Post translational modification
- 1. Definitions of the components: Part 1 – Overview of PTMs 5 3 2 4 1 1. Post-translational modification (PTM): The chemical modifications that take place at certain amino acid residues after the protein is synthesized by translation are known as post- translational modifications. These are essential for normal functioning of the protein. Some of the most commonly observed PTMs include: a) Phosphorylation: The process by which a phosphate group is attached to certain amino acid side chains in the protein, most commonly serine, threonine and tyrosine. Critical role in cell cycle, growth, apoptosis and signal transduction pathways. b) Glycosylation: The attachment of sugar moieties to nitrogen or oxygen atoms present in the side chains of amino acids like aspargine, serine or threonine. Significant affect on protein folding, conformation and stability. c) Acylation: The process by which an acyl group is linked to the side chain of amino acids like aspargine, glutamine or lysine. d) Alkylation: Addition of alkyl groups, most commonly a methyl group to amino acids such as lysine or arginine. Other longer chain alkyl groups may also be attached in some cases. e) Hydroxylation: This PTM is most often found on proline and lysine residues which make up the collagen tissue. It enables crosslinking and therefore strengthening of the muscle fibres.
- 2. Definitions of the components: Part 1 – Overview of PTMs 5 3 2 4 1 2. Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids. 3. Cytosol: A cellular compartment that serves as the site for protein synthesis. 4. Signal sequence: A sequence that helps in directing the newly synthesized polypeptide chain to its appropriate intracellular organelle. This sequence is most often cleaved following protein folding and PTM. 5. Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for post- translational modification of the newly synthesized polypeptide chains. 6. Cleaved protein: The protein product obtained after removal of certain amino acid sequences such as N- or C-terminal sequences, signal sequence etc.
- 3. Types of PTMs PTMs can be categorized as: Trimming Covalent Attachment Protein Folding
- 4. 1 5 3 2 4 Part 1, Step 1 Process of post-translational modification Cytosol Endoplasmic reticulum (ER) P P Glc Glc CH3CH3 Cleaved protein Protein folding & PTMs mRNA Ribosome Protease Removal of certain N- and C-terminal residues As shown in animatio n. First the ‘mRNA’ & ‘ribosome’ must be shown in the ‘cytosol’. The ‘ribosome’ must move across the mRNA as shown and as it moves, the ‘protein’ must appear slowly as though it is growing out of the ribosome (not depicted here). Next, this ‘protein’ must enter the ‘ER’ through the green channels shown. Next, the pie-shaped ‘protease’ must appear which must cut the pink strand followed by the red strand followed by appearance of text on the left. Next, the arrows must appear one at a time with their respective figures on the right. Once the protein has been synthesized by the ribosome from its corresponding mRNA in the cytosol, many proteins get directed towards the endoplasmic reticulum for further modification. Certain N and C terminal sequences are often cleaved in the ER after which they are modified by various enzymes at specific amino acid residues. These modified proteins then undergo proper folding to give the functional protein. Action Description of the action Audio Narration Translated Protein Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
- 5. 1 5 3 2 4 Part 1, Step 2 Different types of PTMs & their modification sites Ser, Thr, Tyr Asn, Ser, Thr Asn, Gln, Lys Lys, Arg Pro, Lys As shown in animatio n. First show the pie chart as depicted. Next, each segment must be highlighted sequentially along with appearance of the corresponding text in the boxes as depicted. There are several types of post translational modifications that can take place at different amino acid residues. The most commonly observed PTMs include phosphorylation, glycosylation, methylation as well as hydroxylation and acylation. Many of these modifications, particularly phosphorylation, serve as regulatory mechanisms for protein action. Action Description of the action Audio Narration
- 6. 1 5 3 2 4 Part 1, Step 3 Increased complexity of proteome due to PTMs A C G G U G C C G U G C A C GA C A C U A C G C A C U Gene sequence Expected protein structure Actual protein structure P CH3 Glc As shown in animatio n. First show the ‘gene sequence’ on top followed by the arrow to the left and the blue structure. This must be zoomed into to show the inset below. The red cross must then appear on this arrow. Next, the arrow to the right must appear followed by the pink structure which must again be zoomed into to show the inset below. The final structure of functional proteins most often does not correlate directly with the corresponding gene sequence. This is due to the PTMs that occur at various amino acid residues in the protein, which cause changes in interactions between the amino acid side chains thereby modifying the protein structure. This further increases the complexity of the proteome as compared to the genome. Action Description of the action Audio Narration
- 7. 1 5 3 2 4 Part 1, Step 4 Phosphorylation reactions Ser R CH2 CH CH3 CH2 Thr Tyr ATP ADP Kinase As shown in animatio n. First show the figure on the top left entering followed by the box below having the various three arrows from “R”. Next the arrow must ease in along with the curved arrow below. Finally, the figure on the right must appear. Phosphorylation of amino acid residues is carried out by a class of enzymes known as kinases that most commonly modify side chains of amino acids containing a hydroxyl group. Phosphorylation requires the presence of a phosphate donor molecule such as ATP, GTP or other phoshorylated substrates. Serine is the most commonly phosphorylated residue followed by threonine and tyrosine. Removal of phosphate groups is carried out by the phosphatase enzyme and thus this forms one of the most important mechanisms for regulation of proteins. Action Description of the action Audio Narration Amino acid residue Phosphorylated residue OHC NH3 + COO- RH OC NH3 + COO- R PO4 3- H th
- 8. 1 5 3 2 4 Part 1, Step 5 Glycosylation reactions Ser/Thr Asn Glycosyl transferase N-linked Glycosylation O-linked Glycosylation Glycosyl transferase Sugar residues N-linked amino acid O-linked amino acid As shown in animatio n. First show appearance of the figure on top let with heading followed by arrow and finally product on right. Similar animation must be carried out for the second reactions. Glycosylation involves the enzymatic addition of saccharide molecules to amino acid side chains. This can be of two types – N-linked glycosylation, which links sugar residues to the amide group of aspargine and O-linked glycosylation, which links the sugar moieties to the hydroxyl groups of serine or threonine. Suitable glycosyl transferase enzymes catalyze these reactions. Sugar residues that are attached most commonly include galactose, mannose, glucose, N- acetylglucosamine, N-acetylgalactosamie as well as fucose. Action Description of the action Audio Narration CONH2 C NH3 + COO- CH2H CONC NH3 + COO- CH2H OHC NH3 + COO- RH OC NH3 + COO- RH th
- 11. Protein Folding Proteins must fold to assume their functional state. Folding can be spontaneous or facilitated by proteins known as Chaperons. The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller hsp40 proteins (not shown), ATP-bound hsp70 molecules grasp their target protein and then hydrolyze ATP to ADP, undergoing conformational changes that cause the hsp70 molecules to associate even more tightly with the target. After the hsp40 dissociates, the rapid rebinding of ATP induces the dissociation of the hsp70 protein after ADP release. Repeated cycles of hsp binding and release help the target protein to refold.
- 12. Ubiquitinylation Proteins are usually modified for selective destruction by cellular proteolytic complexes called proteasomes by attachment of ubiquitin. Ubiquitinylation involves attachment of a small 76 amino acid protein called ubiquitin to the polypeptide chain following which it is degraded by the cellular proteasome machinery. Ubiquitin binds to the lysine residue of the target protein, present in a specific sequence. The tagged protein is then recognized by the proteasomes complex and it gets degraded. Involved in programmed cell death, DNA repair, immune and inflammatory processes. E1= Ubiquitin activating enzyme E2= Ubiquitin conjugating enzyme E3= Ubiquitin ligase Seissler, T et al (2017)
Editor's Notes
- #12: The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller hsp40 proteins (not shown), ATP-bound hsp70 molecules grasp their target protein and then hydrolyze ATP to ADP, undergoing conformational changes that cause the hsp70 molecules to associate even more tightly with the target. After the hsp40 dissociates, the rapid rebinding of ATP induces the dissociation of the hsp70 protein after ADP release. Repeated cycles of hsp binding and release help the target protein to refold
- #13: Figure 1. Schematic representation of the ubiquitin-proteasome system. (A) Transfer of ubiquitin from the ubiquitin-activating enzyme E1 to the ubiquitin-conjugating enzyme E2 followed by its transfer onto the target protein X by the ubiquitin ligase E3. The broken line symbolizes the thiol-ester bond; (B) the 26S proteasome, composed of the 20S barrel and two 19S lids. The ubiquitinated target protein X is recognized by one of the lids and translocated through the barrel where it is degraded by the proteases located on the inside of the β-rings.