title: Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1.
abstract: The multifaceted functions of nitric oxide (NO) in the CNS are defined by the activity of neuronal NO synathase (nNOS). The activities of nNOS are modulated by posttranslational modifications, such as phosphorylation and ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to elucidate whether nNOS is posttranslationally modified by SUMO proteins. Bioinformatic analyses using SUMOplot and SUMOFI predicted that nNOS had potential SUMO modification sites. When HEK293T cells were transiently co-expressed with nNOS and SUMO-1, two bands corresponding to nNOS-SUMO-1 conjugates were detected. In addition, two nNOS-SUMO-1 conjugates were confirmed by an in vitro sumoylation assay using recombinant proteins. Furthermore, nNOS-SUMO-1 conjugates were identified by MALDI-QIT/TOF mass spectrometry. These findings indicate that nNOS is clearly defined as a SUMO-1 target protein both in vitro and at the cellular level. We next characterized specific enzymes in the nNOS-SUMO-1 conjugation cycle at the cellular level. SUMO-1 conjugation of nNOS depended on Ubc9 (E2). The interaction between nNOS and Ubc9 was facilitated by PIASxβ (E3). On the other hand, SUMO-1 was deconjugated from nNOS by SENP1 and SENP2. Overall, this study has newly identified that nNOS is posttranslationally modified by SUMO-1.
authors.affiliation: Laboratory of Molecular and Cellular Neuroscience, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Kagawa 769-2193, Japan. wmatanabe@kph.bunri-u.ac.jp
authors.forename: Masatomo
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kouichi
authors.initials: K
authors.lastname: Itoh
mesh_terms: D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D009154:Mutation; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D058207:Sumoylation; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: C501001:PIAS2 protein, human; D050828:Protein Inhibitors of Activated STAT; D025842:SUMO-1 Protein; D052248:Nitric Oxide Synthase Type I; D044763:Ubiquitin-Conjugating Enzymes; D010450:Endopeptidases; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases; C501833:SENP2 protein, human; D000098843:Ubiquitin-Conjugating Enzyme UBC9