Post translational modifications

Question 1

What are 4 fixed physical post translational modifications?

Answer 1

Glycosylation. Myristoylation. Oxidation. Prenylation.

Question 2

6 Regulatory reversible modifications:

Answer 2

Acetylation. ADP-ribosylation. Methylation. Palmitoylation. Phosphorylation. Ubiquitylation.

Question 3

Example of oxidation PTM (physical). Draw it.

Answer 3

Disulphide cys-cys bonds.

Question 4

Why do disulphide bridges benefit extracellular proteins?

Answer 4

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.

Question 5

Myristoylation: what, on which aa, how? why?

Answer 5

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.

Question 6

(Iso-)Prenylation (lipidation)

Answer 6

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.)

Question 7

CaaX-motif

Answer 7

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.

Question 8

What is KRAS?

Answer 8

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.

Question 9

Myristoylation target sequence

Answer 9

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.

Question 10

What is Arf1?

Answer 10

Lipid vesicle transport regulator protein. N-myristoylation binds this to golgi vesicle membrane.

Question 11

N-linked Glycosylation:

(asparagine linked)

Answer 11

(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.

Question 12

Why doesn’t N-linked glycosylation happen to all Asparagine (N) containing proteins?

Answer 12

Enzyme recognises common NxS/T sequence.

But only happens to proteins sent to endoplasmic reticulum.

Question 13

Generally why don’t intracellular proteins need glycosylation as an indicator of age?

Answer 13

Rapid turnover by proteosomes, regeneration.

Question 14

A common use of glycosylation of proteins:

Answer 14

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

Question 15

Where is Fucose found?

Answer 15

Fucose is a sugar monomer added in the Golgi bodies to the first NAG of the biantennary structure of glycosylated proteins.

Question 16

Why is the cysteine carboxyl group left following prenylation then methylated?

Answer 16

To remove the negative charge of the carboxylic acid, allowing it to interact with the cell membrane.

Phospholipids have negatively charged phosphate heads!

Question 17

How are the 2 N-acetyl glucosamine (NAG) residues added to the asparagine (N) of a protein?

Answer 17

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.)

Question 18

How does HIV use glycosylation?

Answer 18

Has glycosylated surface proteins (e.g. the CD4 binding gp120 protein)

(NxS/T motif containing)

Hence evades immune detection.

Question 19

Palmitoylation:

Answer 19

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.

Question 20

Acetylation?

Answer 20

Regulatory modification of histones, (AcK recognised by bromodomains.)

Acetyl CoA donates acetyl group to (positive) Lysine, K, residues.

(acetyl transferase and deacetylase enzymes)

Question 21

What do bromodomains recognise?

why important?

Answer 21

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.

Question 22

Structure of a nucleosome?

Answer 22

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)

Question 23

Methylation?

(using S-adenosyl methionine)

Answer 23

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)

Question 24

Lysine methylation: who dunnit? who cares?

Answer 24

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.

Question 25

What is recognised by:

Bromodomains?

Chromodomains?

Tudor domains?

Answer 25

Bromodomains recognise acetylated lysines (amino side group)

Chromodomains recognise trimethylated lysines. (Me₃K non-polar)

Tudor domains recognise dimethylated lysines, Me₂K, and symmetrical dimethylated arginine, sMe₂R!

Question 26

Arginine methylation?

Answer 26

Arginine has 2 amino groups on its side chain that can be methylated by RMTs (arginine methyl transferases)

It can be methylated once (MeR) or twice:

Symmetrical methylation sMe₂R is one on each Nitrogen. (recognised by Tudor domains)

Asymmetrical, aMe₂R is 2 on one nitrogen.

Question 27

Role of poly-ADP-ribosylation? (PARylation)

Answer 27

Signals site of DNA damage by linker histone (H1) modification. (on lysines, K, or glutamic acids, E)

( Addition of negatively charged polymers of ADP ribose monomers donated by NAD. Competes with DNA for binding of histones, hence releasing DNA for repair)

Question 28

What do macro domains (and PBZ domains) recognise?

Answer 28

PARylated lysine, K, or glutamic acid, E, residues.

On linker H1 histones.

(bring DNA helicases to unwind DNA for repair)

Question 29

How are proteins targeted for destruction by the proteosome?

Answer 29

By poly-ubiquitylation of Lysine, K, residues.

In compact lysine 48 linked chains.

(contrasted with open, floppy K63 linked chains)

Question 30

What bond connects ubiquitin to a lysine residue?

How is it made?

Answer 30

An “isopeptide” bond from the side chain amino of lysine to carboxyl-terminus of ubiquitin.

Made by ubiquitin ligase (E3) transferring ‘activated’ ubiquitin from ubiquitin conjugating enzyme. (E2)

Question 31

How is Ubiquitin activated?

Answer 31

By E1 ubiquitin activating protein, using ATP.

(before transfer to E2 ubiquitin conjugating enzyme)