Non-Phosphor Based PTMs in Signalling

Question 1

What is ubiquitination?

Answer 1

Also called ubiquitylation, this is the addition of the small and highly stable 76AA protein onto another protein.

Question 2

What is the difference between mono and poly ubiquitination?

Answer 2

Target can be mono or poly-ubiquitinated, with the former generally associated with histone modification and the latter with protein degradation.

Question 3

How can polyubiquitination have various effects?

Answer 3

Chains of ubiquitin can also be build up into different branching patterns/shapes that can be recognised, thus creating a ubiquitin code.

Ubiquitin can be linked to other ubiquitins by various lysines, changing the shape of the chain. The most commonly used ones are Lys11, Lys29, Lys48 and Lys63.

Question 4

What adds ubiquitin to things?

Answer 4

Ubiquitin is added onto lysine residues by a three step process involving three enzymes; E1 (ubiquitin activating enzymes), E2 (ubiquitin conjugating enzymes) and E3 (ubiquitin ligases).

Question 5

What is the first step in ubiquitination?

Answer 5

The first step, activation, involved the ATP dependent E1 priming the ubiquitin by acyl adenylation of its C-terminus. This leads to the ubiquitin forming a thioester linkage between the modified c-terminus and an E1 active site cysteine residue

Question 6

What is the second step in ubiquitination?

Answer 6

The second step is conjugation, in which the ubiquitin is transferred to one of the 35 different E2 proteins by trans(thio)esterification onto an E2 active site cysteine.

Question 7

What is the third step in ubiquitination?

Answer 7

With the ubiquitin properly primed by E1 and E2, E3 carries out the recognition and recruitment of the target protein, thus providing the specificity of the process in the final step; ligation.

E3 binds both the target and E2/ubiquitin and catalyses the transfer of the ubiquitin to the lysine. This occurs when the lysine makes a nucleophilic attach on the thioester bond to create its own isopeptide bond.

Question 8

What degradation pathways can a protein be targeted for by polyubiquitination?

Answer 8

26S Proteasome Pathways

Lysosome Degradation of Membrane Proteins

Question 9

What kind of polyubiquitination targets proteins for the 26S Proteasome Pathway?

Answer 9

An unbranched chain of ubiquitin is created by adding each ubiquitin onto the lasts’ (usually Lys48) residue. This is the way in which E6AP-p53 is marked for degradation.

Question 10

What is the most common form of polyubiquitination in the cell?

Answer 10

Lys48 chains are the most common form of polyubiquitination in the cell, noted for their incredible increase in their levels when the 26S proteasome is inhibited.

Question 11

What other kind of unbranched ubiquitin chains are involved in 26S proteasome targeting?

Answer 11

Lys11 chains are the second most commonly used for proteasome targeting, as they are found on APC/C substrates during mitosis. Lys29 and Lys63 ubiquitin chains are also used but far less frequently.

Question 12

Describe lysosome degradation of membrane proteins. You twat.

Answer 12

This can be mediated by monoubiquitination or by Lys63-linked polyubiquitination. It targets membrane proteins to the clathrin coated pits so that they will be captured into a vesicle during endocytosis and taken to the lysosome for degradation.

Question 13

How is lysosome degradation of membrane proteins used to regulate a signalling pathway?

Answer 13

This is used to downregulate EGF-EGFR signalling by internalising the receptor followed by Cbl-dependent degradation. EGFRvIII, a mutated EGFR found in brain tumours, is however immune to this process.

Question 14

What is the role of deubiquitinases?

Answer 14

Deubiquitinases can be used to remove the polyubiquitin chains from a protein before it is degraded as a method of rescuing proteins that are suddenly needed.

Question 15

What is the NF-kB pathway mechanism?

Answer 15

Activation of this pathway by antigen, cytokines et al, causes phosphorylation of the IKKβ subunit of the IKK complex, causing it to phosphorylate the IκBα.

The IκBα is normally bound to and repressing the NF-κB dimers but upon phosphorylation it dissociates and becomes targeted for degradation by polyubiquitination.

The free NF-kB acts as a TF for cell survival, inflammation and proliferative genes.

Question 16

How does bortezomib work?

Answer 16

Bortezomib (marketed as Velcade) is a drug that is used as a treatment for multiple myeloma. It does this by preventing the degradation of IκBα, an intermediate in the NF-κB signalling pathway that promotes cell survival, proliferation and also inflammation.

Question 17

How can ubiquitination directly regulate proteins?

Answer 17

Regulating Inter-protein Interaction

Regulating Protein Localisation

Regulating Protein Activity

Question 18

What is TGFβR Signalling?

Answer 18

In this pathway Smad2 and Smad4 form a complex which stimulates transcription of TGF-βR target genes.

Question 19

How is ubiquitination used to regulate the TGFβR?

Answer 19

When Smurf2 monoubiquitinates Smad4 is blocks the interaction between it and Smad2. This is reversed by the deubiquitinase USP15, which promotes the transcriptional activity.

Question 20

How is ubiquitination used in DNA Repair Complex Assembly?

Answer 20

Monoubiquitination is vital to the recruitment of complexes involved in replication and DNA repair.

One example of this is the recruitment of DNA Polymerases to the PCNA (Proliferating Cell Nuclear Antigen) during translesion synthesis.

Question 21

When is ubiquitination involved in protein localisation?

Answer 21

PTEN, p53 and FOXO

Question 22

How is ubiquitination used to regulate PTEN?

Answer 22

Whether in the cytoplasm or the nucleus PTEN can stimulate apoptosis by inhibiting the cell-survival factor Akt.

Monoubiquitination of PTEN by NEDD4-1 promotes nuclear import of the phosphatase into the nucleus. NEDD4-1 can however also polyubiquitinated PTEN leading to degradation

Question 23

How is ubiquitination used to regulate p53?

Answer 23

This tumour-suppressing protein can be monoubiquitinated by Mdm2 or MSL2 leading to nuclear export and so loss of tumour suppression.

Some cancers, such as Glioblastoma multiforme present with amplified Mdm2. Higher risk strains of HPV can cause in-nucleus polyubiquitination and subsequent degradation of p53 which can lead to cancer.

Question 24

How is ubiquitination used to regulate protein activity?

Answer 24

Ubiquitination is involved in the regulation of TNFα (Tumour Necrosis Factor) signalling to NFkB.

LUBAC conjugates Met-1 linked linear ubiquitin chains onto the NEMO subunit of IKK, activating it.

NEMO can also bind to Lys63 linked ubiquitin chains, which can be used to bind it to the Receptor Interacting Protein (RIP1) or to the TAK1 Kinase complex which leads to further activation of IKK.

Question 25

What is SUMOylation?

Answer 25

Like ubiquitination SUMOylation is the addition of a small protein to the target. In fact SUMO (Small Ubiquitin-related Modifier) is structurally very similar to ubiquitin, despite sharing only 20% sequence identity.

Question 26

What is the mechanism of SUMOylation?

Answer 26

SUMO is also similar to ubiquitin in that it is added on to proteins in a process mediated by three proteins, though in this instance the SUMO is activated by cleavage of the protein chain at a Gly-Gly motif near the C-terminus.

The rest of the mechanism is more similar to ubiquitin, with formation of an isopeptide bond between the C-terminal glycine of the SUMO and the epsilon amino group of the target lysine.

Question 27

How is SUMOylation used to regulate PTEN?

Answer 27

SUMOylation is very important in the regulation of PTEN. Addition at Lys254 of the PTEN promotes nuclear import, while SUMOylation of Lys 266 is required for full catalytic activity when localised to the plasma membrane.

Question 28

How does lysine acetylation affect proteins in free protein regulation?

Answer 28

The mechanism is the same as in histones – acetylation of the positively charged lysine residues negates the charge thus altering the binding properties and potentially structure of the protein.

Question 29

What substitutions can be used to mimic constant acetylation/deacetylation?

Answer 29

Lysine residues can be mutated to arginine to mimic constant deacetylation and glutamine to mimic the acetyl-lysine.

Question 30

What adds acetyl groups onto lysines?

Answer 30

KATs, known previously only as HATs are responsible for adding acetyl groups onto lysines.

Question 31

What is associated with KAT mutation?

Answer 31

Mutation of two closely related KATs (KAT5A and KAT5B) is responsible for Rubinstein-Taybi Syndrome. These were formerly (and are still commonly) called CREBBP and EP300 respectively.

Question 32

What removes acetylation from lysines?

Answer 32

Lysine Deacetylases (KDACs).

Like KATs, these were formerly referred to as HDACs before their importance in signalling was recognised.

Question 33

What uses are there for KDAC inhibitors?

Answer 33

KDAC inhibitors have been used as anti-cancer drugs, such as Vorinostat which is used to treat Cutaneous T-Cell Lymphoma.

It has also been shown to prevent type I diabetes in mice by protecting the pancreatic beta cells from auto-immune destruction.

The health benefits of KDAC inhibitors have been attributed to the decrease in TF acetylation rather than histone acetylation.

Question 34

What is the role of KATs in the DNA damage response?

Answer 34

Lysine acetylation is used to fine-tune the response to DNA damage, which is controlled by the action of p53.

Question 35

What is the response when DNA is moderately damaged?

Answer 35

p53 is acetylated by a KAT called PCAF at Lys320. This promotes nuclear import allowing it to inhibit growth and possibly stimulate DNA repair, though this remains unclear. The acetylation is thought to promote nuclear import by interfering with the monoubiquitination of p53.

Question 36

What is the response when DNA is severely damaged?

Answer 36

Damage-responsive kinases such as ATM (Ataxia Telangiecstasia Mutated) phosphorylate p53, which promotes acetylation by KAT5A/B.

This acetylation in the C-terminal domain stabilises p53, leading to accumulation of the factor, high transcription of its target genes and consequent apoptosis.

Question 37

How are KDACs involved in FOXO localisation?

Answer 37

Molecules in red wine activate the SIRT KDACs, which remove the acetylation from FOXO1, activating it and binding to target genes.

FOXO has been shown to be involved with ageing in nematodes due to its involvement with the insulin signalling pathway

Question 38

What is an example of acetylation activating an enzyme?

Answer 38

Autoacetylation of the p300 catalytic domain lysine residues increases its acyltransferase activity.

Question 39

What is an example of acetylation inhibiting an enzyme?

Answer 39

When stimulated by growth factor signalling, PCAF acetylates PTEN at lysine residues in the catalytic domain, inhibiting its phosphatase activity.

Question 40

What residues can be methylated?

Answer 40

Lysine and arginine

Question 41

In what ways can arginine be methylated?

Answer 41

Arginine is methylated by PRMT, and can be mono or dimethylated.

Dimethylated arginine can be symmetrical or asymmetrical depending on whether the methyl groups are on the same or on different amino groups.

Question 42

In what ways can lysine be methylated?

Answer 42

Lysine methylation can be mono, di or tri, and is performed by HMT enzymes.

Question 43

How is methylation used to regulate p53?

Answer 43

Methylation of p53 can be on lysine or on arginine residues.

SER 7/9 methylates it, which recruits p300/KAT5B to acetylate it.

Arginine methylation of p53 promotes oligomerisation and nuclear import, which affects its cell cycle arrest function but not its apoptosis regulation.

Question 44

How are prolines hydroxylated?

Answer 44

This is performed by proline hydroxylase domain proteins in an oxygen dependent reaction.

Question 45

What does the nature of the proline hydroxylation reaction enable?

Answer 45

Because of the oxygen-denpendence it is used as an oxygen sensor in the context of HIF (hypoxia inducible factor) proteins.

HIF causes transcription of HIF target genes by binding to heat response elements, which can have widespread effects in the cell.

Question 46

What recognises proline hydroxylation of HIF-1α ?

Answer 46

VHL ubiquitin ligase, which targets HIF for degradation, thus removing the HIF target genes effect in response to higher oxygen levels.

Question 47

What do VHL mutations cause?

Answer 47

VHL mutations cause the disease for which the proteins are named; Vonn-Hippel-Lindau Syndrome, which is characterised by a predisposition for tumours, particularly hemangioblastomas which are a benign but often serious kind of blood vessel neoplasm.

VHL is also frequently mutated in kidney cancer.