2002 Fiscal Year Annual Research Report

細胞内蛋白の特異的分解制御による癌治療法の確立-人工受容体を有するユビキチンリガーゼの作製と応用-

Research Project

Project/Area Number	13557019
Research Institution	Kyushu University
Principal Investigator	中山敬一九州大学, 生体防御医学研究所, 教授 (80291508)
Co-Investigator(Kenkyū-buntansha)	山下順範協和発酵工業株式会社, 東京研究所, 主任研究員中山啓子東北大学, 大学院・医学系研究科, 教授 (60294972) 畠山鎮次九州大学, 生体防御医学研究所, 助教授 (70294973)
Keywords	Myc / ユビキチン化 / キメラタンパク / Mad / Nedd-4
Research Abstract	この研究課題は、人工的なユビキチンリガーゼを作製し、特異的に重要な分子の発現制御を行うという基礎生物学における知見を臨床応用に向けた新たな遺伝子治療への応用を目指し、ユビキチンリガーゼを人工的に改変することによって、新しい基質特異性を有するユビキチンリガーゼを創出し、生体にとって有害な分子を除去するシステムを構築することを目的とする。本研究では特にモデルとして癌遺伝子産物のMycを対象にしている。Mycには特異的に結合する分子Madがあるので、このMadとユビキチンリガーゼであるNedd-4のキメラ分子を作製し、MycがMad/Nedd-4によってユビキチン化を受け、プロテアソームによって分解される系を構築する。本年度は、Mad/Nedd-4キメラタンパクを作製し、その効果を検討したほかに、Nedd-4とは異なる、SCFtypeユビキチンリガーゼでの効果を調べるために、F-boxとMadのキメラタンパクを作製した。これらのキメラタンパクは、in vitroでMycとの結合を確認した後、哺乳動物細胞への発現ベクターを用いて、過剰発現細胞を作製し、そのような細掛こおけるMycの存在量や半減期を測定し、キメラタンパクの効果判定を行った。また、プラークアッセイやコロニーアッセイを使用し、キメラ型ユビキチンリガーゼが存在した場合にMycが過剰発現させても、細胞の癌化能の低下が認められるかを検討した。さらに、Mad/Nedd-4キメラ型ユビキチンリガーゼを発現させた場合、癌細胞が個体レベルでの造腫瘍性を検討するために、ヌードマウスへの移植実験を現在行っている。

Research Products

(18 results)

All Other

All Publications (18 results)

[Publications] Minamishima, Y.A. et al.: "Recovery of liver mass without proliferation of hepatocytes after partial hepatectomy in Skp2-deficient mice"Cancer Res.. 62. 995-999 (2002)
[Publications] Yoshida, K. et al.: "Involvement of p27^<KIP1> degradation by Skp2 in the regulation of proliferation in response to wounding of corneal epithelium"Invest. Ophthalmol. Vis. Sci.. 43. 364-370 (2002)
[Publications] Koguchi, K. et al.: "Modulation of astrocyte proliferation by cyclin-dependent kinase inhibitor p27Kip1"Glia. 37. 93-104 (2002)
[Publications] Ikebe, C. et al.: "Isolation and characterization of a novel F-Box protein Pof10 in fission yeast"Biochem. Biophys. Res. Commun.. 290. 1399-1407 (2002)
[Publications] Yamanaka, A. et al.: "Multiple Skp1-reated proteins in Caenorthabditis elegans, Diverse patterns of interaction with Cullins and F-box proteins"Curr. Biol.. 12. 267-275 (2002)
[Publications] Kanematsu, T. et al.: "Role of the PLC-related, catalytically inactive protein p130 in GABA, receptor function"EMBO J.. 21. 1004-1011 (2002)
[Publications] Miyamoto A. et al.: "Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cδ"Nature. 416. 865-869 (2002)
[Publications] Masuda T. et al.: "Clinical and biological significance of S-phase kinase-associated protein 2(Skp2)gene expression in gastric carcinoma : modulation of malignant phenotype by Skp2 Overexpression, possibly via p27"Cancer Res.. 62. 3819-3825 (2002)
[Publications] Imai Y.et al.: "CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity"Mol. Cell. 10. 55-67 (2002)
[Publications] Garcia-Fernandez. et al.: "Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase Cδ"Oncogene. 21. 7533-7544 (2002)
[Publications] Nakamichi I. et al.: "Formation of Mallory body-like inclusions and cell death induced by deregulated expression of keratin 18"Mol. Biol. Cell. 13. 3411-3451 (2002)
[Publications] Shimoda K. et al.: "Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-α-induced suppression of B lvmphocvte formation"J. Immunol. I. 169. 4707-4711 (2002)
[Publications] Seto Y.et al.: "Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice"J. Immunol. 170. 1077-1083 (2002)
[Publications] Kaneko C. et al.: "Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a"Biochem. Biophys. Res. Commun.. 300. 297-304 (2002)
[Publications] Tomari Sl et al.: "Glomerular differentiation in p27 and p57 double-mutant metanephrol"Anat. Embryol.. 206. 31-36 (2002)
[Publications] Shirane M. et al.: "Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis"Nature Cell Biol.. 5. 28-63 (2002)
[Publications] Shimoda, K. et al.: "Partial impairment of interleukin-12(Il-12)and Il-18 signaling in Tyk2-deficient mice"Blood. 99. 2094-2099 (2002)
[Publications] Ishida N. et al.: "Phosphorylation of p27^<Kip1> on serine 10 is required for its binding to CRM1 and nuclear export"J. Biol. Chem.. 277. 14355-14358 (2002)

2002 Fiscal Year Annual Research Report

細胞内蛋白の特異的分解制御による癌治療法の確立-人工受容体を有するユビキチンリガーゼの作製と応用-

Principal Investigator

中山 敬一 九州大学, 生体防御医学研究所, 教授 (80291508)

Research Products

[Publications] Minamishima, Y.A. et al.: "Recovery of liver mass without proliferation of hepatocytes after partial hepatectomy in Skp2-deficient mice"Cancer Res.. 62. 995-999 (2002)

[Publications] Yoshida, K. et al.: "Involvement of p27^<KIP1> degradation by Skp2 in the regulation of proliferation in response to wounding of corneal epithelium"Invest. Ophthalmol. Vis. Sci.. 43. 364-370 (2002)

[Publications] Koguchi, K. et al.: "Modulation of astrocyte proliferation by cyclin-dependent kinase inhibitor p27Kip1"Glia. 37. 93-104 (2002)

[Publications] Ikebe, C. et al.: "Isolation and characterization of a novel F-Box protein Pof10 in fission yeast"Biochem. Biophys. Res. Commun.. 290. 1399-1407 (2002)

[Publications] Yamanaka, A. et al.: "Multiple Skp1-reated proteins in Caenorthabditis elegans, Diverse patterns of interaction with Cullins and F-box proteins"Curr. Biol.. 12. 267-275 (2002)

[Publications] Kanematsu, T. et al.: "Role of the PLC-related, catalytically inactive protein p130 in GABA, receptor function"EMBO J.. 21. 1004-1011 (2002)

[Publications] Miyamoto A. et al.: "Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cδ"Nature. 416. 865-869 (2002)

[Publications] Masuda T. et al.: "Clinical and biological significance of S-phase kinase-associated protein 2(Skp2)gene expression in gastric carcinoma : modulation of malignant phenotype by Skp2 Overexpression, possibly via p27"Cancer Res.. 62. 3819-3825 (2002)

[Publications] Imai Y.et al.: "CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity"Mol. Cell. 10. 55-67 (2002)

[Publications] Garcia-Fernandez. et al.: "Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase Cδ"Oncogene. 21. 7533-7544 (2002)

[Publications] Nakamichi I. et al.: "Formation of Mallory body-like inclusions and cell death induced by deregulated expression of keratin 18"Mol. Biol. Cell. 13. 3411-3451 (2002)

[Publications] Shimoda K. et al.: "Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-α-induced suppression of B lvmphocvte formation"J. Immunol. I. 169. 4707-4711 (2002)

[Publications] Seto Y.et al.: "Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice"J. Immunol. 170. 1077-1083 (2002)

[Publications] Kaneko C. et al.: "Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a"Biochem. Biophys. Res. Commun.. 300. 297-304 (2002)

[Publications] Tomari Sl et al.: "Glomerular differentiation in p27 and p57 double-mutant metanephrol"Anat. Embryol.. 206. 31-36 (2002)

[Publications] Shirane M. et al.: "Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis"Nature Cell Biol.. 5. 28-63 (2002)

[Publications] Shimoda, K. et al.: "Partial impairment of interleukin-12(Il-12)and Il-18 signaling in Tyk2-deficient mice"Blood. 99. 2094-2099 (2002)

[Publications] Ishida N. et al.: "Phosphorylation of p27^<Kip1> on serine 10 is required for its binding to CRM1 and nuclear export"J. Biol. Chem.. 277. 14355-14358 (2002)

中山敬一九州大学, 生体防御医学研究所, 教授 (80291508)