An Interaction Landscape of Ubiquitin Signaling

Mol Cell. 2017 Mar 2;65(5):941-955.e8. doi: 10.1016/j.molcel.2017.01.004. Epub 2017 Feb 9.


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.

Keywords: DNA damage; TAB2; UCHL3; UbIA-MS; diubiquitin; interaction proteomics; ubiquitin signaling.

MeSH terms

  • Animals
  • Binding Sites
  • Computational Biology
  • Cysteine Endopeptidases / metabolism
  • Databases, Protein
  • Embryonic Stem Cells / metabolism
  • Female
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mass Spectrometry*
  • Mice
  • Neural Stem Cells / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping*
  • Protein Interaction Maps*
  • Proteomics / methods*
  • Signal Transduction*
  • Ubiquitin / metabolism*
  • Ubiquitin Thiolesterase
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination*
  • Uterine Cervical Neoplasms / metabolism
  • Workflow


  • Ubiquitin
  • Ubiquitinated Proteins
  • UCHL3 protein, human
  • Ubiquitin Thiolesterase
  • Cysteine Endopeptidases