Post translational modifications
- 1. TOPIC : POST TRANSLATIONAL EVENTS PRESENTED BY: TEJASWINI PETKAR
- 2. CONTENTS • INTRODUCTION • PROTEIN FOLDING • PROTEOLYTIC CLEVAGE • PROTEIN MODIFICATION THROUGH CHEMICAL MODIFICATIONS PHOSPHORYLATION ACETYLATION GLYCOSYLATION LIPIDATION AMINO ACID MODIFICATION UBIQUITINATION METHYLATION • INTEIN (PROTEIN) SPLICING • CONCLUSION • REFERENCES
- 3. INTRODUCTION Post-translational modification (PTM) refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis. Post-translational modifications (PTMs) mainly occur in the endoplasmic reticulum of the cell but sometimes continue in the golgi body as well. They influence almost all aspects of normal cell biology and pathogenesis. Protein PTMs can also be reversible depending on the nature of the modification; kinases phosphorylate proteins at specific amino acid side chains -a common method of catalytic activation or inactivation. Conversely, phosphatases hydrolyze the phosphate group to remove it from the protein and reverse the biological activity. The characterization of PTMs, although challenging, provides invaluable insight into the cellular functions underlying etiological processes. These processes have also been found critical for initiation or modulation of biological activity,transport and secretion. Technically, the main challenges to studying post-translationally modified proteins are the development of specific detection and purification methods. Fortunately, these technical obstacles are being overcome with a variety of new and refined proteomics technologies.
- 4. o Generally, these post translational modifications influence the structure, stability, activity, cellular localization or substrate specificity of the protein, and interaction with other cellular molecules (proteins, nucleic acids, lipids,cofactors). They hence play a key role in functional proteomics(entire complement of a protein that is or can be expressed by a cell, tissue or organism). o Complexity to proteome for diverse function with limited number of genes occurs by the covalent addition of functional groups to proteins or proteolytic cleavage of regulatory subunits. These modifications include phosphorylation, glycosylation, ubiquitination, methylation, acetylation,..
- 5. PTMs have several significant biological functions which are:- Aid in proper protein folding – for example; few lectin molecules called calnexin(chaperone) binds to glycosylated proteins and assist in its folding in ER. Conferring stability to the protein- glycosylation can modify the stability of the protein by increasing protein half life. It protects the protein against cleavage by proteolytic enzyme by blocking the cleavage sites. Protein sorting or translocation- If phosphorylated mannose residues are present in the protein it always goes to lysosome. Regulate protein activity and function- phosphorylation of protein is a reversible PTM which activates the protein. Increase the diversity and complexity in the proteome significantly.
- 6. Figure .Types of post-translational Modifications (PTMs). source : https://www.ptglab.com/news/blog/post-translational-modifications-an-overview/
- 7. PROTEIN FOLDING In general, protein folding is initiated by interactions between hydrophobic and polar groups, which create a condensation core. It is over this core that the rest of the protein will be arranged until its final shape is acquired. Protein folding gives rise to 3D conformations which is in turn important for biological functions. Generally, defects in their folding determine functional disorders. For this reason, there are quality control systems, which detect defective proteins and rapidly label them for degradation in the proteosome .
- 8. CHAPERONES: Chaperones are proteins that assist the covalent folding or unfolding and the assembly or disassembly of a vast majority of proteins. Chaperone proteins can bind to the nascent polypeptide chain while it is still being synthesized in ribosomes. Chaperones help other proteins fold to their final conformation; some are dedicated to the folding of only one protein, while others are general chaperones which help many different proteins fold. They facilitate and favour interactions on polypeptide surfaces to finally give the specific protein conformation. They can also reversibly bind to hydrophobic regions of unfolded proteins and folding intermediates. They can stabilize intermediate conformations the polypeptide before it reaches its final state, prevent formation of incorrect intermediates and also prevent undesirable or unproductive interactions with other proteins while preventing formation of insoluble protein aggregates. Chaperone proteins also stabilize unfolded chains during transport from the cytosol to its final destination and are involved in the assembly of subunits of oligomeric proteins. In short, all these activities of chaperones help proteins to attain compact and biologically active conformations.
- 10. PROTEOLYTIC CLEVAGE Many proteins are synthesized as precursors which are much bigger in size than the functional ones. Proteolysis occurs when a protease cleaves one or more bonds in a target protein to modify its activity. The events are relatively common in eukaryotes but less frequent in bacteria. This processing may lead to activation, inhibition or destruction of the protein's activity. The attacking protease may remove a peptide segment from either end of the target protein, but it may also cleave internal bonds in the protein that lead to major changes in the structure and function of the protein. Some portions of precursor molecules are removed by proteolysis to liberate active proteins commonly called- trimming, which may in turn occur in golgi apparatus, secretory vesicles and at times after protein secretion. Many proteins are synthesized as inactive precursors, which may be called proproteins or, in the case of enzymes, zymogens. These proproteins are synthesized in one tissue or organ, transported to another tissue or organ and activated there by proteolytic processing. There is no general rule of the thumb for proteolytic processing. It varies according to the protein being processed, location of the protein, and proteases.
- 11. o A variety of digestive proteases are synthesized in the pancreas as inactive proenzymes, so that they don’t start to attack the cells that make them. A few of these are tabulated below : o The above enzymes are active at pH slightly above neutral, corresponding to conditions in the small intestine. o In addition, pepsinogen is made in the stomach, and converted to pepsin, which is normally active at the very low pH in the stomach. Pepsin also acts on large polypeptides. PROENZYME ENZYME FUNCTION Proelastase Elastase Specific for elastin Trypsinogen Trypsin cuts large polypeptides at Arg or Lys Procarboxypeptidase Carboxypeptidase removes C-terminal amino acid from oligopeptides Chymotrypsinogen Chymotrypsin cuts large polypeptides at Phe, Tyr or Trp
- 12. TRYPSIN AS A COMMON ACTIVATOR OF THE PANCREATIC PROENZYMES Trypsinogen is activated by proteolytic cleavage (hydrolysis) of the peptide bond between Lys 6 and Ile 7. The oligopeptide with the first 6 amino acids is lost and Ile 7 becomes the new N-terminal. The positive charge associated with the N-terminal ion-pairs with Asp 194, realigning the peptide loop containing Asp 194. The reaction that activates trypsinogen is induced by the protease enteropeptidase -a membrane protein exposed on the external surface of the mucosal cells that line the duodenum. The neighbours of Asp 194, Gly 193 and Ser 195, make up the nucleophilic catalytic hole in trypsin and chymotrypsin. Trypsin is also responsible for activating chymotrypsinogen, procarboxypeptidase and elastase. Trypsin cuts chymotrypsinogen between Arg 15 and Ile 16, at a location which is structurally equivalent to Lys6 and Ile7 in trypsinogen. Self-exposure to chymotrypsin results in additional cleavages occurring at Leu13, Tyr146 and Asn148. The dipeptides Ser14-Arg15 and Thr147- Asn148 are lost, but the three large oligopeptides remain held together by disulfide bonds. The final product is a-chymotrypsin
- 13. Conformations of Chymotrypsinogen (Red) and Chymotrypsin (Blue) The electrostatic interaction between the carboxylate of aspartate 194 and the α-amino group of isoleucine 16, essential for the structure of active chymotrypsin, is possible only in chymotrypsin.
- 14. CHEMICAL MODIFICATION Most of the proteins that are translated from mRNA undergo chemical modifications before becoming functional in different body cells. These chemical changes that occur after a protein has been produced, can impact the structure, electrophilicity and interactions of proteins. The chemical modifications of PTMs can be roughly classified: Based on the addition of chemical groups- phosphorylation, acetylation, hydroxylation, methylation Based on the addition of complex groups- glycosylation, AMPylation, lipidation. Based on the addition of polypeptides- Ubiquitination Based on the cleavage of proteins- Proteolysis
- 15. PHOSPHORYLATION Phosphorylation is the most common and important mechanism of acute and reversible regulation of protein function. Covalent attachment of a phosphate group (negative charges) to an amino acid side chain of a protein can cause a structural change, for example, by attracting a cluster of positively charged side chains. Such a change occurring at one site in a protein can in turn alter the protein’s conformation elsewhere. Since phosphorylation involves attachment or detachment of phosphate group hence the switch on/ off of the function, it thus plays an important role in regulatory mechanism. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle. Bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair.
- 16. The reverse reaction of phosphorylation is called dephosphorylation, and is catalyzed by protein phosphatases. Protein kinases and phosphatases work independently and in a balance to regulate the function of proteins. The largest group of kinases is those that phosphorylate either serines or threonines and as much are termed serine/threonine kinases. In animal cells, serine, threonine and tyrosine are the amino acids subjected to phosphorylation. The ratio of phosphorylation of the three different amino acids is approximately 1000/100/1 for serine/threonine/tyrosine (Edelman AM et al., 1987). Fig; phosphorylation of the amino acid serine
- 18. ACETYLATION Acetylation refers to addition of acetyl group usually at the N-terminus of the α protein. It is involved in several biological functions, including protein stability, location, synthesis, apoptosis, cancer and DNA stability. Protein acetylation in mitochondria has taken center stage, revealing that 63% of mitochondrially localized proteins contain lysine acetylation sites. In humans, 80–90% of all proteins become co-translationally acetylated at their N α-termini of the nascent polypeptide chains. These reactions are catalyzed by N-acetyltransferases (NATs) . Acetylated lysine residues were first discovered in histones regulating gene transcription. Acetylation and deacetylation of histones form a critical part of gene regulation. For example, acetylation of histones reduces the positive charge on histone, reducing its interaction with the negatively charged phosphate groups of DNA, making it less tightly wound to DNA and accessible to gene transcription.
- 20. GLYCOSYLATION Glycosylation involves addition of an oligosaccharide termed ‘glycan’ to either a nitrogen atom (N-linked glycosylation) or an oxygen atom (O-linked glycosylation). N-linked glycosylation occurs in the amide nitrogen of asparagine, while the O-linked glycosylation occurs on the oxygen atom of serine or threonine. These in turn lead to the synthesis of glycoproteins. Vitamin K dependent carboxylation of glutamic acid residues in certain clotting factors is an example of the role of protein glycosylation.
- 21. Proteins are glycosylated for several reasons: Carbohydrates present on the surface of cells secrete proteins. Selective addition of saccherides to specific protein residues convey more structural stability or function to the native protein structure. They have critical roles in protein sorting, immune recognition, receptor binding, inflammation, and pathogenicity. For example, N-linked glycans on an immune cell can dictate how it migrates to specific sites. Similarly, it can also determine how a cell recognizes ‘self’ and ‘non-self’. Some glycoproteins are more stable once they have polysaccharides attached, others for cell recognition and communication. Still some proteins simply refuse to fold properly without their accompanying side chains. o Since glycosylation is thought to be largely a function of the Golgi apparatus, experiments into the role of glycosylation in protein conformation in vivo are difficult to design.
- 23. LIPIDATION Protein lipidation is an important co- or posttranslational modification in which lipid moieties are covalently attached to proteins. Proteins are covalently modified with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins. Lipidation markedly increases the hydrophobicity of proteins, resulting in changes to their conformation, stability, membrane association, localization, trafficking, and binding affinity to their co-factors. Protein lipidation, including cysteine prenylation, N-terminal glycine myristoylation, cysteine palmitoylation, and serine and lysine fatty acylation, occurs in many proteins in eukaryotic cells and regulates numerous biological pathways, such as membrane trafficking, protein secretion, signal transduction, and apoptosis. Lipidation can be further subdivided into : prenylation, N- myristoylation, palmitoylation, and glycosylphosphatidylinositol (GPI)-anchor addition.
- 24. PRENYLATION: Prenylation involves the addition of isoprenoid moiety to a cysteine residue of a substrate protein . It also be termed as the addition of a single 15-carbon farnesyl or single or dual 20-carbon geranyl geranyl moieties to one or two cysteines near the C- terminus of target proteins catalyzed by farnesyltransferases or by protein geranlygeranyl transferases. It is critical in controlling the localization and activity of several proteins that have crucial functions in biological regulation. These isoprenyl anchors promote not only protein-membrane ,but also protein-protein interactions. Several diseases are correlated to this PTM, like cancer and premature aging disorders. Protein prenylation occurs also in a wide range of parasites, leading to the use of protein farnesyl transferase inhibitors in protozoan parasitic diseases.
- 25. N- MYRISTOYLATION: Protein N-myristoylation refers to the irreversible addition of myristic acid to proteins through an amide linkage to an N-terminal glycine residue. This occurs mostly in eukaryotic or viral proteins. This PTM facilitates in turn the interaction with membranes or a hydrophobic protein domain. Usually myristoylation acts with other posttranslational modifications like palmitoylation , or in combination with positively charged residues in order to enhance membrane-protein interactions. Myristoylation is involved in several critical cellular processes, such as signaling pathways, apoptosis and extracellular protein export. Several diseases are linked to N-myristoylation like cancer, epilepsy, Alzheimer's disease and viral as well as bacterial infections and alternatively, during apoptosis.
- 26. PALMITOYLATION: It has no single sequence requirement outside of the presence of a cysteine residue. Although a number of fatty acids (including stearate, oleate, and even polyunsaturated fatty acids ) can be incorporated by this mechanism, the most common is 16-carbon, saturated palmitic acid, thus S-acylation is often referred to as ‘palmitoylation’. Nearly all palmitoylated proteins are modified by attachment of the fatty acid to a cysteine residue via thioester linkage (S-palmitoylation). Two families of enzymes regulate the palmitoylation/ depalmitoylation process: Palmitoyltransferases (PATs), which catalyze the attachment of a palmitate from CoA to specific cysteines, and Acyl Protein Thioesterases (APTs), which remove the palmitate acyl chain. Palmitoylation occurs both in soluble and membrane proteins playing a critical role in the regulation of key biological processes, such as protein membrane trafficking, signaling, cell growth and development. It is a key feature of numerous signal transducers. Aberrant palmitoylation is associated to a variety of human diseases including neurological disorders (e.g., Huntington disease's or Alzheimer's disease) and cancer.
- 27. GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)- ANCHOR: Approximately 1% of all eukaryotic proteins are modified by a complex lipid structure known as the GPI anchor. Assembly of the anchor and transfer to the protein occurs in the ER. In GPI-anchor addition, the carboxyl-terminal signal peptide of the protein is split and replaced by a GPI anchor. It is mediated by nearly two dozen different enzymes and loss of critical enzymes in the GPI biosynthetic pathway is lethal in organisms ranging from yeast to parasites to mice. The saturated nature of the fatty acids attached to PI enhances insertion of GPI- anchored proteins into lipid raft domains rich in cholesterol and sphingolipid, where they participate in a wide variety of signal transduction pathways. The structural complexity of the GPI anchor as well as its heterogeneous nature have made it difficult to define its exact biological functions.
- 28. UBIQUITINATION Ubiquitination involves addition of a protein found, termed ‘ubiquitin’, to the lysine residue of a substrate. Ubiquitin is a small (8.5 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e. it occurs ubiquitously. It was discovered in 1975, New York by Gideon Goldstein and further characterized throughout the 1970s and 1980s. The addition of ubiquitin to a substrate protein is called ubiquitination or less frequently ubiquitylation. Ubiquitination affects proteins in many ways: it can mark them for degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions. Monoubiquitinated proteins may influence cell tracking and endocytosis. Depending on mono- and polyubiquitination and on how ubiquitin chains are linked together, post-translational modifications of cellular proteins by covalent attachment of ubiquitin and ubiquitin-like proteins are involved in transcriptional regulation, receptor internalization, DNA repair, stabilization of protein complexes and autophagy.
- 30. Ubiquitylation is a three step process (activation, conjugation, and ligation) whereby: first, the ubiquitin is activated by a ubiquitin-activating enzyme (E1), then, conjugated to a ubiquitin-conjugating enzyme (E2), finally transferred by a ubiquitin-ligase enzyme (E3) to a substrate molecule via an isopeptide bond with an internal lysine. Overview of the ubiquitin-mediated protein degradation pathway. Reprinted from European Journal of Cancer 40, A.M. Burger and A.K. Seth, The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications, PP.2217-2229, 2004, PMID: 15454246
- 31. This reversible modification is implicated in the regulation of several cellular processes, like protein degradation, cell cycle division, the immune response, lysosomal trafficking and control of insulin. The aberration of ubiquitylation is linked to human pathologies varying from inflammatory neurodegenerative diseases to different forms of cancers. Despite the availability of several ubiquitin-protein ligase complex structures, the ubiquitylation reaction mechanism is still poorly understood.
- 32. Fig :Roles of ubiquitination and sumoylation. Polyubiquitinated proteins are targeted to the proteasome for degradation via E1, E2, and E3 enzymes. Dubs (deubiquitination enzymes) reverse this process. Ubiquitin targets a number of proteins involved in different cellular processes (blue lines). (Sumoylation contributes to similar processes (red lines) but modifies proteins by attaching SUMO via E1, E2, and E3 enzymes, and this modification generally activates a protein. Sumoylation is reversible by ULP proteases. These modifications are often triggered by specific environmental stimuli, and the cellular processes they regulate contribute to fungal pathogenicity.)
- 33. METHYLATION Methylation refers to addition of a methyl group. It is a type of alkylation. Protein methylation can occur on arginine, lysine, histidine, proline, and carboxyl groups. About 1–2% of genes in a variety of prokaryotic and eukaryotic organisms encode methyltransferases and a large fraction of these are specific for modifying protein substrates. The addition of a methyl group 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 of proteins can occur on multiple amino acids on proteins, including arginine, lysine, histidine, etc. Methylation is mediated by methyltransferases. Methylation of histones is mechanically linked to other types of histone modifications, such as acetylation, phosphorylation, and monoubiquitylation; combinations of these modifications cooperate to regulate chromatin structure and transcription by stimulating or inhibiting binding of specific proteins.
- 34. The flexible N-terminal and C-terminal ends of histones are known to contain lysine modifications important for the coupling of histones to changes in chromatin organization and the epigenetic control of gene expression. Methylated proteins, as well as methylation regulatory enzymes are involved in several human diseases such as cancer, cardiovascular diseases, multiple sclerosis and neurodegenerative disorders . Thus, the inhibition of these enzymes with small molecules could be an effective therapeutic means of intervention.
- 37. INTEIN (PROTEIN) SPLICING An intein (internal -protein) is an intervening polypeptide domain which is precisely excised from a precursor polypeptide during protein splicing. Inteins can be considered as intervening sequences in certain proteins which can be compared to the introns of mRNAs. They have to be removed and exteins ligated concomitantly in appropriate order by the formation of a peptide bond to yield an active protein product . Intein mediated protein splicing is spontaneous; it requires no external factor or energy source, only the folding of the intein domain. There are more than 200 inteins identified to date; sizes range from 100–800 amino acids. Inteins are also functional in exogenous contexts and can be used to chemically manipulate virtually any polypeptide backbone. Given this, protein chemists have exploited various facets of intein reactivity to modify proteins in myriad ways for both basic biological research as well as potential therapeutic applications. For example, the side reactions of protein splicing, N- and C-extein cleavage, can both be enhanced by the introduction of specific point mutations in the conserved splicing motifs.
- 38. Fig: A comparison between RNA splicing and protein splicing. In the mechanism of RNA Coding sequence splicing, shown on the left, the transcription of the coding sequence immediately precedes the RNA splicing. RNA splicing thus occurs at the level of the RNA precursor and the intron is excised before translation of the RNA to the 5’ 3’ protein. In contrast, protein splicing, shown on the right, occurs at the level of the protein. In I protein splicing, a polypeptide domain called an intein is excised from the middle region of a precursor polypeptide to give two proteins that are not colinear with the coding sequence.
- 39. SELECTED EXAMPLES OF PTMS OF PROTEINS THROUGH THEIR AMINO ACIDS AMINO ACID PTM(S) Amino- terminal amino acid Glycosylation, acetylation, myristoylation, formylation Carboxy terminal amino acid Methylation, ADP ribosylation Arginine Methylation Aspartic acid Phosphorylation , hydroxylation Cysteine (-SH) Cysteine(-S-S-) formation, selenocysteine formation, glycosylation Glutamic acid Methylation, γ- carboxylation Histidne Methylation, phosphorylation Lysine Acetylation, methylation, hydroxylation, biotinylation Methionine Sulfoxide formation Phenylalanine Glycoxylation, hydroxylation Proline Glycoxylation, hydroxylation Serine Phosphorylation, glycosylation Threonine Phosphorylation, methylation, glycosylation Tryptophan Hydroxylation Tyrosine Hydroxylation, phosphorylation, sulfonylation, iodination
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