DUB labelling

>> profile activity of DUBs in cell extract
>> DUB remove covalently bound ubiquiin moieties from protein substrates, thereby controlling many cellular functions. Alterations in these processes contribute to pathologies
>> post-translational modification of priteins by ubiquitin is a reversible process
>> ubiquitylation is carried out by a cascade of E1, E2, E3 ubiquitinating enzmes
>> apart from mono- and milto-ub, future ubiquitlation diversity is achieved by the assembly of poly ub chains through ispeptide bond formation between C-terminal glycine and one of the seven K residues of Ub.

>> modification of target proteins by poly-Ub chans linked through K48 serves as degratoin signals..
shuttles the Ubed protein through a series of Ub-binding proteins to the proteasome for degraation
>> K63 –> regulation of signaling evermts.

>> all 7 lysine residues on Ub can be used to create poly-Ub chains
>> activity based probes
>>Electrophilic probes based
on the ubiquitin scaffold, such as Ub-vinyl methylester (VME)
or Ub-vinyl sulfone (VS) act as Michael acceptors towards the
catalytic thiol of the DUB active site, which enables their covalent
capture and activity-based protein profiling (ABPP), a concept
successfully applied to other proteases.[9]

Profiling ubiquitin linkage specificities of DUB with branched ubiquitin isopeptide probes

branched Ub isopeptide activity-based probes (UIPPs) –> to profile ubiquitin (Ub) linkage specificity of DUBs in cell extracts


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