Post-Translational Modification

Post Translational modification.

1. POSTTRANSLATIONAL MODIFICATIONS PROTEOLYTIC CLEAVAGE AND COVALANT MODIFICATIONS

2. Introduction: Post-translational Modifications is a biochemical mechanism in which amino acid recidue in proteins are covalently modified. It can occur on the amino acid side chains or at the proteins C- or N- termini. Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modifications Other forms of PTM consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue.

3. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. During translation, about 30-40 polypeptide residues are relatively protected by the ribosome (tunnel T and exit sites E1 and E2 in the large subunit). Once the polypeptide chain emerges from the ribosome it starts to fold and can be subject to post-translational modifications.

4. Why post-translational processing? Adds functionality Effects targeting R egulate protein activity and protein interactions Increases mechanical strength Changes recognition Important component in cell signaling

5. Types of Post-translation modiications 1.Proteolytic Cleavage 2.Covalent modification 3.Protein splicing 4. Noncovalent modifications: folding, addition of co-factors. 5. Involvement of molecular chaperones in 1, 2, and 3.

6. 1.Proteolytic Cleavages It involves proteolytic removal of their leading Met (or fMet ) residue shortly after it emerging from the ribosomes . Many proteins which are involved in a wide vareity of biological processes, are synthesized as inactive precursors that are activated under proper conditions by limited proteolysis Inactive proteins that are activated by removal of polypeptides are called proproteins , whereas the excised polypeptides are termed propeptides .

7. Propeptides Direct Collagen Assembly collagen, a major extracellular component of connective tissue, is a fibrous triple-helical protein whose polypeptides each contain the amino acid sequence ( Gly -X-Y) n where X is often Pro, Y is often 4-hydroxyproline ( Hyp ), and n=340. The polypeptides of procollagen differ from those of the mature protein by the presence of both N-terminal and C-terminal propeptides of 100 residues whose sequences, for the most part, are unlike those of mature collagen. The N- and C-terminal propeptides of procollagen are respectively removed by amino- and carboxyprocollagen peptidases.

8. An inherited defect of aminoprocollagen peptidase in cattle and sheep results in a bizarre condition, dermatosparaxis , that is characterized by extremely fragile skin. An analogous disease in humans, Ehlers– Danlos syndrome VII, is caused by a mutation in one of the procollagen polypeptides that inhibits the enzymatic removal of its aminopropeptide .

9. Signal Peptides Are Removed from Nascent Proteins by a Signal Peptidase: The SRP binds a ribosome synthesizing a signal peptide to a protein pore known as the translocon that is embedded in the membrane [the rough endoplasmic reticulum (RER) in eukaryotes and the plasma membrane in bacteria] and conducts the signal peptide and its following nascent polypeptide through the translocon . Proteins bearing a signal peptide are known as proproteins or, if they also contain propeptides , as preproproteins . Signal Peptides are specifically cleaved from the nascent polypeptide by a membrane-bound signal peptidase.

10. Polyproteins polypeptides that contain the sequences of two or more proteins. many polypeptide hormones the proteins synthesized by many viruses, including those causing polio and AIDS and ubiquitin , a highly conserved eukaryotic protein involved in protein degradation. Specific proteases post- translationally cleave polyproteins to their component proteins, presumably through the recognition of the cleavage site sequences.

11. 2.Covalent Modification The proteins synthesized in translation are subjected to many covalent changes. By these modifications in the in amino acids, the proteins may be converted to active form or inactive form. Types of Covalent Modifications: Phosphorylation Hydroxylation Glycosylation Methylation

12. a. Collagen Assembly Requires Chemical Modification Collagen biosynthesis is illustrative of protein maturation through chemical modification. As the nascent polypeptides pass into the RER of the fibroblasts that synthesized them, the Pro and Lys residues are hydroxylated to Hyp , 3-hydroxy-Pro, and 5-hydroxy-Lys. The enzymes that do so are sequence specific: Prolyl 4-hydroxylase and lysyl hydroxylase act only on the Y residues of the Gly -X-Y sequences, whereas prolyl 3-hydroxylase acts on the X residues but only if Y is Hyp . Glycosylation , which also occurs in the RER, subsequently attaches sugar residues to Hyl residues.

13. The folding of three polypeptides into the collagen triple helix must follow hydroxylation and glycosylation . Folding is also preceded by the formation of specific interchain disulfide bonds between the carboxylpropeptides . The procollagen molecules pass into the Golgi apparatus where they are packaged into secretory vesicles and secreted into the extracellular spaces of connective tissue. The aminopropeptides are excised just after procollagen leaves the cell and the carboxypropeptides are removed.

14. The collagen molecules then spontaneously assemble into fibrils, which suggests that an important propeptide function is to prevent intracellular fibril formation. The collagen molecules in the fibrils spontaneously cross-link.

15. Phosphorylation : Reversible protein modifications. A phosphate is added by a specific kinase and later removed by a specific phosphatase . Phosphorylation may either increase or decrease the activity of the proteins. Physiologically relevant examples are the phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from pancreas.

16. The enzymes that phosphorylate proteins are termed kinases and those that remove phosphates are termed phosphates. ATP+protein -> phosphoprotein+ADP The level of tyrosine phosphorylation is minor, the important of phosphorylation of this amino acid is profound. An example, the activity of numerous growth factor receptors is controlled by tyrosine phosphorylation . Enzymes called protein kinases catalyse phosphorylation while protein phosphatases are responsible for dephosphorylation . Known as Metabolism.

17. 2. Hydroxylation: During the formation of Collagen, the amino acids proline and lysine are respectively converted to hydroxyproline and hydroxylysine . The hydroxylation occurs in the endoplasmic reticulum and requires vitamin C.

18. 3. Glycosylation : The attachment of carbohydrate moiety is essential for some proteins to perform their functions. The compex carbohydrate moiety is attached to amino acids, serine and threonine (O-linked) or to aspargine (N-linked), leading to the synthesis of glycoproteins . Vitamin K dependent carboxylation of glutamic acid residues in certain clotting factors is also a post- translaional modifications.

19. Examples of Post-translational modifications through their amino acids Amino Acids Post- translatonal Modifications Amino-terminal amino acids Glycosylation , acetylation , myristoylation , formylation . Carboxy terminal amino acids Methylation , ADP- ribosylation Arginine Methylation Aspartic acids Phosphorylation , hydroxylation Cysteine (-SH) Cyteine (-S-S-) formation, selenocysteine formation, glycosylation . Glutamic acids Methylation , ᵞ - carboxylation Histidine Methylation , biotinylation .

20. Lysine Acetylation , methylation , hydroxylation, biotinylatiom Methionine Sulfoxide formation Phenyl alanine Glycosylation , hydroxylation Proline Hydrxylation , glycosylation Serine Phosphorylation , glycosylation Threonine Phosphorylation , methylation , glycosylation Tryptophan Hydroxylation Tyrosine Hydroxylation, phosphorylation , sulphonylation , iodination

21. 4. Protein Methylation : Post translational methylation of proteins occurs on nitrogen and oxygen. Activated methyl donar for these rections is S- adenosylmethionine . The most common methylations are on the ƹ-amine of the R-group of lysine residues and the guanidino moiety of the of the R-group of arginine . Methylation of lysine residues in histones in nucleosome is an important regulator of chromatin structure and consequently of transcriptional activity.

22. Nitrgen methylations are found on the imidozole ring of histidine and R-group amides of glutamates and aspartate . Methylation of the oxygen of the R-group carboxylates of glutamate and aspartate also takes place and forms esters. Proteins can also be methylated on the thiol R-group of cysteine .

23. 3. Protein Splicing: Inteins and Exteins Protein splicing is a post-translational modification process in which an internal protein segment (an intein ) excises itself from a surrounding external protein, which it ligates to form the mature extein . Over 500 putative inteins , ranging in length from 100 to 1650 residues, have so far been identified in archaebacteria , eubacteria , single-celled eukaryotes, and viruses.

24. Protein splicing Attack by the N-terminal intein residue ( Ser,Thr , or Cys ; shown in Fig. 32-73 as Ser) on its preceding carbonyl group, yielding a linear ( thio )ester intermediate. 2. A transesterification reaction in which the ￢OH or ￢SH group on the C- extein’s N-terminal residue attacks the above ( thio )ester linkage, thereby yielding a branched intermediate in which the N- extein has been transferred to the C- extein .

25. 3. Cleavage of the amide linkage connecting the intein to the C- extein by cyclization of the intein’s C-terminal Asn or Gln . The succinimide ring of the excised intein then spontaneously hydrolyzes to regenerate Asn (or iso-Asn ). 4. Spontaneous rearrangement of the ( thio )ester linkage between the ligated exteins to yield the more stable peptide bond.

26. Most Inteins Encode a Homing Endonuclease All inteins contain polypeptide inserts forming so-called homing endonucleases . The break initiates the double-strand break repair of the DNA via recombination. Most inteins mediate a highly specific transposition or “homing” of the genes that insert them in similar sites. The protease activity excises the intein from the host protein, thereby preventing deleterious effects on the host, whereas the endonuclease activity assures the mobility of the intein gene.

27. 4.Non-covalent modifications Addition of metal ions and co-factors: Calcium (Ca++): very important intra-cellular messenger, i.e. calmodulin Magnesium (Mg++): ATP enzymes Copper (Cu++), Nickel (Ni+), Iron (Fe++) Zinc (Zn++): Zinc finger domains are used for DNA recognition Modifications involving tertiary structure (protein fold) Enzymes called molecular chaperones are responsible for detecting mis -folded proteins. Chaperones only bind mis -folded proteins that exhibit large hydrophobic patches on their surfaces.

28. A zinc finger domain: Zn++ is bound by two cysteine and two histidine residues. Zinc finger domains interact in the major groove with three consecutive bases from one strand of duplex Bform DNA.

29. Chaperones: Mediate folding and assembly. Do not convey steric information. Do not form part of the final structure. Suppress non-productive interactions by binding to transiently exposed portions of the polypeptide chain. First identified as heat shock proteins ( Hsp ). Hsp expression is elevated when cells are grown at higher-than-normal temperatures. Stabilize proteins during synthesis. Assist in protein folding by binding and releasing unfolded/ mis -folded proteins. Use an ATP-dependent mechanism 5.Chaperone-Assisted Protein Folding

30. Major types of chaperones: Hsp60, Hsp70 , Hsp90 (cytoplasm, ER, chloroplasts, mitochondria): thought to bind and stabilize the nascent polypeptide chain as it is being extruded from the ribosome. also involved in "pulling" newly synthesized polypeptide into ER lumen.

31. Refference : 1.Biochemistry- Donald voet / Jadith G. Voet , 4 th edition, page no:1403-1408 2.Principles of Biochemistry- A Lehninger , David L. Nelson and M.M.Cox CBS pub. 1993 page no.235-236 3.Biotechnology- Usathyanarayana , Arunabha publisher page no:56