Isopeptidases that specifically remove ubiquitin or ubiquitin-like molecules from polypeptide adducts are emerging as key regulatory enzymes in a multitude of biochemical pathways. We have developed a set of tools that covalently target the active site of ubiquitin or ubiquitin-like deconjugating enzymes. We have used epitope-tagged ubiquitin and ubiquitin-like derivatives in immunoprecipitation assays to identify active proteases by mass spectrometry (MS/MS). The epitope tag confers the ability to conduct an immunoblot-based profiling assay for active isopeptidases in cell extracts. We have applied a ubiquitin-based probe in the structural analysis of the ubiquitin hydrolase UCH-L3 in its ligand-bound state. We describe the use of these electrophilic derivatives of ubiquitin and ubiquitin-like molecules in the identification, activity profiling, and structural analysis of these proteases. These tools can be used to rapidly profile activity of multiple Ub/UBL-specific proteases in parallel in cell extracts. We also show that in vitro these probes can be conjugated onto parts of the Ub/UBL conjugating machinery.
ASJC Scopus subject areas
- Molecular Biology