Distribution and Paralog Specificity of Mammalian DeSUMOylating Enzymes

• Type of Material: Article
• Authors: Nagamalleswari Kolli, Emory University; Jowita Mikolajczyk, Burnham Institute for Medical Research; Marcin Drag, Burnham Institute for Medical Research; Debaditya Mukhopadhyay, National Institutes of Health; Nela Moffatt, Emory University; Mary Dasso, National Institutes of Health; Guy Salvesen, Burnham Institute for Medical Research; Keith D Wilkinson, Emory University
• Language: English
• Date: 2010-09-01
• Publisher: Portland Press
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © The Authors Journal compilation © 2010 Biochemical Society
• Final Published Version (URL): http://www.biochemj.org/bj/430/bj4300335.htm
• Title of Journal or Parent Work: Biochemical Journal
• ISSN: 0264-6021
• Volume: 430
• Issue: 2
• Start Page: 335
• End Page: 344
• Grant/Funding Information: This work was supported by National Institutes of Health Grant 5RO1GM066355.
• Keywords: substrate specificity; SUMO; SENP; desumoylation; paralogs
• Research Categories: Chemistry, Biochemistry

Abstract

The covalent attachment of the small ubiquitin-like protein modifier (SUMO) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalyzed by SUMO-specific processing proteases (SENPs). Mammals contain four SUMO paralogs and six SENP enzymes. Our studies describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal SENP catalytic domains (cSENPs). We first characterized the reaction of each endogenous SENP and their catalytic domains (cSENP) with HA-tagged SUMO1 and SUMO2 vinyl sulfones (HA-SUMO-VS), active site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogs, while all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1 or SUMO2-amidomethyl coumarin (SUMO-AMC) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by KM and kcat while cSENP7 works only on SUMO2. Compared to cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity indicating that paralog specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggests that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.