common Examples of Post-Translational Modifications

Characteristics of Post-translational Modifications

  • The process is enzyme mediated, as discussed above.

  • It can occur at any stage: This means that some proteins are modified co-translationally for example in disulfide bonding, some once folding is completed and others once they are localized (many proteins, for example collagens, are processed in an inactive form and are activated by enzymatic removal of a pro-domain once they reach their destination).

  • This can be reversible depending on the nature of the modification. For example, the activities of kinases and phosphatases in which one enzyme adds a phosphate group to a protein while the other removes the phosphate group through hydrolysis. This reversible enzymatic activity often acts as an “on-off switch” for the biological activity of the protein.

Glycosylation

This is the addition of a carbohydrate or sugar moiety to proteins and this ranges from simple monosaccharide modifications of nuclear transcription factors to the complex branched polysaccharide chains of cell surface receptors. There are two types of glycosylation; the N-linked glycosylation, which occurs in the form of Asparagine-linked oligosaccaharide and the O-linked glycosylation in the form of serine/threonine-linked oligosaccharide are both major structural components of many cell surfaces and secreted proteins. Glycosylations are often required for correct peptide folding and can increase protein stability and solubility and protect against degradation.

Phosphorylation

Phosphorylation is the addition of a phosphate (PO4) group to a serine, tyrosine or threonine residue in a peptide chain, though it can occur on other residues in prokaryotes. The addition or removal of a phosphate group can alter protein conformation (and therefore function) by locally altering the charge and hydrophobicity where it is added. It plays an important role in regulating many important cellular processes such as cell cycle, growth, apoptosis (programmed cell death) and signal transduction pathways. For example, in signalling, kinase cascades are turned on or off by reversible phosphorylation either by addition or removal of a phosphate group.

Protein phosphorylation overview

N-Acetylation

This process involves the transfer of an acetyl group to nitrogen and it occurs almost in all eukaryotic proteins. It has both reversible and irreversible mechanisms. Methionine aminopeptidase (MAP) is an enzyme responsible for N-terminal acetylation which results in the cleavage of N-terminal methionine before replacing the amino acid with an acetyl group from acetyl-coA by the enzyme N-acetyltransferase. Acetylation helps in protein stability, protection of the N-terminus and the regulation of protein-DNA interactions in the case of histones.

Lipidation

Lipidation attaches a lipid group, such as a fatty acid, covalently to a protein. In general, lipidation helps in cellular localization and targeting signals, membrane tethering and as mediator of protein-protein interactions. Important types include palmitoylation which creates a thioester link between long-chain fatty acids and cysteine residues, N-myristorlation of glycine residues which plays a role in membrane targeting and GPI-anchor addition which links a glycosyl-phosphatidylinositol (GPI) to and extracellular protein to mediate its attachment to the plasma membrane.

Ubiquitination

The addition of ubiquitin (an 8kDa polypeptide consisting of 76 amino acid residues) linked to an amine group of lysine in target protein via its C-terminal glycine. Poly-ubiquitinated proteins are targeted for destruction which leads to component recycling and the release of ubiquitin. An example of this is in the cell cycle where ubiquitination marks cyclins for destruction at defined time points.

Protein with a polyubiquitin chain

S-Nytrosylation

This process adds a nitrosyl group to a protein and is used by cells to stabilize proteins, regulate gene expression and provide nitric acid donors. It is also responsible for the generation, localization, activation and catabolism of S-nitrothiols (formed from nitric oxide and free cysteine residues). The S-nitrothiols (SNOs) are under tight regulation and can be dinitrosylated by caspases (enzymes that mediate apoptosis) in response to extra or intracellular cues. When this happens, the SNOs are denitrosylated which leads to apoptosis.

Methylation

This process involves the transfer of one carbon methyl group to either nitrogen or oxygen to amino acid side chains of proteins, called an N or O-methylation respectively. The enzymes responsible for this process are the methyltransferases while S-adenosyl methionine (SAM) is the primary methyl donor. Methylation is best known in the context of direct methylation of DNA, however epigenetics can also be regulated by methylation of proteins. Methylation of histones, a type of DNA binding protein, can regulate DNA availability for transcription.

Proteolysis

  1. Proteolysis is the breaking apart of the peptide bond in a protein which can happen anywhere in a protein. There are 11,000 protease enzymes with a varied array of specificity, localization, length of activity and mechanism of peptide bond cleavage. Proteolysis is a thermodynamically favourable and irreversible reaction and is therefore under tight regulatory control. The control mechanisms include regulation by cleavage in either cis or trans and compartmentalization.

Degradative proteolysis is important as it removes unassembled protein subunits and misfolded proteins and also maintains protein concentration at homeostatic concentrations. Some proteases are classified based on their site of action like the aminopeptidases which act on amino terminus and carboxipeptidases which act on carboxy terminus of a protein respectively. Others are classified based on the active site group of a protease that are involved in proteolysis. These proteases include; serine proteases, cysteine proteases, aspartic acid proteases and zinc metalloproteases.

Proteolysis can also release useful cleavage fragments and remove autoinhibitory domains from proteins. This regulation can prevent fibrous and polymer proteins from assembling in inappropriate locations and keep proteins which could otherwise have damaging effects like enzymes and growth factors inactive until they reach their target location. It is also used to remove features of a protein which are not needed in the mature form, particularly targeting signal sequences and the N-terminal methionine.

Proteolysis

Disulfide Bonding

Disulfide bonds are covalent bonds formed between two cysteine residues (R-S-S-R). These bonds contribute to the correct folding of proteins as other elements of secondary structure are fixed in place around them and because they are very strong bonds they stabilise the protein, for this reason small secreted proteins tend to have a lot of disulfide bonds. Although usually between residues of the same protein they can occur between cysteines of different proteins, for example the growth factor TGF-beta1 forms a disulfide bond with LTBP1 which prevents it from signalling.

Others

Other forms of post translational modifications include: tyrosine sulfation, proline hydroxylation, hydroxyproline arabinosylation and nitration of tyrosine.

 

Detecting and Quantifying Post-Translational Modifications

The study of post-translational modifications has been faced with the challenges of developing specific detection, purification and quantification methods. In recent times, these challenges are being overcome by the development of a variety of new and refined methods in proteomics. These methods include the use of MALDI/MS (Matrix-assisted Laser Desorption/Ionization Mass Spectrometry, the combination of ECD (Electron Capture Dissociation) and peptide fragmentation with new generations of high-sensitivity FTMS (Fourier-Transform Mass Spectrometry).

Bioinformatics methods to predict modification sites in-silico has also become a promising area of development. The development of databases of protein modifications with reference to the already existing protein and genomic database is thought to be a significant way in which the detection and quantification of PTMs could also be improved.

Conclusion

The study of post-translational modifications has brought about great insights into the fields of proteomics, protein functions in relation to gene functions. The in-depth study of some diseases such as heart diseases, cancer, neurodegenerative diseases and diabetes can be improved with the current advances in method development for the determination and quantification of PTMs.

References

  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. Mann, M and Jensen, O., N (2003) Proteomic analysis of post-translational modifications. Nature Biotechnol. 21, 255-261.

  4. Matsubayashi, Y (2012) Recent advances in research on small post-translationally modified peptide signals in plants. Genes to Cells 17, 1-10.

  5. Ralp, A. Bradshaw and Albert, E. Stewart (1994) Analysis of protein modifications: Recent advances in detection, characterization and mapping. Curr. Opin. Biotechnol. 5(1), 85-93.

  6. Yang X., J and Seto E (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell. 31, 449-61.

 

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