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Biochim Biophys Acta. 2012 Nov;1823(11):2029-37. doi: 10.1016/j.bbamcr.2012.05.014. Epub 2012 May 19.

Detection of ubiquitin-proteasome enzymatic activities in cells: application of activity-based probes to inhibitor development.

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  • 1Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK.

Abstract

BACKGROUND:

Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors.

SCOPE OF REVIEW:

Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation.

MAJOR CONCLUSIONS:

Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors.

GENERAL SIGNIFICANCE:

Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Copyright © 2012 Elsevier B.V. All rights reserved.

PMID:
22613766
[PubMed - indexed for MEDLINE]
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