title: A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.
abstract: Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.
authors.affiliation: Biophotonics Collaboration, School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews KY16 9SS, Scotland, UK.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Martin
authors.forename: Neil
authors.initials: N
authors.lastname: Hattersley
authors.forename: Ifor D W
authors.initials: ID
authors.lastname: Samuel
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
authors.forename: Michael H
authors.initials: MH
authors.lastname: Tatham
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational; D025842:SUMO-1 Protein / Q000737:chemistry / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D061988:Proteasome Inhibitors; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; C054562:yellow fluorescent protein, Bacteria