Deubiquitinating enzymes (DUBs) reverse the process of ubiquitination, and number nearly 100 in humans. In principle, DUBs represent promising drug targets, as several of the enzymes have been implicated in human diseases. The isopeptidase activity of DUBs can be selectively inhibited by targeting the catalytic site with drug-like compounds. Notably, the mammalian 26S proteasome is associated with three major DUBs: RPN11, UCH37, and USP14. Because the ubiquitin 'chain-trimming' activity of USP14 can inhibit proteasome function, inhibitors of USP14 can stimulate proteasomal degradation. We recently established a high-throughput screening (HTS) method to identify small-molecule inhibitors specific for USP14. The protocols in this article cover the necessary procedures for preparing assay reagents, performing HTS for USP14 inhibitors, and carrying out post-HTS analysis. Curr. Protoc. Chem. Biol. 4:311-330 © 2012 by John Wiley & Sons, Inc.
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