Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: synthesis of active site-directed probes.
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.
Ovaa
Huib
H
Netherlands Cancer Institute, Division of Cellular Biochemistry, Amsterdam, The Netherlands.
Galardy
Paul J
PJ
Ploegh
Hidde L
HL
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017353:Gene Deletion; D015335:Molecular Probes; D010957:Plasmids; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015335:Molecular Probes; D025801:Ubiquitin