title: OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis.
abstract: Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1' and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.
authors.forename: Tycho E T
authors.initials: TE
authors.lastname: Mevissen
authors.forename: Manuela K
authors.initials: MK
authors.lastname: Hospenthal
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.forename: Paul R
authors.initials: PR
authors.lastname: Elliott
authors.forename: Masato
authors.initials: M
authors.lastname: Akutsu
authors.forename: Nadia
authors.initials: N
authors.lastname: Arnaudo
authors.forename: Reggy
authors.initials: R
authors.lastname: Ekkebus
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Kulathu
authors.forename: Tobias
authors.initials: T
authors.lastname: Wauer
authors.forename: Farid
authors.initials: F
authors.lastname: El Oualid
authors.forename: Stefan M V
authors.initials: SM
authors.lastname: Freund
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
authors.forename: David
authors.initials: D
authors.lastname: Komander
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008958:Models, Molecular; D010051:Ovarian Neoplasms / Q000201:enzymology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D014452:Ubiquitins; C545663:YOD1 protein, human; D013869:Thiolester Hydrolases; D010450:Endopeptidases