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論文
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Yokota, Shinichi ; Higashi, Eriko ; Fukami, Tatsuki ; Yokoi, Tsuyoshi ; Nakajima, Miki
概要:
金沢大学医薬保健研究域薬学系<br />Human CYP2A6 is responsible for the metabolism of nicotine and coumarin as well as the metabolic act
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ivation of tobacco-related nitrosamines. Earlier studies revealed that CYP2A6 activity was increased by dietary cadmium or cruciferous vegetables, but the underlying mechanisms remain to be clarified. In the present study, we investigated the possibility that Nrf2 might be involved in the regulation of CYP2A6. Real-time RT-PCR analysis revealed that the CYP2A6 mRNA level in human hepatocytes was significantly (P < 0.01, 1.4-fold) induced by 10 μM sulforaphane (SFN), a typical activator of Nrf2. A computer-based search identified three putative antioxidant response elements (AREs) in the 5′-flanking region of the CYP2A6 gene at positions -1212, -2444, and -3441, termed ARE1, ARE2, and ARE3, respectively. Electrophoretic mobility shift assays demonstrated that Nrf2 bound only to ARE1. Luciferase assays using HepG2 cells revealed that the overexpression of Nrf2 significantly increased the reporter activities of the constructs containing a 30-bp fragment that included ARE1. However, the activity of the construct containing the intact 5′-flanking region (-1 to -1395) including ARE1 was not increased by the overexpression of Nrf2. In contrast, when the reporter construct was injected into mice via the tail vein, the reporter activity in the liver was significantly (P < 0.05, 1.9-fold) increased by SFN (1 mg/head) administration. In conclusion, we found that human CYP2A6 is regulated via Nrf2, suggesting that CYP2A6 is induced under oxidative stress. © 2010 Elsevier Inc. All rights reserved.
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論文
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村上, 清史 ; Murakami, Seishi
概要:
HBV X蛋白(HBx)の核内標的であるRPB5の機能解析を目的とした研究で得られた主な成果は、1)基本転写因子TFIIFとRPB5の特異的結合を見い出し、アラニン置換でスキャンした結果、RAP30のY124とQ131がRPB5結合に必須な
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残基として特定された。培養細胞内で内在性PolIIが野生型のRAP30を含むTFIIFと会合したがRAP30のY124A叉はQ131A変異を持つTFIIFとは会合せず、PolIIとTFIIFの会合にRAP30とRPB5の結合が必須である(JBC,2001)。2)RPB5の2重鎖DNA結合能を見い出した。アラニン置換変異による解析から、DNA結合能に必須な4アミノ酸残基を特定した。その内T111及びS113の2残基は、DNA結合への直接寄与が示唆された。結晶モデルでプロリン残基(P80とP112)の寄与が予測されたが、我々はこれらの残基の影響を認めなかった。3)RMPは細胞質と核に共に見い出された。RMPの細胞内局在には、C端側にある核移行シグナル(NLS)とN端側のcoiled coilドメインの細胞質局在シグナル(CLS)が重要な寄与をする(投稿準備中)。4)RPB5との相互作用の局面で、HBVX蛋白に拮抗する新規蛋白RMPの機能解析を目的として、酵母Two-hybrid法でRMPに相互作用する蛋白を選択した。その結果、RMP自体が単離され、繰り返し単離された候補のcDNAは、DNAメチル化に関わりcorepressor機能が報告されている蛋白であった。培養細胞でこれらの蛋白とRMPとの特異的結合を確認した。<br />RPB5 at the tip of the lower jaw of pol II is involved in activated transcription and interacts with basal transcription factors and Hepatitis B Virus X protein (HBx). A crystal model of pol II predicted RPB5 is close to DNA downstream to transcription start site. 1) RPB5 was found to bind to recombinant TFIIF reconstituted consisting of RAP30 and RAP74. The central parts of RAP30 and RPB5 are critical for the binding. By scanning clustered, then point alanine-substitution mutants of RAP30, two residues, Y124 and Q131, were found to be critical for the RPB5-binding. Endogenous pol II was recruited to TFIIF consisting of wild RAP30 but not mutant RAP30 at aa124 or at aa131. 2) We found that RPB5 retains ability to bind to double-stranded DNA. The 4 amino acid residues in the exposed domain of RPB5 were identified to be critical for the DNA- binding analyzed by scanning alanine substitution mutants of the exposed domain. Among them, T111 and SI13 would be directly involved in the DNA-binding. 3) RMP (RPB5-mediating protein), a functional antagonist of HBx, has a coiled-coil domain at the N-terminal, RPB5-binding region, and the C-terminal region responsible for co- repressor activity. The coiled coil domain harboring cytoplasmic localization signal (CLS) and a NLS located at the C-terminal region are both important for cytoplasmic and nuclear localization of RMP. 4) To analyze function(s) of RMP, RMP-interacting partners were searched by yeast two hybrid screening. RMP, by itself, was screened and found to interacts with RMP in vitro and in mammalian cells. Another candidate, repeatedly screened out, has been reported to be a corepressor and involved in DNA methylation. Biological outcome of the interactions remains to be examined.<br />研究課題/領域番号:11480200, 研究期間(年度):1999-2001<br />出典：「RNAポリメラーゼサブユニット5を介した転写活性化機構と新規転写補助因子RMP」研究成果報告書 課題番号11480200(KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
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