2005 Fiscal Year Final Research Report Summary
Analysis of membrane protein complex using baculoviral display
| Project/Area Number |
16310147
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | The University of Tokyo |
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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)
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| 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)
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| Project Period (FY) |
2004 – 2005
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| Keywords | baculovirus / membrane protein / GPCR / γ-secretase / lipid raft / nicastrin / peptide transporter / FRET |
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| 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.
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[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.
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Journal Title
J Pathol. Apr;208(5)
Pages: 662-672
Description
「研究成果報告書概要(和文)」より
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[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.
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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
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[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
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Journal Title
J Pathol. Apr; 208(5)
Pages: 662-672
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
Protein Expr Purif. September26
Pages: 130-135
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Expression of the LXR{alpha} Protein in Human Atherosclerotic Lesions2005
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
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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
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[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.
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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
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[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.
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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
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[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.
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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.
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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
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[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.
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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
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[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.
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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
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