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The Analysis of Proteolytic Enzymes for SREBP and Its Application to Drug Discovery

Research Project

Project/Area Number 11557080
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section展開研究
Research Field Metabolomics
Research InstitutionThe University of Tokyo

Principal Investigator

KODAMA Tatsuhiko  Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Professor., 先端科学技術研究センター, 教授 (90170266)

Co-Investigator(Kenkyū-buntansha) KAWABE Yosiki  Chugai Pharmaceutical Co., LTD., Senior Scientist., 創薬研究所, 研究員
HAMAKUBO Takao  Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Associate Professor., 先端科学技術研究センター, 助教授 (90198797)
川邊 良樹  中外製薬(株), 創薬研究所, 研究員
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 2000: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥8,900,000 (Direct Cost: ¥8,900,000)
KeywordsSREBP / protease / cholesterol / SCAP / SSD / S1P / baculovirus / cathepsin B / HMG-CoA還元酵素 / LDL受容体 / HMG-COA還元酵素 / SIP
Research Abstract

We have been reported that SREBP-2 (sterol regulatory element-binding protein-2) play a key role in the regulation of cholesterol butt not fatty acid metabolism by establishing cell lines in which human SREBP-2 could be induced by IPTG.We also constructed a sensitive assay method for detection of SREBP cutting enzymes using internally quenched fluorogenic peptide substrate, the amino acid sequence of which is the same as the predicted cutting site. By this assay system, we purified neprilysin and cathepsin B as cleaving enzymes in hamster liver microsome fraction. These enzymes were considered to be degradative enzymes for SREBPs in endoplasmic reticulum. The soluble part of human site 1 protease (S1P) was expressed in CHO cells and purified with his-tag affinity column. As the activity of soluble S1P was hardly detectable by the above assay method, we developed a new expression system using the baculovirus. By the baculovirus expression system, we have succeeded to express whole S1P protein and detected Ca^<++> dependent enzymatic activities primarily on S1P expressed budded viruses. We have also detected the expression of membrane proteins such as SREBP-2, SCAP (SREBP cleavage activating protein) and HMG-CoA reductase in viral fractions of culture supernatant of transfected Sf9 cells. This expression system is considered to be a useful tool for expressing membrane proteins. On the other hand, we analyzed genomic structures of S1P, SCAP, and HMG-CoA reductase, and found that exon-shuffling may have occurred in evolutionally step in those proteins that have sterol sensing domain (SSD).

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] Nakajima T,Ota N,Kodama T,Emi M: "Isolation and radiation hybrid mapping of a highly polymorphic CA repeat-sequence at the SREBP cleavage-activating protein (SCAP) locus."J Hum Genet. 44(5). 350-1 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Morimoto K,Wada T,Hinagata J,Kodama T: "VXFD in the cytoplasmic domain of macrophage scavenger receptors mediates their efficient internalization and cell-surface expression."Biol Pharm Bull. 22(10). 1022-6 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shirai H,Murakami T,Yamada Y,Doi T,Kodama T: "Structure and function of type I and II macrophage scavenger receptors."TMech Ageing Dev. 111. 107-21 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Takaku M,Wada Y,Jinnouchi K,Kodama T: "An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation."Arterioscler Thromb Vasc Biol. 19(10). 2330-2339 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamaguchi M,Shirai H,Sato R,Kawabe Y,Kodama T: "Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes."Biochem Biophys Res Commun. 258(3). 542-7 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Terauchi Y,Kodamam T, et al.: "Increased insulin sensitivity and hypoglycemia in mice lacking the p85a subunit of phophoinositide 3 kinase"Nature Genetics. 21. 230-235 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawabe Y,Suzuki T,Hayashi M,Hamakubo T,Sato R and Kodama T.: "The Physiological role of sterol regulatory element- binding protein-2 in cultured human cells"Biochim Biophys Acta. 1436. 307-318 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamaguchi M,Shirai H,Sato R,Kawabe Y,Fukuda R,Kodama T,Hamakubo,T: "Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes."Biochem Biophys Res Commun. May19 258(3). 542-7 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Takaku M,Wada Y,Jinnouchi K,Takeya M,Takahashi K,Usuda H,Naito M,Kurihara H,Yazaki Y,Kumazawa Y,Okimoto Y,Umetani M,Noguchi N,Niki E,Hamakubo T,Kodama T: "An In Vitro Cocultrue Model of Transmigrant Monocytes and Foam Cell Formation."Arterioscler Thromb Vasc Biol. 19(10). 2330-2339 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T,Ota N,Kodama T,Emi M: "Isolation and radiation hybrid mapping of a highly polymorphic CA repeat sequence at the SREBP cleavage-activating protein (SCAP) locus."J Hum Genet. 44. 350-1 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T,Hamakubo T,Kodama T,Inazawa J,Emi M.: "Genomic structure and chromosomal mapping of the human sterol regulatory element binding protein (SREBP) cleavage- activating protein (SCAP) gene."J Hum Genet.. 44. 402-7 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T,Iwaki K,Kodama T,Inazawa J,Emi M.: "Genomic structure and chromosomal mapping of tthe human Site- 1 protease (S1P) gene"J Hum Genet. 45. 212-217 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Liao HS,Kodama T,Geng YJ.: "Expression of class A scavenger receptor inhibits apoptosis of macrophages triggered by oxidizedlow density lipoprotein and oxysterol."Arterioscler Thromb Vasc Biol. 12. 212-217 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T,Iwaki K,Hamakubo T,Kodama T,Emi M.: "Genomic structure of the gene encoding human 3- hydroxy-3 - methyl- glutaryl coenzyme A reductase : comparison of ex on/intr on organization of sterol-sensing domains among four related genes."J Hum Genet. 45. 284-9 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Babaev VR,Gleaves LA,Carter KJ,Suzuki H,Kodama T,Fazio S,Linton MF.: "Reduced ather osclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A."Arterioscler Thromb Vasc Biol. 20. 2593-9 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawabe Y, Suzuki T, Hayashi M, Hamakubo T, Sato R and Kodama T.: "The Physiological role of sterol regulatory element-binding protein-2 in cultured human cells"Biochim Biophys Acta. 1436. 307-318 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamaguchi M, Shirai H, Sato R, Kawabe Y, Fukuda R, Kodama T, Hamakubo T: "Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes."Biochem Biophys Res Commun. 258 (3). 542-7 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Takaku M, Wada Y, Jinnouchi K, Takeya M, Takahashi K, Usuda H, Naito M, Kurihara H, Yazaki Y, Kumazawa Y, Okimoto Y, M, Noguchi N, Niki E, Hamakubo T, Kodama T: "An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation."Arterioscler Thromb Vasc Biol. 19 (10). 23302339 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T, Ota N, Kodama T, Emi M: "Isolation and radiation hybrid mapping of a highly polymorphic CA Repeat sequence at the SREBP cleavage-activating protein (SCAP) locus."J Hum Genet. 44. 350-1 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T, Hamakubo, Kodana T, Inazawa J, Emi M.: "Genomic structure and chromosomal mapping of the human sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) gene."J Hum Genet.. 44. 402-7 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T, Iwaki K, Kodama T, Inazawa J, Emi M.: "Genomic structure and chromosomal mapping of the human Site-1 protease (S1P) gene"J Hum Genet. 45. 212-217 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Liao HS, Kodama T, Geng YJ.: "Expression of class A scavenger receptor inhibits apoptosis of macrophages triggered by oxidized low density lipoprotein and oxysterol.."Arterioscler Thromb Vasc Biol. 12. 196875 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T, Iwaki K, Hamakubo T, Kodama T, Emi M.: "Genomic structure of the gene encoding human 3-hydroxy-3-methyl-glutaryl coenzyme A reductase : comparison of exon/intron organization of sterol-sensing domains among four related genes."J Hum Genet. 45. 284-9 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Babaev VR, Gleaves LA, Carter KJ, Suzuki H, Kodama T, Fazio S.Linton MF.: "Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A."Arterioscler Thromb Vasc Biol. 20. 2593-9 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima T,Iwaki K,Kodama T,Inazawa J,Emi M.: "Genomic structure and chromosomal mapping of the human Site-1 protease (S1P) gene"J Hum Genet. 45. 212-217 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Liao HS,Kodama T,Geng YJ.: "Expression of class A scavenger receptor inhibits apoptosis of macrophages triggered by oxidized low density lipoprotein and oxysterol. "Arterioscler Thromb Vasc Biol.. 12. 1968-75 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakajima T,Iwaki K,Hamakubo T,Kodama T,Emi M.: "Genomic structure of the gene encoding human 3-hydroxy-3-methyl-glutaryl coenzyme A reductase : comparison of exon/intron organization of sterol-sensing domains among four related genes."J Hum Genet.. 45. 284-9 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Babaev VR,Gleaves LA,Suzuki H,Kodama T,: "Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A."Arterioscler Thromb Vasc Biol.. 20. 2593-9 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakajima T,Ota N,Kodama T,Emi M: "Isolation and radiation hybrid mapping of a hiphly polymorphic CA repeatsequence at the SREBP cleavating protein (SCAP) locus"J Hum Genet. 44(5). 350-351 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Morimoto K,Wada Y,Hinagata J,Kodama T: "VXFD in the cytoplasmic domain of macrophage scavenger receptors mediates their efficient internalization and cell-surface expression"Biol Pharm Bull. 22(10). 1022-1026 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Shirai H,Murakami T,Yamada Y,Doi T,Kodama T: "Structure and function of type I and ll macrophage scavenger receptors"Tmech Ageing Dev. 111. 107-121 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Takaku M,Wada Y,Jinnouchi K,Kodama T: "An In Vitro Coculture Model of lransmigrant Monocytes and Foam Cell Formation"Arterioscler Thromb Vasc Biol. 19(10). 2330-2339 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yamaguchi M,Shirai H,Sato R,Kawabe Y,Kodama T: "Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes"Biochem Biophys Res Commun. 258(3). 542-547 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tarauchi Y,Kodamam T,et al.: "Increased insulin sensitivity and hypoglycemia in mice lecking the p85a subunit of phophoinositide 3 kinase"Nature Genetics. 21. 230-235 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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