RRC ID 18520
Author Watanabe M, Itoh K.
Title Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1.
Journal Biochim. Biophys. Acta
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.
Volume 1814(7)
Pages 900-7
Published 2011-7
DOI 10.1016/j.bbapap.2011.04.006
PII S1570-9639(11)00092-6
PMID 21545853
MeSH Binding Sites Cysteine Endopeptidases / metabolism Endopeptidases / metabolism HEK293 Cells Humans Immunoblotting Immunoprecipitation Mutation Nitric Oxide Synthase Type I / genetics Nitric Oxide Synthase Type I / metabolism* Protein Binding Protein Inhibitors of Activated STAT / metabolism Protein Processing, Post-Translational* SUMO-1 Protein / genetics SUMO-1 Protein / metabolism* Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Substrate Specificity Sumoylation Transfection Ubiquitin-Conjugating Enzymes / metabolism
IF 3.79
Times Cited 9
WOS Category BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells 293 (RCB1637)