title: Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets.
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.
authors.affiliation: Department of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Dennis
authors.initials: D
authors.lastname: Flierman
authors.affiliation: Department of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Gerbrand J
authors.initials: GJ
authors.lastname: van der Heden van Noort
authors.affiliation: Department of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Reggy
authors.initials: R
authors.lastname: Ekkebus
authors.affiliation: Department of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Tycho E T
authors.initials: TE
authors.lastname: Mevissen
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Manuela K
authors.initials: MK
authors.lastname: Hospenthal
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Komander
authors.affiliation: Department of Cell Biology II, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address: h.ovaa@nki.nl.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D008223:Lymphoma / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D014452:Ubiquitins; D010447:Peptide Hydrolases