Abstract
SummaryUbiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents.
Highlights
Ubiquitin (Ub), a 76 amino acid post-translational modifier, is at the center of a large number of cellular processes
It was shown that triazole-based polyUb chains and activity-based probes are well tolerated as isopeptide mimics (Dresselhaus et al, 2013; McGouran et al, 2013; Schneider et al, 2014; Weikart et al, 2012)
We showed previously that the mUband purified by reverse-phase high performance liquid chro- PA probe reacts with a large set of Deubiquitylating enzymes (DUBs) present in these cells matography (RP-HPLC) (Figure 2B; 1)
Summary
Ubiquitin (Ub), a 76 amino acid post-translational modifier, is at the center of a large number of cellular processes. Target proteins can be covalently modified with Ub on either a lysine residue on the protein surface or on the N terminus. Ub is activated by an E1 enzyme, forming a thioester bond via its C-terminal carboxylate. Ub can be coupled to another Ub molecule via any of its seven lysine residues or its N terminus to yield Ub chains. Deubiquitylating enzymes (DUBs) can reverse ubiquitylation by cleaving the (iso)-peptide bond between the C-terminal carboxylate of Ub and the substrate. 100 human DUBs are known so far, and some DUBs have been shown to exhibit linkage and substrate specificity in the deubiquitylation reaction (Clague et al, 2013; Faesen et al, 2011; Komander et al, 2009; Mevissen et al, 2013)
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