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
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
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
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
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 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 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
