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    Non-hydrolyzable Diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by S2 Pockets

    Flierman, D. and van der Heden van Noort, G.J. and Ekkebus, R. and Geurink, P.P. and Mevissen, T.E.T. and Hospenthal, Manuela and Komander, D. and Ovaa, H. (2016) Non-hydrolyzable Diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by S2 Pockets. Cell Chemical Biology 23 (4), pp. 472-482. ISSN 2451-9456.

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    Abstract

    Ubiquitin 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.

    Metadata

    Item Type: Article
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Depositing User: Administrator
    Date Deposited: 09 Jun 2016 14:55
    Last Modified: 09 Jun 2016 14:55
    URI: http://eprints.bbk.ac.uk/id/eprint/15482

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