Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: Synthesis of active site-directed probes

Huib Ovaa, Paul J. Galardy, Hidde L. Ploegh

Research output: Contribution to journalReview article

14 Scopus citations

Abstract

The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.

Original languageEnglish (US)
Article number32
Pages (from-to)468-478
Number of pages11
JournalMethods in enzymology
Volume399
DOIs
StatePublished - Dec 19 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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