Week 4.1 Ubiquitin

Question 1

What is ubiquitin?

Answer 1

Ubiquitin is a small 76 amino acid protein that is covalently attached to other proteins, and acts as a signal for ‘something to happen’

It has a comma like structure;

Shown bellow

Question 2

Where is ubiqutin found?

Is it found in prokaryotes?

Answer 2

Ubiquitin found in all eukaryotes, it is highly conserved.

You dont find it in prokaryotes - but you do find things that are precursors to it.

Question 3

Where is it covalentlyattached? and by what?
What does it mostly attach to on other proteins? what else?

What are the two names fro the process of attachment?

Answer 3

Ubiquitin is covalently attached by a Glycine at C-terminnus - G76

It mostly attaches to lysine (K) in the protein substrates - but also found on Methionine (M), Cysteine (C) and Threonien (T).

Process refered to as ubiquitination or ubiquitylation

Question 4

Ubiquitylation involves a cascade of reactions mediatied by distinct enzymes

What are the three steps?
And what are there names?

How many variations of each enzymes?

Answer 4

1. Activation - E1 aka activation enzyme
2. Conjugation - E2 conjugation enzyme
3. Ligation - E3 ligation enzyme

There are about 2 enzymes encoding E1, 50 enzymes for E2 and over 600 for the ligation enzyme E3

Question 5

What question did the discovery of ubiquitin answer?

What was found to be the reason?

Answer 5

Why does intracellular proteolysis requrie ATP

Didnt seem to make energetic sense because there is alot of energy in amide bonds.

The reason that intracellular proteolysis consumes ATP is because it is very dependent on the process of ubiquitylation. and the process of ubiquitylation consumes ATP

Question 6

Process of ubiquitylation

Activation process

What does the E1 used to synthesize C-terminal adnylate?

What does E1 adenylate then do?

Answer 6

E1 uses ATP to synthesize C-terminal adnylate

E1 adenylate then acts as a substrate to form E1- ubiqutin thiol ester

Question 7

Ubiquitylation

Conjugation…

Where is the activated ubiquitin transferred to?

… and ligation

Where is the ubiqutin then transferred too?

Answer 7

The activated ubiquitin is transferred from the E1 to the E2 via thiol ester linkage

Then it is transferred to the lysine residue in the substrate via the actin of the ligase enzyme E3.

Question 8

Ubiquitylation

What is the critical enzyme in the ubiquitylation process?

What do ligases bind to, what does it therefore control?

What are the two basic classes of ligases?

Answer 8

E3 ubiquitin ligase

Ligases bind substrates and therefore controls specificity.

The ligation step is the most highly regulated

There are two distinct ligases with distinct mechanisms

1. RING/U-box type
2. HECT type

Question 9

RING-type (U-box)

What do they bind to?

Answer 9

Act as scaffold protein

The bind to both the substrate and the E2 and bring them together into the right orientation relative to each other to allow the transition to the substrate

Question 10

HECT-type

How does this work?

Answer 10

1st it takes the ubq off the E2 by forming thioester link and then transferes it to the substrate

Question 11

Ubiquitin ligases

There is a lot of diversity

What are the methods of regulation?

Answer 11

Can be single polypeptides or form part of large complexes

Has multiple stratergies for recognizing and binding to substrates

Regulated by many factors including other PTMs such as phosphorylation, oxidation and glycosylation and properties such as the folding status of substrate

Question 12

Ubiquitylation is a dynamic process there are a class of enezymes that are called De-ubiquinating enzymes (DUBs)

Why?

Answer 12

They are responsible for the removal ubiquitin - in a cyclcal pattern

Analogous to phosphorylation

Question 13

What was ubiquitin originally identified for?

But what is it now known for?

Answer 13

Originally identified due to role in intracellular proteolysis

Now known that ubiquitin is involved in regulating a huge range of cellular processes

Question 14

How is ubiquitin a multi-functional signal?

Given that the structure of ubiqutin is highly consereved how can it have mutliple signals?

Answer 14

By being able to have a range of attachment methods

Monoubiquitylation

Multi-site monoubiquitylation

Key one: Polyubiquitylation

Question 15

Ubiquitin is a multi-functional signal

When do you get?

What two main di-Ub forms are there?

Answer 15

You get a polymer with a different structure, depending on what is linking

K48 di-ubiquitin - is a very closed conformation

Wherease K63 is in contrast

Structually they are very different

Question 16

Ubiquitin - a multifunctional signal

If you have different signalling molecuels you also need effectors

What do the effector proteins contain?

Answer 16

Effector proteins contain a Ub-binding domain UBD

UBD is attached to a large protein -
e.g UBA, MIU, CUE

Have different binding specificities

Question 17

Example of Ub-binding domain

Particular hydrophobic patch is always on the same place on the K48 di-Ub

What is the UBD called? what is it recognised by?

Answer 17

UBD –> The UBA binding domain - recognised by specific receptors that can recognise hydrophobic patch

Also…the structure changes significantly with pH

Question 18

Polyubiquitylation

What determines the type and length of chain?

What are E4 enzymes?

What is chain editing?

Answer 18

Type and length of chain is determiend by E3 alone or E3 in combination with specific E2

A class of E3 enzymes are called E4 enzymes they are essentially modified Ub-ligases, that extend the pre-existing ubiquitylation - i.e extend the Ub chain

Chain editing: Chains can be editied by action of De-Ub/E3 complex

Deubiquitylation and Ubiquitylation

Question 19

What types of deubiquitylation enzymes are there?

Answer 19

1. Linkage specific
2. Exo vs endo cleavage
3. Substrate specific
4. Mono-deubiqutination

(Spasser and Brik 2012)

Question 20

Polyubiquitylation

Proteomic analysis of WT yeast, looking at relative proportions of these different linkage chains

Answer 20

K48 and K11 are involved in proteolysis

Reletive abundance reflects what is going on in that cell

Question 21

Ubiquitin

So just how important is it?

(this is Dr. Sul’s area of research)

What proteins are potential substrates for ubiquitylation?

What organelle stability is controlled by ubiquitin?

What mutations cause disease related to ubiquitin?

Answer 21

Theoretically every protein that is translated on a cystosolic ribosome is a potential substrate for ubiquitylation

The stability of the mitochondria as an organelle is controlled via ubiquitin

Huge numbers of diseases where ubiquitin is involved - often due to mutations in E3

Question 22

What does ubiquitin do? (go over what Ub does and in context of disease)

What was the first identified function of ubiquitin? what was the proteasome called?

What is it now known to be involved in?

Answer 22

First function identifieid was the role it plays in controlling intracellular proteolysis via the 26S proteasome

Now known to be involved in many more processes - many of which have nothing do to with proteasome and/or proteolysis

Question 23

The 26S proteasome

This was the first function identified as the ubiquitin
What is 26S proteasome often refferred to as?

How large is the complex?
Where is it found?
What is is responsible for?

Answer 23

26S proteasome is often reffered to as the cellular bin

It is a large complex #700kDa
It is found in the nucleus and cytoplasm

It is responsible for the majoritiy of cellular proteolysis

Question 24

The 26S proteasome

What is the core protease

What is the regulatory particle

Answer 24

20S core protease partical which is essentially a hollow tube

and

19S regulatory particle that consists of a lid and base

Question 25

What can the 26S particle be split into? What does the 20S protease form? How many distinct types of protease activity does the 20S particle have and why?

Answer 25

The 26S particle can be readily split into a 19S regulatory particle and a 20S core protease 20S particle forms 4 stacked rigns of 7 alpha and beta-subunits forming a hollow cylinder The core protease 20S has 5 different types of protease activity which ensures that anything that enters gets completely degraded

Question 26

What contains the ubiquitin receptor proteins? What does it bind strongly to? What does the base of the 19S particle have? What else is also part of the 19S particle? What does it do?

Answer 26

19S regulatory particle contains lid with the ubiquitin receptor proteins It binds strongly to K48 and K11 linked chains of at least 4 units in length The base of the 19S particle has a reverse chaperone subunit that unfolds proteins and feeds them into 20S core protease The 19S particle also has DUB which removes and recyles ubiquitin before degradation.

Question 27

The blue is the Ub protein V It is recognised by the lid on the proteasome V Modified protein is unfolded V Passed through the core chamber V Ub then removed and recyled and re-used Thus the same Ub protein can be used many times

Question 28

The 26S proteasome and disease What is the huge role that the 26S proteasome has? What is it essential for?\*hint;week 5 What sort of target is it?

Answer 28

The 26S proteasome has a huge role in removing damaged and unwanted proteins It is essential for protein quality control From a disease perspective it is essential for cell survival - mice gene knock-out embryo lethal - It is a potential therapeutic target

Question 29

26S proteasome and disease Going to talk about the regulation of p53 and The site of action of Velcade

Question 30

26S proteasome and p53 What is one of the most important tumor suppressor proteins in humans? What is its role? What does it eliminate, and how?

Answer 30

One of the most important tumor suppressor proteins in humans is p53 It plays a critical role in the response to cellular stresses such as **DNA damage** and **oncogene activation** p53 eliminates potentially cancerous cell by initiatng cell-cyle arrest and ultimately leads to **apoptosis**

Question 31

The way that p53 works

Answer 31

p53 when it is not activated is found in the cytosol V When it is activated it moves into the necleus V Binds to various promoter regions on various gene to initiante transcription and gene expression

Question 32

What is the regulation of p53 like? How many roles foes it have? What is the major regulatory protein?

Answer 32

Regulation of p53 is very tightly controlled, dont want cancer cells but dont went normal cells apopotosis Multiple roles for ubiquitin and proteasome The major regulatory protein is **MDM2**

Question 33

What type of Ub-ligase i.e E3 is MDM2 regulatory protein? How many ways does it regulate p53? What are they?

Answer 33

MDM2 is a RING-type Ub-ligase MDM2 regulates p53 by three ways 1. It inhibits transcription activity of p53 by binding to it 2. It mediates p53 nuclear export -into the cytosol 3. causes degredation by the 26S proteasome

Question 34

Mutations in MDM2 is linked to what percentages of all cancers? Which cancers in particular What is increased MDM2 activity correlated with? (What does more MDM2 mean) What does MDM2 regulate? In mouse gene-knock out is it embryo lethal? When is it viable?

Answer 34

Mutations in MDM2 is linked to 10\_20% of all cancers (particularly in bones, salviary glands and hodgkin lymphoma) Increased MDM2 activity is strongly correlated with **metastasis** More MDM2, less p53, less apoptosis **Major role of p53 is in the regulation of p53, mouse gene KO is embryo lethal - BUT - viable if p53 knocked-out first**

Question 35

What about 26S proteasome as a drug target? The 26S proteasome is absolutely essential for cell viability What is essential in rapidly dividing cells? Stabilization of apoptoic factors such as p53 should?

Answer 35

Maintaing protein quality is essential espeically in rapidly dividing cells Stabilization of apoptoic factors such as p53 shoudl kill cancer cells The 26S proteasome makes a good drug target for these reasons - if you want to kill cancer cells

Question 36

Currently there is only one drug on the market that targets 26S proteasome - what is this drug called? When was this therapeutic proteasome inhibitor approved? What does boronic acid peptide inhibit? What is ti used for?

Answer 36

*Velcade* is the trade name for the drug botezomib First therapeutic proeasome inhibitor first approval by FDA in 2003 then by NICE in 2009 Boronic acid peptide analogue, specifically inhibits 20S protease activity Currently used as treatment for multiple myeloma and some forms of lymphoma

Question 37

26S proteasome as a drug target What sort of effefct does botezomib have? Where is proteasome? What happens if there is no proteasome? What drugs might there be in the future

Answer 37

Botezomib has a very broad spectrum ihibitor Proteasome is essentially in all cells No proteasome - accumulation of Ub conjugates, reduces free Ub pool, all Ub-mediate processes inhibited Drugs that target specific ligases likely to be available in future - less of target effects