title: An Interaction Landscape of Ubiquitin Signaling.
abstract: Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands. Electronic address: x.zhang@science.ru.nl.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands.
authors.forename: Arne H
authors.initials: AH
authors.lastname: Smits
authors.affiliation: Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, the Netherlands; Department of Chemical Immunology, Leiden University Medical Center, Albinusdreef 2, Leiden 2333ZA, the Netherlands.
authors.forename: Gabrielle B A
authors.initials: GB
authors.lastname: van Tilburg
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands.
authors.forename: Pascal W T C
authors.initials: PW
authors.lastname: Jansen
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Makowski
authors.affiliation: Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, the Netherlands; Department of Chemical Immunology, Leiden University Medical Center, Albinusdreef 2, Leiden 2333ZA, the Netherlands. Electronic address: h.ovaa@lumc.nl.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands. Electronic address: michiel.vermeulen@science.ru.nl.
authors.forename: Michiel
authors.initials: M
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D001665:Binding Sites; D019295:Computational Biology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D030562:Databases, Protein; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D058953:Neural Stem Cells / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D054875:Ubiquitination; D002583:Uterine Cervical Neoplasms / Q000378:metabolism; D057188:Workflow
chemical_list: D025801:Ubiquitin; D057149:Ubiquitinated Proteins; C509806:UCHL3 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases
keywords: DNA damage; TAB2; UCHL3; UbIA-MS; diubiquitin; interaction proteomics; ubiquitin signaling