What is the role of PTM?
Diversify the human proteome by at least a factor of 10.
- over 200 PTMs
- 25k genes = ~100k
- x10 by PTM = ~1 million
W/o them, cannot survive
What are the 2 types of covalent modifications of proteins?
1) enzyme-assisted covalent addition/elimination of a chemical group
2) covalent cleavage of peptide fragments in protein driven by professes or, less frequently by auotocatalytic cleavage
What do kinases, phosphatases, ligases + transferase?
Add/remove functional groups, proteins, lipids or sugars to or from amino acid sequences
What do proteases do?
Cleave peptide bonds to remove specific sequence or regulatory subunits
How many genes are involved in encoding enzymes that intervene in PTM of proteins?
How many genes are involved in encoding enzymes involved in glycosylation?
5%
1%
What is the function of PTMs?
- protein folding
- protein stability
- exo and endocytosis
- apoptosis
- differentiation
- cell division
- housing of prosthetic groups (adding haem group to haemaglobin)
- vesicular trafficking (tell cell where to transport protein)
- ligand binding e.g. notch (receptor on cell surface may bind diff ligands - dependent on PTM; evident in notch signalling - can bind to delta or jagged > diff in binding depends on PTM of notch)
- single transduction (>ligand>phosphorylation of residues>activate cascade)
- gene expression (histones PTM by methylation - depends if genes are transcribed or not)
What adds and removes phosphates?
Add = kinase
Remove = phosphatases
Depends which one activates
What kind of a PTM is phosphorylation?
- a reversible PTM
- regulates protein function
- adds phosphate to serine, threonine or tyrosine
What type of PTM is glycosylation?
- the most abundant and diverse PTM
- more than 50% of human proteome is glycosylated (many secreted proteins, cytosolic and nucleus proteins)
- all membrane proteins are glycosylated
What is altered in cancers?
Glycosylation
= change in phenotype + interaction of cells and trafficking
What is the glycocalyx?
- produced by sugars
- sugary coat
- involved in protection of epithelium e.g. in gut
- 1st thing any cell or growth factor encounters
What are the most common types of glycosylation?
1) N-linked g
2) O-linked g
What are the 4 most common types of PTM?
1) glycosylation
2) phosphorylation
3) methylation
4) proteolysis
5) acetylation: addition of acetyl groups to histones and lysines
6) ubiquitination = mod of protein with a highly conserved 76 AA polypeptide ubiquitin - tell protein to move
What are the two types of covalent modifications of proteins?
1) enzyme-assisted covalent addition/elimination of a chem group
2) covalent cleavage of peptide fragments in protein driven by proteases or, less frequently by autocatalytic cleavage
What is the function of PTM?
To diversify human proteome
- over 200 PTM
- 25k genes = ~100k proteins
- with PTM = x10 so ~1 million
When do PTMs occur?
- at any step in the “life cycle” of a protein eg:
1) modifies after translation to mediate proper folding or stability, or to direct nascent protein to distinct cellular compartments
2) to activate/inactivate catalytic activity or to otherwise influence the biological activity of the protein eg. Surface, cytoplasm, nucleus
3) proteins covalently linked to tags that target a protein for degradation
4) often modified through a combo of post-translational cleavage and addition of functional groups through a step-wise mechanism of protein maturation or activation
What are pro-protein concertases?
Family of proteins that activate other proteins.
(Many proteins inactive when first synthesised bc of chains of AAs that block their activity. PP remove these chains and activate the protein)
Artenstein and Opal (2011)
How does proteolysis work?
By pro-protein convertases, which induce the activation of proteins/peptides
What is glycosylation vital for?
The function of proteins and glycolipids, cells and organisms
What type of glycans are found in the cell?
1) proteoglycans
2) glycoproteins
3) glycosphingolipids
???
What is N-linked glycosylation?
- initiated in the ER
- initial sugars added en bloc to asparagine in the sequence Asp-N-Ser/Thr (potential site for N-linked glycosylation)
What is O-linked glycosylation?
- initiated in golgi
- O-GlcNAc* can be added in the cytoplasm
- sugars are added singly and sequentially (to serine or threonine)
- Many nuclear and cytoplasmic proteins have this single sugar attached (the O-GlcNAc)
How does proteolysis work by proprotein convertases to induce the active form of insulin (protein)?
1) signal sequence on preproinsulin is cleaved* off in the ER = proinsulin
2) proinsulin = inactive PP.
• c-chain removed + disulphide bonds formed within the ER —> keep chain A & B together
3) formation of insulin = active
*cleavage occurs in the secretory granules of pancreatic beta cells
• preproinsulin stored in secretory granules until required
(2 PROTEOLYSIS STEPS RESULTING IN ACTIVE INSULIN PP)
What are the steps in insulin signalling?
1) Insulin binds to receptor = phosphorylation of insulin chains
2) allows phosphorylation of the Shc protein
3) causes a cascade down MAPKK pathway = differential gene expression
OR
2) phosphorylation of IRS > phos of PI3K > phos of PDK1/2
3) causes AKT pathway to be activated = movement of GLUT-4 to the surface = enhances glucose transport
