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Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

Abstract

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins.

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Figure 1: Qualitative DUB specificity analysis.
Figure 2: Schematic description of UbiCRest analysis.
Figure 3: UbiCRest analysis to determine ubiquitin linkage types on substrates.
Figure 4: UbiCRest analysis to test for heterotypic polyubiquitin.

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Acknowledgements

We thank the present and past members of the Komander laboratory for reagents and critical comments on the manuscript. This work was supported by the Medical Research Council (U105192732), the European Research Council (309756), the Lister Institute for Preventive Medicine, the EMBO Young Investigator Program (all to D.K.); and by a Marie Curie Initial Training Network 'UPStream' grant (to T.E.T.M.).

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M.K.H., T.E.T.M. and D.K. designed the method and wrote the manuscript.

Corresponding author

Correspondence to David Komander.

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Competing interests

D.K. is part of the DUB Alliance that includes Cancer Research Technology and FORMA Therapeutics, and is a consultant for FORMA Therapeutics. A patent application has been filed for the described method. Boston Biochem/Biotechne distribute an enzyme kit for performing UbiCRest analysis.

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Hospenthal, M., Mevissen, T. & Komander, D. Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest). Nat Protoc 10, 349–361 (2015). https://doi.org/10.1038/nprot.2015.018

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