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

Journal article

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.

Authors: Kramer, H; Nicholson, B; Kessler, B; Altun, M

• Publication status: Published
• Journal: Biochimica et biophysica acta
• Volume: 1823
• Issue: 11
• Pages: 2029-2037
• Publication date: 2012-11-01
• DOI: 10.1016/j.bbamcr.2012.05.014
• EISSN: 1879-2596
• ISSN: 0006-3002
• Language: English
• Keywords: Proteasome Endopeptidase Complex; Animals; Drug Design; Molecular Probes; Humans; Ubiquitin