Chemistry & Biology
Volume 20, Issue 12, 19 December 2013, Pages 1447-1455
Journal home page for Chemistry & Biology

Article
Deubiquitinating Enzyme Specificity for Ubiquitin Chain Topology Profiled by Di-Ubiquitin Activity Probes

https://doi.org/10.1016/j.chembiol.2013.10.012Get rights and content
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Highlights

  • Synthesis of Di-ubiquitin-based active site probes representing all eight linkages

  • Mass spectrometric profiling of DUB-Ub linkage preference in whole cell extracts

  • Activity-based Di-Ub probe screen for DUB specificity toward poly-Ub linkages

  • DUBs detected with a preference for noncanonical linkages over K48/K63-linked Ub

Summary

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.

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Present address: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden