Post translational modifications Flashcards
What are 4 fixed physical post translational modifications?
Glycosylation. Myristoylation. Oxidation. Prenylation.
6 Regulatory reversible modifications:
Acetylation. ADP-ribosylation. Methylation. Palmitoylation. Phosphorylation. Ubiquitylation.
Example of oxidation PTM (physical). Draw it.
Disulphide cys-cys bonds.
Why do disulphide bridges benefit extracellular proteins?
Stabilise them, making them heat and protease resistant and structurally resilient.
Intracellular environment is too reducing for disulphide bonds, which are made by oxidation in the endoplasmic reticulum.
Myristoylation: what, on which aa, how? why?
Myristic acid (fatty acid). Attaches by amide bond to N terminal of Glycine. (G) (following removal of methionine residue from n-terminus)
N-myristoylation allows hydrophilic proteins to anchor into the lipid bilayer.
…..S/TxxxGM sequence recognised.
(Iso-)Prenylation (lipidation)
Addition of a geranyl-geranyl or farnesyl (e.g. Ras) hydrophobic group to a sulphur of a cysteine residue in a CaaX-motif at Carboxyl terminus.
By thioether bond.
(aaX later removed and Remaining carboxyl terminus methylated.)
(Allows anchoring to cell membrane. Important in protein protein and protein membrane interactions.)
CaaX-motif
Target of prenylation at carboxyl terminus of protein. C = cysteine. a = Alanine or other small aliphatic amino acid (e.g. non-aromatic hydrocarbon side chain). X could be a variety of amino acids, which determines whether it is targeted by farnesyltransferase or geranylgeranyltransferase.
What is KRAS?
Product of KRAS proto-oncogene. A GTPase usually anchored to cell membrane by prenyl group. Essential to normal cell signalling but mutations can lead to cancers. Farnesyltransferase inhibitors trialled as cancer treatment, failed as geranylgeranyl added instead.
Myristoylation target sequence
At the N-terminus. Serine or Threonine, 3amino acids then glycine then methionine. S/TxxxGM. Methionine removed from end. Myristic acid added by amide bond.
What is Arf1?
Lipid vesicle transport regulator protein. N-myristoylation binds this to golgi vesicle membrane.
N-linked Glycosylation:
(asparagine linked)
(3 other types of glycosylation, most are poorly understood. )
This is the most common: attachment, to Asparagine (N) amino group, of 2 N-acetyl glucosamines (NAGs) with long branched chains of mannose ‘antennae’.
Further modifications occur in ER and then Golgi bodies.
Why doesn’t N-linked glycosylation happen to all Asparagine (N) containing proteins?
Enzyme recognises common NxS/T sequence.
But only happens to proteins sent to endoplasmic reticulum.
Generally why don’t intracellular proteins need glycosylation as an indicator of age?
Rapid turnover by proteosomes, regeneration.
A common use of glycosylation of proteins:
As a marker for the age of the proteins.
Extracellular proteins are checked for presence of sialic acids at end of their antennae by receptors on lymphocytes (e.g.T or NK cells).
If they are not there they are degraded
Where is Fucose found?
Fucose is a sugar monomer added in the Golgi bodies to the first NAG of the biantennary structure of glycosylated proteins.
Why is the cysteine carboxyl group left following prenylation then methylated?
To remove the negative charge of the carboxylic acid, allowing it to interact with the cell membrane.
Phospholipids have negatively charged phosphate heads!
How are the 2 N-acetyl glucosamine (NAG) residues added to the asparagine (N) of a protein?
To be glycosylated a protein bearing the common NxS/T motif is sent to the **endoplasmic reticulum. **
2 NAG residues are bound to the side group nitrogen of Asn, N, residue by N-glycosidic bond.
(they themselves are connected by a B-1,4-glycosidic bond.)
How does HIV use glycosylation?
Has glycosylated surface proteins (e.g. the CD4 binding gp120 protein)
(NxS/T motif containing)
Hence evades immune detection.
Palmitoylation:
Regulatory modification adding palmitic acid to sulfur on Cysteine residues (cysteine/leucine clusters) by reversible thioester bond.
Reversibly targets proteins between membrane bound and cytosolic locations.
Acetylation?
Regulatory modification of histones, (AcK recognised by bromodomains.)
Acetyl CoA donates acetyl group to (positive) Lysine, K, residues.
(acetyl transferase and deacetylase enzymes)
What do bromodomains recognise?
why important?
Acetyl lysine residues. AcK.
Often on histone H3 tails.
Polybromo protein contains many bromodomains and targets a chromatin remodelling complex to areas rich in AcK.
Structure of a nucleosome?
DNA wrapped around 4 different types of histone proteins. (2 of each kind)
Histone H3s are important as their N-terminus ends project out, and can be modified. (e.g. acetylated, methylated and phosphorylated)
(linker, H1, histones on DNA between nucleosomes, can be PARylated)
Methylation?
(using S-adenosyl methionine)
Methyltransferases transfer methyl groups from S-adenosyl methionine.
Onto arginines or lysines.
Maintains positive charge of amino acids, important for interactions with certain domains.
(demethylases remove methyl groups)
Lysine methylation: who dunnit? who cares?
Lysine positive side chain amino groups (on histone H3 N-terminal tail) methylated by lysine methyl transferases, KMTs. (multiple times)
Me2K recognised by Tandem tudor domains (interested in remaining Hydrogen)
(Non-polar) Me3K recognised by hydrophobic pocket of **Chromo domains. **(but also with surrounding negative residues!)
Both of these domains commonly found in proteins that interact with chromatin.
