Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets

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.

Novel Diubiquitin Probes Expand the Chemical Toolkit to Study DUBs

“…In this issue of Cell Chemical Biology, Flierman et al (2016) develop a new family of novel nonhydrolyzable diubiquitin probes that will be valuable tools to study how DUBs achieve specificity. In this issue of Cell Chemical Biology, Flierman et al (2016) develop a new family of novel nonhydrolyzable diubiquitin probes that will be valuable tools to study how DUBs achieve specificity.…”

“…The novel probes developed by Flierman et al (2016) are an exciting addition to the existing toolkit that will allow us to study DUBs that use additional S2 site interactions. The novel probes developed by Flierman et al (2016) are an exciting addition to the existing toolkit that will allow us to study DUBs that use additional S2 site interactions.…”

Genetically Directed Production of Recombinant, Isosteric and Nonhydrolysable Ubiquitin Conjugates

“…Solid‐phase peptide synthesis (SPSS) approaches for preparing nonhydrolysable Ub conjugates have also been described, but these require specialist expertise and are not generally applicable . Although conjugates formed with triazole‐based linkages have been employed for biological studies, the behaviour towards receptors or DUBs that recognise the linkage itself or that specifically “sense” the inter‐ubiquitin distance in a particular linkage is unknown. Furthermore, there is no experimental structure of a nonhydrolysable conjugate, and a comparison of the affinity towards DUBs and Ub receptors relative to their native counterparts has not been investigated.…”

Release of Enzymatically Active Deubiquitinating Enzymes upon Reversible Capture by Disulfide Ubiquitin Reagents

“…The majority of DUBs that could be labeled in cell extract could also be labeled after elution with reducing agent. Notably, the 56 kDa DUB USP14 (65 kDa after binding of TMR‐Ub‐PA)3a,3c,3f, 10 was not retrieved or not active after elution. This is likely due to the absence of proteasome after washing the resin.…”