Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes

de Jong, A., Merkx, R., Berlin, I., Rodenko, B., Wijdeven, R.H.M., El Atmioui, D., Yalçin, Z., Robson, C.N., Neefjes, J.J. and Ovaa, H. (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. ChemBioChem, 13(15), pp. 2251-2258. (doi: 10.1002/cbic.201200497)

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Publisher's URL: http://dx.doi.org/10.1002/cbic.201200497

Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Rodenko, Dr Boris
Authors: de Jong, A., Merkx, R., Berlin, I., Rodenko, B., Wijdeven, R.H.M., El Atmioui, D., Yalçin, Z., Robson, C.N., Neefjes, J.J., and Ovaa, H.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:ChemBioChem
Publisher:Wiley - VCH Verlag GmbH & Co. KGaA
ISSN:1439-4227
ISSN (Online):1439-7633
Published Online:25 September 2012
Copyright Holders:Copyright © 2012 Wiley -VCH Verlag GmbH and Co. KGaA
First Published:First published ChemBioChem 13(15):2251-2258
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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