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Lookup NU author(s): Professor Craig Robson
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Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
Author(s): de Jong A, Merkx R, Berlin I, Rodenko B, Wijdeven RHM, El Atmioui D, Yalcin Z, Robson CN, Neefjes JJ, Ovaa H
Publication type: Article
Publication status: Published
Journal: ChemBioChem
Year: 2012
Volume: 13
Issue: 15
Pages: 2251-2258
Print publication date: 25/09/2012
ISSN (print): 1439-4227
ISSN (electronic): 1439-7633
Publisher: Wiley - V C H Verlag GmbH & Co. KGaA
URL: http://dx.doi.org/10.1002/cbic.201200497
DOI: 10.1002/cbic.201200497
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