title: Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes.
abstract: Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
authors.forename: Joanna F
authors.initials: JF
authors.lastname: McGouran
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
authors.forename: Selina R
authors.initials: SR
authors.lastname: Gaertner
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
authors.forename: Mikael
authors.initials: M
authors.lastname: Altun
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK. Electronic address: holger.kramer@dpag.ox.ac.uk.
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: benedikt.kessler@ndm.ox.ac.uk.
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
mesh_terms: D019943:Amino Acid Substitution; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D015335:Molecular Probes; D011994:Recombinant Proteins; D025801:Ubiquitin; D064570:Ubiquitin-Specific Proteases; D008239:Lysine