What processes is ubiquitin involved in?
proteasomal degradation
apoptosis
immune response
transcription and translation
cell cycle
…
Different functions are distinguished by different ubiquitinylation patterns:
different ubiquitinylation sites in proteins
8 different ways to combine two ubiquitins
different Ub-chain lengths
How are substrates ubiquitinylated and deubiquitinylated?
E1 binds Ub (thioester)
transfer to E2 (trans-thioesterification)
transfer to substrate by Ub ligase (2 types)
RING-type
HECT/RBR-type
Deubiquitinylation: Deubiquitinylases (DUBs)
Describe the mechanism of E1 enzymes.
Overall goal: Ub-activation and transfer to E2
Three binding sites: ATP, Ub, E2
E1 adenylates ubiquitin (-> Ub-AMP)
Ubiquitin is transferred to E1 (-> E1-Ub)
thioesterification
Ubiquitin is transferred from E1 to E2
trans-thioesterification
Describe the mechanism of E2 and E3 enzymes.
Overall goal: Binding of Ub and transfer to target protein (directly or via E3).
E2 catalyzes different reactions depending on the ligase it interacts with.
RING:
RING-E3 does not bind Ub directly but helps E2 to find the target protein
builds a scaffold to enforce favorable conformation for E2-Ub-target interaction
1-step mechanism
E3 is responsible for specificity
HECT
Ub is transferred from E2 to E3 (thioester), then transfer from E3 to the target protein
2-step mechanism
has 2 lobes with flexible spacer
E2 is responsible for specificity
RBR
RING domain and catalytic cysteine
mechanism resembles RING (scaffold, fixates E2) and HECT (binds Ub via thioester)
Name educts, intermediates and products of DUB reactions.
What kinds of DUBs are there?
Generally: 7 families (6 cysteine proteases, 1 metalloprotease)
Draw a substrate protein that is ubiquitinated at a serine residue and at a lysine residue.
Describe in what sense the E1-catalyzed reaction resembles a step during protein synthesis. What is the common principle that renders both reactions irreversible?
E1 reaction:
ARS reaction:
Common principle:
Can you think of why the ubiquitination system may have evolved as a catalytic cascade (rather than single enzymes catalyzing ubiquitination)?
Separation of reaction steps into enzyme families
Reduced evolutionary cost than singular enzyme catalyzing series of complex chemical reactions.
Regulation on several levels.
Mainly E3 ligases and DUBs require specificity in substrate recognition
system is versatile and specific
Ubiquitination sites on cellular proteins are typically identified by a trypsin-mediated digest of the sample followed by mass spectrometry. Use a cartoon representation of a ubiquitinated substrate to explain schematically which ubiquitin-derived digestion products are searched for.
Hint: You need to look up the amino acid sequence preference of trypsin and the amino acid sequence of ubiquitin. Note that trypsin does not cleave after a ubiquitinated residue.
Trypsin cleaves after arginine and lysine.
Ubiquitin has LRLRGG-motive at C-terminus (bound to lysine residue of the substrate).
Trypsin cannot cleave behind the substrate-lysine directly because of the Ub, but the R in the Ub C-terminus is cleaved
—> Ub is cleaved but GG remains bound to the substrate
—> +112 Da in the mass spectrum
If you had the opportunity to develop drugs against ubiquitin-system enzymes, which family would you choose and why? Discuss!
How can the different families of ubiquitinylating enzymes potentially be inhibited?
E1
ATP-competitive compounds
catalytic cysteine-reactive compounds
mechanism-based inhibitors
E3
substrate recognition blockers
global activity blockers
compounds that reprogram activity
proteolysis-targeting chimeras (PROTACs)
Do you expect the ubiquitin system will furnish as many drugs as protein kinases and why? What are the fundamental differences between these groups of targets in terms of drug discovery? Discuss!
Ubiquitin system bears great drug potential
part of many crucial processes within the cell
What are specific challenges in developing PROTACs for therapeutic applications? Consider molecular size, dosage, and requirements with regard to the target protein and ligase?
PROTACs: proteolysis-targeting chimeras
bind both target protein and E3 ligase
target protein is ubiquitinylated and degraded
Challenges:
optimization of both “ends” and the linker
linker should not be too long, E3 must still reach the target protein
“ends” must bind properly but must also release again
Lipinski’s rule of five
small (<500 kDa) to enter the cell easily
should be hydrophobic (to enter the cell) but also have H-bonds
ligase and target need to be present in the same cell/tissue
hook-effect: when overdosed, monovalent interactions with ligase or target
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