X染色体上の新規ガン抑制遺伝子MEFに関する研究

• Project/Area Number: 17790068
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Biological pharmacy
• Research Institution: Kumamoto University
• Principal Investigator: メリー・アン スイコ 熊本大学, 大学院・医学薬学研究部, 助手 (20363525)
• Project Period (FY): 2005 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000); Fiscal Year 2005: ¥1,900,000 (Direct Cost: ¥1,900,000)
• Keywords: Myeloid elf-1 like factor(MEF) / 癌抑制遺伝子 / 翻訳後修飾 / SUMO / 転写因子 / プロモーター活性 / lysozyme / perforin / Myeloid elf-1 like factor (MEF) / プロモーター / Sp1 / DNAメチル化

Research Abstract

Meyeloid-elf-1 factor(MEF)は663アミノ酸からなる転写因子で,85アミノ酸からなるDNA結合部位(ETSドメイン)をもつ転写因子の1つである.本研究では,MEFの翻訳後修飾機構の解明を主目的とし,近年,その制御機構が脚光をあびるSUMO化修飾に関して,種々の検討を行った.まず,MEFがSUMO化されるか否かを検討するため,大腸菌内に基質タンパク質と,SUMO化関連タンパク質(SUMO, E1,E2)を過剰発現させることで,SUMO化タンパク質を簡便,大量に調整した.pET30a-MEFとpTE1E2S1またはpTE1E2S2をBL21(DE3)STAR株にco-transformationした後,IPTGの添加によりタンパク質発現を誘導した.talon beadsを用いて,大腸菌lysateからMEFタンパク質を回収し,anti-MEF antibodyを用いてWestern blottingを行った.その結果,SUMO化関連酵素を含む大腸菌lysateでは,高分子量のMEFが観察された.従って,過剰発現させたSUMOによって,MEFはSUMO化されることが示唆された,次に,HEK293細胞にLysozymeおよびPerforinプロモーターをもつルシフェラーゼコンストラクトとMEFもしくは,MEF+SUMO1+Ubc9を導入し,それぞれのプロモーター活性を測定した.MEFの導入により,Lysozymeプロモーターの活性は上昇したが,MEFと共にSUMOを導入すると,それぞれのプロモニター活性がMEF単独導入時に比較して3分の1程度にまで減少した.従って,プロモーター活性に対するMEFの転写活性化能は,SUMO化よって低下することが示唆された.

Research Products

• [Journal Article] Curcumin enhances cystic fibrosis transmembrane regulator expression by down-regulating calreticulin. (2007)
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• [Journal Article] Calreticulin Facilitates the Cell Surface Expression of ABCG5/G8 (2006)
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  • Journal Title: Biochem. Biophys. Res. Commun. 347 Pages: 67-75
• [Journal Article] Calreticulin negatively regulates the cell surface expression of CFTR. (2006)
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  • Journal Title: J Biol Chem 281 Pages: 12841-12848
• [Journal Article] SUMO down-regulates the activity of Elf4/Myeloid Elf-1-like factor. (2006)
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  • Journal Title: Biochem. Biophys. Res. Commun 348 Pages: 880-888
• [Journal Article] Bafilomycin A1-Sensitive Pathway is required for the Maturation of Cystic Fibrosis Transmembrane Conductance Regulator. (2006)
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  • Journal Title: Biochem. Biochim. Acta(Mol. Cell Res.) 1763 (10) Pages: 1017-1023
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  • Journal Title: FASEB J 20(6) Pages: 782-784
• [Journal Article] Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation. (2006)
  • Author(s): Sato T, Ando Y, Susuki S, Mikami F, Ikemizu S, Nakamura M, Suhr O, Anraku M, Kai T, Suico MA, Shuto T, Mizuguchi M, Yamagata Y, Kai H.
  • Journal Title: FEBS Lett. 580(2) Pages: 491-496
• [Journal Article] Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells. (2006)
  • Author(s): Shuto T, Furuta T, Oba M, Xu H, Li JD, Cheung J, Gruenert DC, Uehara A, Suico MA, Okiyoneda T, Kai H.
  • Journal Title: FASEB J. 20(6)
• [Journal Article] Sp1-dependent regulation of Myeloid Elf-1 like factor in human epithelial cells. (2005)
  • Author(s): Koga T, Suico MA, Nakamura H, Taura M, Lu Z, Shuto T, Okiyoneda T, Kai H.
  • Journal Title: FEBS Lett. 579(13) Pages: 2811-2816
• [Journal Article] Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression. (2005)
  • Author(s): Koga T, Harada H, Shi TS, Okada S, Suico MA, Shuto T, Kai H.
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• [Journal Article] Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells. (2005)
  • Author(s): Shuto T, Kato K, Mori Y, Viriyakosol S, Oba M, Furuta T, Okiyoneda T, Arima H. Suico MA, Kai H.
  • Journal Title: Biochem Biophys Res Commun. 338(3) Pages: 1402-1409
• [Journal Article] Vitamins K1 and K2 potentiate hyperthermia by down-regulating Hsp72 expression in vitro and in vivo. (2005)
  • Author(s): Shimohara S, Murakami T, Morikawa M, Matsuo J, Nagayama S, Shuto T, Suico MA, Okiyoneda T, Yamatsu I, Mizushima T, Shimasaki T, Kai H.
  • Journal Title: Int J Oncol. 27(6) Pages: 1527-1533