title: Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: crystallography, activity profiling, and protease identification.
abstract: 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.
authors.affiliation: Department of Pathology, Harvard Medical School (NRB), Boston, Massachusetts, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Galardy
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D003461:Crystallography; D006801:Humans; D007150:Immunohistochemistry; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D025801:Ubiquitin; D010447:Peptide Hydrolases