title: Ubiquitin C-terminal hydrolases cleave isopeptide- and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers.
abstract: Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on Ub homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the eight Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesized isopeptide-linked Ub from lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs in vivo.
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: John S
authors.initials: JS
authors.lastname: Bett
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Maria Stella
authors.initials: MS
authors.lastname: Ritorto
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Richard
authors.initials: R
authors.lastname: Ewan
authors.affiliation: †Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Ellis G
authors.initials: EG
authors.lastname: Jaffray
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
authors.affiliation: ‡MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, England, U.K.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Thimo
authors.initials: T
authors.lastname: Kurz
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
authors.affiliation: †Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Tatham
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D011108:Polymers; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D043222:Ubiquitin Thiolesterase