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Study for Ineractions between functional domains of neuronal nitric oxide synthase and cellular proteins

Research Project

Project/Area Number 14580640
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionTOHOKU UNIVERSITY

Principal Investigator

SAGAMI Ikuko  Tohoku University, Institute of Multidisciplinary Research for Advanced Material, lecturer, 多元物質科学研究所, 講師 (10143033)

Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
Keywordsnitric-oxide synthase / calmodulin / caveoline / signal transduction / site-directed mutagenesis / protein-protein interactions / autoinhibitory domain
Research Abstract

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O2 activation and NO formation. On the other hands, caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated by using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (82-101:CaV1p1) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an 1C50 value of 1.8 []M. Mutations of F584 and W587 within a caveolin-binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences m the mechanism of inhibition by caveolin for nNOS as compared to those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of an autoinhibitory domain deletion mutant (delta40), and a C-terminal truncation mutant (deltaC33), suggesting that CaV1p1 inhibits interdomain electron-transfer induced by CaM from the reductase domain to the oxygenase domain.

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Bengea, S.: "Analysis of the Kinetics of CO Binding to Neuronal Nitric Oxide Synthase by Flash Photolysis : Dual Effects of Substrates, Inhibitors, and Tetrahydrobiopterin"J.Inorg.Biochem. (印刷中). (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yadav, J.: "Arg410 near the heme proximal ligand of neuronal nitric oxide synthase is critical for both substrate recognition and electron transfer"Chem.Letters. (印刷中). (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sato, Y.: "Identification of caveolin-1-interacting sites in neuronal nitric oxide synthase : molecular mechanism for inhibition of NO formation"J.Biol.Chem.. 279・10. 8827-8836 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Takahashi H.: "Critical Role of Val567 in Substrate Recongnition by Neuronal Nitric Oxide Synthase for NO Formation Activity"Chem.Letters. 32・11. 998-999 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yadav, J.: "Cyanide Binding Study of Neuronal Nitric Oxide Synthase : Effects of Inhibitors and Mutations at the Substrate Binding Site"J.Inorg.Biochem.. 94. 25-30 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Bengea, S.: "CO Binding to the Isolated Oxygenase Domain of Neuronal Nitric Oxide Synthase : Effects of Inhibitors and Mutations at the Substrate-binding Site"J.Inorg.Biochem. 94. 343-347 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 佐上郁子: "NO合成酵素の構造と機能:主に電子伝達のメカニズムについて"生化学. 75・5. 351-358 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 佐上郁子: "P450の分子生物学 分担「他のヘムーチオーレートタンパク質の構造と機能」(大村恒雄, 石村 巽, 藤井義明 編)"講談社サイエンティフィク. 255(67-73) (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Bemgea S., et al.: "Analysis of the Kinetics of CO Binding to Neuronal Nitric Oxide Synthase by Flash Photolysis : Dual Effects of Substrates, Inhibitors, and Tetrahydrobiopterin"J.Inorg.Biochem.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yadav, J., et al.: "Arg410 near the Heme Proximal Ligand of Neuronal Nitric Oxide Synthase Is Critical for Both Substrate Recognition and Electron Transfer"Chem.Letters. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sato Y., et al.: "Identification of caveolin-1-interacting sites in neuronal nitric oxide synthase : molecular mechanism for inhibition of NO formation"J.Biol.Chem.. 279-10. 8827-8836 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Takahashi H., et al.: "Critical Role of Val567 in Substrate Recognition by Neuronal Nitric Oxide Synthase for NO Formation Activity"Chem.Letter. 32-11. 998-999 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yadav, J., et al.: "Cyanide binding study of neuronal nitric oxide synthase : effects of inhibitors and mutations at the substrate binding site"J.Inorg.Biochem. 94. 25-30 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Bengea, S., et al.: "CO binding to the isolated oxygenase domain of neuronal nitric oxide synthase : effects of inhibitors and mutations at the substrate-binding site"J.Inorg.Biochem. 94. 343-347 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sagami, I., et al.: "Structure-function relationships of NO synthase : electron transfer reaction"Sekagaku. 75-5. 351-358 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sagami, I.(Ohmura, T, Ishimura, Y, fujii, Y., eds.): "Structures and functions of other heme-thiolate proteins"Molecular Biology of Cytochrome P450 (Koudannsya Scientifics Press). 7 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Simona Bengea, Ikuko Sagami, Toru Shimizu: "CO Binding to the Isolated Oxygenase Domain of Neuronal Nitric Oxide Synthase :"J.Inorganic Biochemistry. 94. 343-347 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jyoti Yadav, Ikuko Sagami, Toru Shimizu: "Cyanide Binding Study of Neuronal Nitric Oxide Synthase :"J.Inorganic Biochemistry. 95. 25-30 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Yuko Sato Y., Ikuko Sagami, Toru Shimizu: "Identification of caveolin-1-interaction sites in neuronal nitric oxide synthase : molecular mechanism for inhibition of NO formation"J.Biol.Chem. (In press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Simona Bengea, Ikuko Sagami, Toru Shimizu: "CO Binding to the Isolated Oxygenase Domain of Neuronal Nitric Oxide Synthase"J. Inorganic Biochemistry. (in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Jyoti Yadav, Ikuko Sagami, Toru Shimizu: "Cyanide Binding Study of Neuronal Nitric Oxide Synthase"J. Inorganic Biochemistry. (in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 佐上 郁子: "部位特異的アミノ酸置換"新タンパク質科学実験法(大島泰郎、鈴木紘一、藤井義明、村松喬編). 2. 160-173 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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