title: In Vitro Characterization of Chain Depolymerization Activities of SUMO-Specific Proteases.
abstract: SUMO-specific proteases, known as Ulps in baker's yeast and SENPs in humans, have important roles in controlling the dynamics of SUMO-modified proteins. They display distinct modes of action and specificity, in that they may act on the SUMO precursor, mono-sumoylated, and/or polysumoylated proteins, and they might be specific for substrates with certain SUMO paralogs. SUMO chains may be dismantled either by endo or exo mechanisms. Biochemical characterization of a protease usually requires purification of the protein of interest. Developing a purification protocol, however, can be very difficult, and in some cases, isolation of a protease in its pure form may go along with a substantial loss of activity. To characterize the reaction mechanism of Ulps, we have developed an in vitro assay, which makes use of substrates endowed with artificial poly-SUMO chains of defined lengths, and S. cerevisiae Ulp enzymes in crude extract from E. coli. This fast and economic approach should be applicable to SUMO-specific proteases from other species as well.
authors.affiliation: Institute for Genetics, Biocenter, University of Cologne, 50674, Cologne, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Eckhoff
authors.affiliation: Institute for Genetics, Biocenter, University of Cologne, 50674, Cologne, Germany. j.dohmen@uni-koeln.de.
authors.forename: R Jürgen
authors.initials: RJ
authors.lastname: Dohmen
mesh_terms: D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D058207:Sumoylation
chemical_list: C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D006639:Histidine; C085706:FLAG peptide; D010450:Endopeptidases; C405949:ULP2 protein, S cerevisiae
keywords: Desumoylation; In vitro assay; Protease; SUMO chain; Ulp/SENP