Week 4.1 Ubiquitin Flashcards
What is ubiquitin?
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
Where is ubiqutin found?
Is it found in prokaryotes?
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.
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?
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
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?
- Activation - E1 aka activation enzyme
- Conjugation - E2 conjugation enzyme
- Ligation - E3 ligation enzyme
There are about 2 enzymes encoding E1, 50 enzymes for E2 and over 600 for the ligation enzyme E3
What question did the discovery of ubiquitin answer?
What was found to be the reason?
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
Process of ubiquitylation
Activation process
What does the E1 used to synthesize C-terminal adnylate?
What does E1 adenylate then do?
E1 uses ATP to synthesize C-terminal adnylate
E1 adenylate then acts as a substrate to form E1- ubiqutin thiol ester
Ubiquitylation
Conjugation…
Where is the activated ubiquitin transferred to?
… and ligation
Where is the ubiqutin then transferred too?
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.
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?
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
- RING/U-box type
- HECT type
RING-type (U-box)
What do they bind to?
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
HECT-type
How does this work?
1st it takes the ubq off the E2 by forming thioester link and then transferes it to the substrate
Ubiquitin ligases
There is a lot of diversity
What are the methods of regulation?
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
Ubiquitylation is a dynamic process there are a class of enezymes that are called De-ubiquinating enzymes (DUBs)
Why?
They are responsible for the removal ubiquitin - in a cyclcal pattern
Analogous to phosphorylation
What was ubiquitin originally identified for?
But what is it now known for?
Originally identified due to role in intracellular proteolysis
Now known that ubiquitin is involved in regulating a huge range of cellular processes
How is ubiquitin a multi-functional signal?
Given that the structure of ubiqutin is highly consereved how can it have mutliple signals?
By being able to have a range of attachment methods
Monoubiquitylation
Multi-site monoubiquitylation
Key one: Polyubiquitylation
Ubiquitin is a multi-functional signal
When do you get?
What two main di-Ub forms are there?
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
Ubiquitin - a multifunctional signal
If you have different signalling molecuels you also need effectors
What do the effector proteins contain?
Effector proteins contain a Ub-binding domain UBD
UBD is attached to a large protein -
e.g UBA, MIU, CUE
Have different binding specificities
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?
UBD –> The UBA binding domain - recognised by specific receptors that can recognise hydrophobic patch
Also…the structure changes significantly with pH
Polyubiquitylation
What determines the type and length of chain?
What are E4 enzymes?
What is chain editing?
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
What types of deubiquitylation enzymes are there?
- Linkage specific
- Exo vs endo cleavage
- Substrate specific
- Mono-deubiqutination
(Spasser and Brik 2012)
Polyubiquitylation
Proteomic analysis of WT yeast, looking at relative proportions of these different linkage chains
K48 and K11 are involved in proteolysis
Reletive abundance reflects what is going on in that cell
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?
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
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?
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
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?
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
The 26S proteasome
What is the core protease
What is the regulatory particle
20S core protease partical which is essentially a hollow tube
and
19S regulatory particle that consists of a lid and base
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?
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
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?
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.
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
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?
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
26S proteasome and disease
Going to talk about the regulation of p53
and
The site of action of Velcade
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?
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
The way that p53 works
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
What is the regulation of p53 like?
How many roles foes it have?
What is the major regulatory protein?
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
What type of Ub-ligase i.e E3 is MDM2 regulatory protein?
How many ways does it regulate p53? What are they?
MDM2 is a RING-type Ub-ligase
MDM2 regulates p53 by three ways
- It inhibits transcription activity of p53 by binding to it
- It mediates p53 nuclear export -into the cytosol
- causes degredation by the 26S proteasome
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?
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
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?
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
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?
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
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
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
