Ubiquitin Signalling (2) Flashcards
What are the two types pf DUBs?
Metallo
Thiol
Name the family of metallo DUBs
JAMM
Name the families of thiol DUBs
UCH OTU USP Josephin MINDY ZUP1
What are metallo DUBs?
They have a metal ion at its catalytic site
What are thiol DUBs?
Use a cysteine based mechanism to cleave Ub chains
What are the functions of DUBs?
To process precursors to generate pre-Ub
To recycle Ub
Cleave Ub off substrates and complexes
why is Ub recycled?
It is a stable compound and therefore requires a lot of energy to degrade it so it is cleaved off by the DUBs and recycled back into the cellular pool of Ubs
What happens if the cellular pool of Ubs is too low?
It is toxic to the cell
How do cancer therapies manipulate the Ub cellular pool and why?
they inhibit the proteasome -> proteasomic stress -> stop the degradation of proteins -> deplete the cellular pool of Ub
This toxicity induces cell death
What is the subcellular location of DUBs?
Is very dynamic, they need to be with the protein which needs to be cleaved
Some sit at the mitochondria and regulate mitophagy
What do DUBs have to discriminate between?
Ub and Ub-like modifiers
What does USP21 cleave and how does it discriminate
Ub and Ub-like modifier, ISG15
It binds to the C-terminal tail and recognises the conserved arginine
Nedd8 has an alanine here so is not recognised but ISG15 has an arginine and therefore is
What are the 2 ways for DUBs to cleave and what are each of the DUBs called?
ExoDUB - cleaves the chain from one end down to the substrate
Endocleavage - It cleaves the middle of the chain
What is fasted form of cleavage?
Endocleavage
What do distal Ub bind to?
S1 or S2
What does the proximal Ub bind to?
S1’ or S2’
Where is the distal Ub located?
At the N terminus
Where is the proximal Ub located?
At the C-terminus
What is the binding sites like of those which have Ub linkage specificity
They will have a defined S1 site but not a defined S1’ site
Which DUBs have Ub linkage specificity?
OTU
JAMM
Josephin
MINDY
Which DUBs have substrate or sequence specificity?
Ubp8 (SAGA)
What DUBs have nonspecificity?
USP
UCH
What are the defined S1’ Ub binding sites?
Ub-assisted catalysis
Conformational dynamics
S1’ site on UBD in cis
S1’ site on UBD in trans
What additional Ub-binding sites are there?
S2 site on catalytic domain
Additional UBD on cis
S2’ site on catalytic domain
What are the three components of the catalytic thiol?
Catalytic cysteine
A histidine
A negatively charged asparagine or aspartate usually
What is the catalytic mechanism for Cys DUBs?
Cysteine causes a nucleophilic attack -> forms a negatively charged tetrahedral intermediate -> stabilised by an oxyanion hole -> hydrolysis and release of one of the Ub -> formation of a second acyl enzyme intermediate -> deactylation (water attack) -> form a tetrahedral intermediate -> distal Ub release
What is the catalytic mechanism of metallo DUBs?
Hydroxyl group of water molecule will induce a nucleophillic attack on a carbon-carbon between the two Ub -> Tetrahedral intermediate -> collapse of intermediate -> release the two Ub -> regeneration of Apo state (regenerate the catalytic site)
What are the 3 components in metallo DUB catalytic site?
Znc
Negatively charged residue
Histidine
Name a DUB which uses an E3 ligase to induce degradation
E20
How does E20 induce degradation
It cleaves K63 linked chains (which have a positive signal) so that there is only 1 Ub left on the substrate -> E3 ligase will add more Ub in a K48 linkage so that it is targeted for degradation
What are pseudo DUBs?
DUBs which have lost their catalytic function
What is the role of pseudo DUBs?
They bind to the Ub and prevent its cleavage - therefore protecting the cleavage
How are DUBs regulated?
Post translational modifications
Regulation at the transcription or translation level
Some only work in complexes
Auto-processes - can proteolyse itself and therefore inactivate itself
Allosteric activation
Scaffold proteins
Oxidation
What post translational modifications can regulate DUBs?
Sometimes need phosphorylation in their catalytic site to be activated
Some get ubiquitylated and therefore inhibited
Some are SUMOylated and activated
How are DUBs regulated by oxidation?
Under oxidising conditions, the thiol turns into a sulphuric acid and inhibits it
Reversible
How are DUBs regulated by scaffold proteins?
Some proteins have different functions depending on what other protein they bind to
Give an example of a DUB which is regulated by scaffold proteins
UCH-L5
How is UCH-L5 regulated by scaffold proteins?
If it is bound to Rpn13 via a DEUBAD domain it activates the proteasome
If it binds to a INO80G complex via the DEUBAD domain, UCH-L5 is inhibited
What is the degradation cycle of the proteasome?
Ub signal is recognised by Ub-binding receptors on the lid of the RP subunit -> unstructured region of the peptide bind to the ATPase -> pulls peptide through the whole particle -> Ub is cleaved by DUBs -> released back into the Ub cellular pool
What are ubiquitin receptors?
Proteins which are located at the proteasome but have a recognition site for Ub so they can bring the ubiquitylated protein to the proteasome
Give an example of a Ub receptor
DSK2
Name 3 DUBs which sit at the proteosome
UCH-37
Rpn11
USP14
Which is the last enzyme to see the peptide before it goes into the catalytic core of the proteosome?
Rpn11
How and what is the role of Rpn11?
To cleave the last Ub
When the peptide is being pulled through by the ATPase, Rpn11 binds to the Ub -> causes a conformational change in Insert-1 -> activation and therefore cleavage of Ub
Why is it important to have a DUB which is inactive until the peptide has reached the catalytic core?
Reaching the catalytic core is time consuming and therefore if you release it at the beginning it will not reach the core and the peptide will be released back into the cell and not degraded
What therapeutic technique is being exploited in neurodegenerative diseases?
Inhibit DUBs at the proteasome so the peptide has more of a change to reach the catalytic core and be degraded
What cancer is proteasomal inhibition currently in clinical trials for?
Multiple myeloma
