Analysis of membrane protein complex using baculoviral display

• Project/Area Number: 16310147
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: The University of Tokyo
• Principal Investigator: HAMAKUBO Takao The University of Tokyo, Research Center for Advanced Science and Technology, Professor, 先端科学技術研究センター, 教授 (90198797)
• Co-Investigator(Kenkyū-buntansha): SAKIHAMA Toshiko The University of Tokyo, Research Center for Advanced Science and Technology, Specially Appoimted Associate Professor, 先端科学技術研究センター, 科学技術振興特任教員(特任助教授) (70187061) ; MASUDA Kazuyuki The University of Tokyo, Research Center for Advanced Science and Technology, Specially Appoimted Research Associate, 先端科学技術研究センター, 科学技術振興特任教員(特任助手) (20345151)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥15,700,000 (Direct Cost: ¥15,700,000); Fiscal Year 2005: ¥6,600,000 (Direct Cost: ¥6,600,000); Fiscal Year 2004: ¥9,100,000 (Direct Cost: ¥9,100,000)
• Keywords: baculovirus / membrane protein / GPCR / γ-secretase / lipid raft / nicastrin / peptide transporter / FRET / 核内受容体 / 磁性ビーズ / プロテオミクス / 転写複合体 / HNF4α / ラフト / スタチン / アルツハイマー

Research Abstract

Membrane proteins such as peptide transporter G-protein coupled receptors (BLT1, CCR2, CCR5, Edg1, AT1a, AT2, D1, D5) and their complex (reoeptor-Gs-adenylyl cyclase) or active γ-secretase complex (presenillin-APH1-nicastrin-PEN2) have been successfully recovered in baculovirus fraction. The expressed membrane proteins were considered to be active on the baculovirus membrane. Interestingly, γ -secretase acitivity was recovered only when all 4 components were expressed on the baculovirus. Using these membrane protein expressed baculovirus, specific monoclonal antibodies (Mabs) were generated against membrane proteins including GPCR. These Mabs are usable not only for western blotting but also FACS analysis and immunohistochemistry. Some of them have shown the neutralizing activity against ligand-receptor mediated cAMP production or the antibody dependent cell cytotoxic activities. Also we showed the importance of lipid micro- environment of membrane and geranyl-geranylation for γ-secretase acitivity in generating β-amyloid from its precursor. One molecule FRET(fluorescence resonance energy transfer) was succefully constructed using RGS (regulator of G- protein signalling) and Gi which recognized G- protein activation by alminium fluoride.
These results indicate that the baculovirus display system of membrane proteins or their complex is useful not only for high throughput screening assay system but also for making active Mabs for therapeutic use.

Research Products

• [Journal Article] Three-dimensional structure of the gamma-secretase complex. (2006)
  • Author(s): OguraT., MioK., HayashiI., MiyashitaH., KopanR., KodamaT., Hamakubo T., Iwastubo T., Tomita T., Sato C.
  • Journal Title: Biochem Biophys Res Commun. May5:343(2) Pages: 525-534
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay. (2006)
  • Author(s): Daigo K, Sugita S, Mochizuki Y, Iwanari H, Hiraishi K, MiyanoK, Kodama T, Hamakubo T.
  • Journal Title: Anal Biochem. Apr15;351(2) Pages: 219-228
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. (2006)
  • Author(s): TanakaT., JiangS., HottaH., TakanoK., IwanariH., SumiK., DaigoK., OhashiR., SugaiM., MidorikawaY., HippoY., WatanabeA., UchiyamaY., HasegawaG., ReidP., AburataniH., IkegameC., UmezuH., HirayamaY., HamakuboT., SakaiJ., NaitoM., KodamaT.
  • Journal Title: J Pathol. Apr;208(5) Pages: 662-672
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Three-dimensional structure of the gamma-secretase complex. (2006)
  • Author(s): Ogura T, Mio K, Hayashi I, Miyashita H, Fukuda R, Kopan R, Kodama T, Hamakubo T, Iwastubo T, Tomita T, Sato C.
  • Journal Title: Biochem Biophys Res Commun. May 5;343(2) Pages: 525-534
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay. (2006)
  • Author(s): Daigo K, Sugita S, Mochizuki Y, Iwanari H, Hiraishi K, Miyano K, Kodama T, Hamakubo T.
  • Journal Title: Anal Biochem. Apr 15;351(2) Pages: 219-228
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. (2006)
  • Author(s): Tanaka T, Jiang S, Hotta H, Takano K, Iwanari H, Sumi K, Daigo K, Ohashi R, Sugai M, Ikegame C, Umezu H, Hirayama Y, Midorikawa Y, Hippo Y, Watanabe A, Uchiyama Y, Hasegawa G, Reid P, Aburatani H, Hamakubo T, Sakai J, Naito M, Kodama T
  • Journal Title: J Pathol. Apr; 208(5) Pages: 662-672
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Three-dimensional structure of the γ-secretase complex. (2006)
  • Author(s): Ogura T, Mio M, Hayashi I, Miyashita H, Fukuda R, Kodama T, Hamakubo T, Iwastubo T, Tomita T, sato C
  • Journal Title: Biochemical and Biophysical Research Communications (印刷中)
• [Journal Article] A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay. (2006)
  • Author(s): Daigo K, Sugita S, Mochizuki Y, Iwanari H, Hiraishi K, Miyano K, Kodama T, Hamakubo T.
  • Journal Title: Anal Biochem. (印刷中)
• [Journal Article] Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. (2006)
  • Author(s): Tanaka T, Jiang S, Hamakubo T, Sakai J, Naito M, Kodama T.
  • Journal Title: J Pathol. (印刷中)
• [Journal Article] Recovery of functional peptide transporter PepT1 in budded baculovirus fraction. (2005)
  • Author(s): Saitoh R, Ohtomo T, Ito Y, Nezu J, Kimura N, Funahashi S, Aso Y, Ohizumi I, Kodama T, Hamakubo T, Tsuchiya M.
  • Journal Title: Protein Expr Purif. September26 Pages: 130-135
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Histone methyltransferases G9a and GLP form heteromeric complexex and are both crucial for methylation of euchromatin at He-K9. (2005)
  • Author(s): Tachibana M, Ueda J, Fukuda M, TakedaN, OhtaT, IwanariH, Sakihama T, Kodama T, Hamakubo T.
  • Journal Title: Genes Dev. Apr 1;19(7) Pages: 815-826
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Association of active gamma-secretase complex with lipid rafts. (2005)
  • Author(s): UranoY, Hayashi I, Isoo N, Reid PC, Shibasaki Y, Noguchi N, Tomita T, IwatsuboT, HamakuboT, Kodama T.
  • Journal Title: J Lipid Res. May46(5) Pages: 904-912
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Expression of the LXR{alpha} Protein in Human Atherosclerotic Lesions (2005)
  • Author(s): Watanabe Y, Jiang S, Takabe W, Ohashi R, Tanaka T, Uchiyama Y, Katsumi K, Iwanari H, Noguchi N, Naito M, Hamakubo T, Kodama T
  • Journal Title: Arterioscler Thromb Vasc Biol. Mar;25(3) Pages: 622-627
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Vascular endothelial growth factor- and thrombin-induced termination factor, down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. (2005)
  • Author(s): MinamiT, HoriuchiK, MiuraM, AbidMR, TakabeW, NoguchiN, KohroT, GeX, AburataniH, HamakuboT, KodamaT, Aird WC.
  • Journal Title: J Biol Chem. Nov26;279(48) Pages: 50537-50554
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Recovery of functional peptide transporter PepT1 in budded baculovirus fraction. (2005)
  • Author(s): Saitoh R, Ohtomo T, Ito Y, Nezu J, Kimura N, Funahashi S, Aso Y, Ohizumi I, Kodama T, Hamakubo T, Tsuchiya M.
  • Journal Title: Protein Expr Purif. September;46(1) Pages: 130-135
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. (2005)
  • Author(s): Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, Sakihama T, Kodama T, Hamakubo T, Shinkai Y.
  • Journal Title: Genes Dev. Apr 1;19(7) Pages: 815-826
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Association of active gamma -secretase complex with lipid rafts. (2005)
  • Author(s): Urano Y, Hayashi I, Isoo N, Reid PC, Shibasaki Y, Noguchi N, Tomita T, Iwatsubo T, Hamakubo T, Kodama T.
  • Journal Title: J Lipid Res. May; 46(5) Pages: 904-912
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of the LXR{alpha} Protein in Human Atherosclerotic Lesions. (2005)
  • Author(s): Watanabe Y, Jiang S, Takabe W, Ohashi R, Tanaka T, Uchiyama Y, Katsumi K, Iwanari H, Noguchi N, Naito M, Hamakubo T, Kodama T.
  • Journal Title: Arterioscler Thromb Vasc Biol. Mar;25(3) Pages: 622-627
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Vascular endothelial growth factor- and thrombin-induced termination factor, down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. (2005)
  • Author(s): Minami T, Horiuchi K, Miura M, Abid MR, Takabe W, Noguchi N, Kohro T, Ge X, Aburatani H, Hamakubo T, Kodama T, Aird WC.
  • Journal Title: J Biol Chem. Nov 26;279(48) Pages: 50537-50554
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles. (2005)
  • Author(s): Ikuo Hayashi, Yasuomi Urano, Rie Fukuda, Nariko Isoo, Tatsuhiko Kodama, Takao Hamakubo, Taisuke Tomita, Takeshi Iwatsubo
  • Journal Title: J Biol Chem. Sep 3;279(36) Pages: 38040-38046
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Recovery of functional peptide transporter PepT1 in budded baculovirus fraction. (2005)
  • Author(s): Saitoh R, Ohtomo T, Ito Y, Nezu J, Kimura N, Funahashi S, Aso Y, Ohizumi I, Kodama T, Hamakubo T
  • Journal Title: Protein Expr Purif September 26 Pages: 130-135
• [Journal Article] Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. (2005)
  • Author(s): Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, Sakihama T, Kodama T, Hamakubo T, Shinkai Y.
  • Journal Title: Genes Dev. April 1;19 Pages: 815-826
• [Journal Article] Association of active gamma -secretase complex with lipid rafts. (2005)
  • Author(s): Urano Y, Hayashi I, Isoo N, Reid PC, Shibasaki Y, Noguchi N, Tomita T, Iwatsubo T, Hamakubo T, Kodama T.
  • Journal Title: J Lipid Res. May46(5) Pages: 904-912
• [Journal Article] Expression of the LXR{alpha} Protein in Human Atherosclerotic Lesions (2005)
  • Author(s): Watanabe Y, Jiang S, Takabe W, Ohashi R, Tanaka T, Uchiyama Y, Katsumi K, Iwanari H, Noguchi N, Naito M, Hamakubo T, Kodama T
  • Journal Title: Arterioscler Thromb Vasc Biol. March 25 Pages: 622-627
• [Journal Article] Association of active gamma-secretase complex with lipid rafts. (2005)
  • Author(s): UranoY, HayashiI, IsooN, ReidPC, ShibasakiY, NoguchiN, TomitaT, IwatsuboT, HamakuboT, KodamaT
  • Journal Title: J Lipid Res. Feb 16(Epub ahead of print)
• [Journal Article] Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles. (2004)
  • Author(s): Hayashi I, Urano Y, Fukuda R, Isoo N, Kodama T, Hamakubo T, Tomita T, Iwatsubo T.
  • Journal Title: J Biol Chem. Sep3;279(36) Pages: 38040-38046
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A Comparison of Differences in the Gene Expression Profiles of Phorbol 12-myristate 13-acetate Differentiated THP-1 Cells and Human Monocyte-derived Macrophage. (2004)
  • Author(s): Kohro T, Tanaka T, Murakami T, Wada Y, Aburatani H, Hamakubo T, Kodama T
  • Journal Title: J Atheroscler Thromb. 11(2) Pages: 88-97
  • NAID: 10012921563
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. (2004)
  • Author(s): HippoY, WatanabeK, WatanabeA, MidorikawaY, Yamamoto, IharaS, TokitaS, IwanariH, ItoY, NakanoK, NezuJ, TsunodaH, YoshinoT, OhizumiI, TsuchiyaM, OhnishiS, MakuuchiM, HamakuboT, KodamaT, Aburatani H.
  • Journal Title: Cancer Res. Apr 1;64(7) Pages: 2418-2423
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interaction between Hex and GATA Transcription Factors in Vascular Endothelial Cells Inhibits flk-1/KDR-mediated Vascular Endothelial Growth Factor Signaling. (2004)
  • Author(s): Minami T, MurakamiT, Horiuchi K, Miura M, Noguchi T, Miyazaki J, Hamakubo T, Aird WC, Kodama T.
  • Journal Title: J Biol Chem. May14;279(20) Pages: 20626-20635
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A Comparison of Differences in the Gene Expression Profiles of Phorbol 12-myristate 13-acetate Differentiated THP-1 Cells and Human Monocyte-derived Macrophage. (2004)
  • Author(s): Kohro T, Tanaka T, Murakami T, Wada Y, Aburatani H, Hamakubo T, Kodama T.
  • Journal Title: J Atheroscler Thromb. 11(2) Pages: 88-97
  • NAID: 10012921563
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. (2004)
  • Author(s): Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S, Tokita S, Iwanari H, Ito Y, Nakano K, Nezu J, Tsunoda H, Yoshino T, Ohizumi I, Tsuchiya M, Ohnishi S, Makuuchi M, Hamakubo T, Kodama T, Aburatani H.
  • Journal Title: Cancer Res. Apr 1;64(7) Pages: 2418-2423
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Interaction between Hex and GATA Transcription Factors in Vascular Endothelial Cells Inhibits flk-1/KDR-mediated Vascular Endothelial Growth Factor Signaling. (2004)
  • Author(s): Minami T, Murakami T, Horiuchi K, Miura M, Noguchi T, Miyazaki J, Hamakubo T, Aird WC, Kodama T.
  • Journal Title: J Biol Chem. May 14;279(20) Pages: 20626-20635
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of the LXR{alpha} Protein in Human Atherosclerotic Lesions. (2004)
  • Author(s): Watanabe Y, Jiang S, Takabe W, Ohashi R, Tanaka T, Uchiyama Y, Katsumi K, Iwanari H, Noguchi N, Naito M, Hamakubo T, Kodama T.
  • Journal Title: Arterioscler Thromb Vasc Biol. Dec 29(Epub ahead of print)
• [Journal Article] Vascular endothelial growth factor-and thrombin-induced termination factor, down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. (2004)
  • Author(s): Minami T, Horiuchi K, Miura M, Abid MR, Takabe W, Noguchi N, Kohro T, Ge X, Aburatani H, Hamakubo T, Kodama T, Aird WC.
  • Journal Title: J Biol Chem. Nov 26;279(48) Pages: 50537-50554
• [Journal Article] Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles. (2004)
  • Author(s): Ikuo Hayashi, Yasuomi Urano, Rie Fukuda, Noriko Isoo, Tatsuhiko Kodama, Takao Hamakubo, Taisuke Tomita, Takeshi Iwatsubo
  • Journal Title: J Biol Chem. Sep 3;279(36) Pages: 38040-38046
• [Journal Article] A Comparison of Differences in the Gene Expression Profiles of Phorbol 12-myristate 13-acetate Differentiated THP-1 Cells and Human Monocyte-derived Macrophage. (2004)
  • Author(s): Kohro T, Tanaka T, Murakami T, Wada Y, Aburatani H, Hamakubo T, Kodama T.
  • Journal Title: J Atheroscler Thomb. 11(2) Pages: 88-97
  • NAID: 10012921563
• [Journal Article] Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. (2004)
  • Author(s): Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S, Tokita S, Iwanari H, Ito Y, Nakano K, Nezu J, Tsunoda H, Yoshino T, Ohizumi I, Tsuchiya M, Ohnishi S, Makuuchi M, Hamakubo T, Kodama T, Aburatani H
  • Journal Title: Cancer Res. Apr 1;64(7) Pages: 2418-2423