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post translational modifications of protein

Post-translational modifications (PTMs) are chemical modifications of proteins that occur after translation. PTMs play a key role in regulating protein function by modifying activity, localization, and interactions. The main types of PTMs discussed are phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis. These modifications are identified through techniques like mass spectrometry, HPLC, radioactive labeling, and gel electrophoresis. PTMs are important for processes like cell signaling, growth, and apoptosis.

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Post Translational Modifications of Proteins
• It is the chemical modification of protein after its translation. • Key role in functional Proteomics. • They regulate activity, localization and interaction with other cellular molecules such as proteins, nucleic acids, lipids and cofactors. Introduction
• Phosphorylation • Glycosylation • Ubiquitination • S-Nitrosylation • Methylation • N-Acetylation • Lipidation • Proteolysis Types of Post Translational Modifications of Proteins
• Addition of phosphate group to a protein. • Principally on serine, threonine or tyrosine residues. • Also known as Phospho regulation. • Critical role in cell cycle, growth, apoptosis and signal transduction pathways. Phosphorylation Protein kinases ATP + protein ———————> phosphoprotein + ADP
Phosphorylation
Example
• The covalent attachment of oligosaccharides • Addition of glycosyl group or carbohydrate group to a protein. • Principally on Asparagine, hydroxylysine, serine or threonine. • Significant effect on protein folding, conformation, distribution, stability and activity. Glycosylation
Example
• N-Linked glycans – attached to nitrogen of Asparagine or arginine side chains. • O-Linked glycans – attached to hydroxy oxygen of serine,threonine • Phospho glycans – linked through the phosphate of serine. • C-Linked glycans – Rare form, Sugar is added to a carbon on tryptophan side chain. Classes of Glycans
• Ubiquitin is a small regulatory protein that can be attached to the proteins and label them for destruction. • Effects in cell cycle regulation, control of proliferation and differentiation, programmed cell death (apoptosis), DNA repair, immune and inflammatory processes and organelle biogenesis. Ubiquitination
Ubiquitin cycle
• Nitrosyl (NO) group is added to the protein. • NO a chemical messanger that reacts with free cysteine residues to form S-nitrothiols. • Used by cells to stabilize proteins, regulate gene expression. S-Nitrosylation
• Addition of methyl group to a protein. • Usually at lysine or arginine residues. • Binds on nitrogen and oxygen of proteins • Methyl donor is S-adenosylmethionine (SAM) • Enzyme for this is methyltransferase • Methylation of lysine residues in histones in DNA is important regulator of chromatin structure Alkylation/Methylation
Example Where SAM (S-adenosyl methionine) is converted into SAH(S-adenosyl homocysteine)
• Addition of acetyl group to the nitrogen. • Histones are acetylated on lysine residues in the N-terminal tail as a part of gene regulation. • Involved in regulation of transcription factors, effector proteins, molecular chaperons and cytoskeletal proteins. • Methionine aminopeptidase (MAP) is an enzyme responsible for N-terminal acetylation N-Acetylation
Example
Where, HDACs = Histone deactyllase , KATs = N-acetyltransferase.
• Lipidation attachment of 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. Lipidation
• C-terminal glycosyl phosphatidylinositol (GPI) anchor • N-terminal myristoylation • S-palmitoylation • S-prenylation Types of lipidation
C-terminal glycosyl phosphatidylinositol (GPI) anchor • GPI anchors tether cell surface proteins to the plasma membrane • GPI-anchored proteins are often localized to cholesterol- and sphingolipid-rich lipids, which act as signaling platforms on the plasma membrane.
N-myristoylation • It is the attachment of myristoyl group a 14- carbon saturated fatty acid (C14) to a protein. • It is facilitated by N-myristoyltransferase (NMT) and uses myristoyl-CoA as the substrate.
S-palmitoylation • It is addition of C16 palmitoyl group from palmitoyl-CoA • Palmitoyl acyl transferases (PATs) enzyme favors this step. • Reversed by thioesterases
S-prenylation • Addition of a farnesyl (C15) or geranylgeranyl (C20) group to proteins. • Enzyme involved in this reaction is farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II).
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
Disulfide Bonding
• Cleavage of peptide bonds by proteases. • Examples of Proteases- Serine Proteases, Cysteine Proteases, Aspartic acid Proteases. • Involved in Antigen processing, Apoptosis, Cell signalling Proteolysis
• Mass spectrometry • HPLC analysis • Incorporation of radioactive groups by addition to growing cells – e.g., 75Se-labeling and chromatographic isolation of proteins • Antibody cross-reactivity – e.g., antibody against phosphotyrosine • Polyacrylamide gel electrophoresis (PAGE) Identification of modifications
Identification of modifications small-molecule modifications can affect not only the activity, but also the structure of proteins, much as ligands such as ATP can affect the activity and structure of proteins use 2D gel electrophoresis to detect modified proteins in whole-cell (or partly purified) lysates O-GlcNAc is an abundant modification of nucleocytoplasmic proteins. Nucleo-cytoplasmic proteins from HeLa cells were immunopurified with an O-GlcNAc-specific antibody and stringently washed, and the O-GlcNAc- containing proteins were specifically eluted with free GlcNAc. The resulting proteins were separated on two-dimensional gels and visualized by silver staining. pI, isoelectric point; MW, molecular weight. From Wells et al. (2001) Science 291, 2376-8.
• Jensen, O., N (2004) Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Current Openings in bio-chemistry. 8, 33-41 • Mann, M and Jensen, O., N (2003) Proteomic analysis of post- translational modifications. Nature Biotechnology. 21, 255-261. • Matsubayashi, Y (2012) Recent advances in research on small post-translationally modified peptide signals in plants. Genes to Cells 17, 1-10. • Ralp, A. Bradshaw and Albert, E. Stewart (1994) Analysis of protein modifications: Recent advances in detection, characterization and mapping. 5(1), 85-93. • Walsh C. (2006) Posttranslational modification of proteins : Expanding nature's inventory. Englewood, Colo.: Roberts and Co. Publishers. xxi, 490 p. p. • Gaston B. M. et al. (2003) S-nitrosylation signaling in cell biology. Mol Interv. 3, 253-63. • Jaffrey S. R. and Snyder S. H. (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE. 2001, pl1. • 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. References
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