title: Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system.
abstract: SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner.
authors: Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Science and Technology Corporation, CREST Research Project, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; Department of Chemistry and Biochemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors: Mikako
authors: M
authors: Hirohama
authors: Zhang Initiative Research Unit, RIKEN, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors: Arnout R D
authors: AR
authors: Voet
authors: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan.
authors: Takeaki
authors: T
authors: Ozawa
authors: Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.
authors: Hisato
authors: H
authors: Saitoh
authors: Department of Chemistry and Biochemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors: Yoichi
authors: Y
authors: Nakao
authors: Zhang Initiative Research Unit, RIKEN, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors: Kam Y J
authors: KY
authors: Zhang
authors: Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address: akihiro-i@riken.jp.
authors: Akihiro
authors: A
authors: Ito
authors: Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Science and Technology Corporation, CREST Research Project, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors: Minoru
authors: M
authors: Yoshida
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D006801:Humans; D008027:Light; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C583727:SIMC1 protein, human; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D008156:Luciferases
keywords: Assay; Protein–protein interactions; SIM; SUMOylation