Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes

Chem Biol. 2013 Dec 19;20(12):1447-55. doi: 10.1016/j.chembiol.2013.10.012. Epub 2013 Nov 27.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • HEK293 Cells
  • Humans
  • Lysine / chemistry
  • Lysine / genetics
  • Lysine / metabolism*
  • Models, Molecular
  • Molecular Probe Techniques
  • Molecular Probes / chemistry
  • Molecular Probes / genetics
  • Molecular Probes / metabolism
  • Protein Processing, Post-Translational
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism*
  • Ubiquitin-Specific Proteases / metabolism*
  • Ubiquitination


  • Molecular Probes
  • Recombinant Proteins
  • Ubiquitin
  • Ubiquitin-Specific Proteases
  • Lysine