Xenobiotic-Metabolism and Steroidogenic Enzyme Cytochrome P450 and Their Regulation---Bioinformatic and Functional Genomic Approach (II)

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Project title

Code/計畫編號

NSC97-2627-B019-001

Translated Name/計畫中文名

環境異質代謝與固醇合成酵素細胞色素P450及其調控---生物資訊與功能基因體學探討---(總計畫):生物資訊與功能基因體學探討(II)

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

陳逸然
張大慈

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1651405

Year

2008

Start date/計畫起

01-08-2008

Expected Completion/計畫迄

01-07-2009

Bugetid/研究經費

1827千元

ResearchField/研究領域

生物技術(理)

Cytochrome P450 是一群含有原血紅素（heme）輔基的單氧氧化酶，參與生物體內各項的氧化代謝反應，普遍存在於各種生物個體中。這些酵素大致可分為體內內生性物質氧化代謝以及環境異質類與藥物代謝兩種，後者包括有CYP1~3類，在哺乳類動物中其基因的表現分別受到AHR、CAR 與PXR等細胞核接受器（nuclear receptor）的調控。在另一方面，參與固醇類荷爾蒙的合成酵素細胞色素P450，包括CYP11 (P450scc)、CYP17、CYP19 與CYP21，其中CYP11 是固醇合成反應中的速率決定因子。細胞核接受器SF-1 控制各固醇類荷爾蒙的合成酵素細胞色素P450 基因的表現。在本計畫中，我們結合生物資訊分析以及各種功能性基因體學的方法來探討環境異質代謝與固醇合成酵素細胞色素P450 的結構、調控與生物功能。在去年第一期的研究中，我們著重在CYP1C1的調控機制，而在今年第二期的研究中我們則著重在CYP3A65調控機制上。我們首先利用生物資訊的方法分析了CYP3A65基因上游5.5 Kb的區域，發現了6個AHR::ARNT的辨識序列（XRE），另外也含有21個腸道專一性轉錄子CdxA的辨識序列以及9個HNF-1/3辨識區域，這些序列可能與控制CYP3A65組織專一性的表現有關。為了進一步了解CYP3A65基因受TCDD影響的機制，我們利用Tol2系統，建立CYP3A65-GFP轉殖基因品系斑馬魚，發現從2 dpf時期起可以在消化道出現明顯的GFP螢光的表現，表現部位與時間均與原生性 CYP3A65基因的活性相同。當我們以morpholino弱化AHR2後會抑制GFP的表現，但是並不會抑制另一消化道的標示基因IFABP的表現，顯示AHR2確實參與了CYP3A65的調控，但是並不影響消化道的發育。另外我們將AHR2基因弱化後，發現並不會對PXR的表現產生顯著的影響，顯示AHR與PXR兩者間並無相互的關連性。目前我們正在探討PXR對CYP3A65基因的功能，並且將在第三年中利用生物資訊的方法，比對各物種間CYP3A基因調控的保守性，藉以了解該基因的演化進程，尤其想要了解AHR在整個CYP3A基因演化進程中所扮演的角色。 The Cytochrome P450 super-family is a fundamental requirement for the viability of most life, with Cytochrome P450 proteins having been identified in organisms ranging from bacteria to man. These enzymes may be subdivided into those that metabolise purely endogenous chemicals, and those that are involved in xenobiotic metabolism. Of the latter group it consists of highly conserved CYP1-3 sub-families and they are regulated by nuclear receptors aryl hydrocarbon recpetor (AHR), constitutive androstane receptor (CAR) and pregnane-X receptor (PXR), respectively. Of the steroidogenic enzymes, it consists of CYP11 (P450SCC), CYP17, CYP19 (aromatase) and CYP21 sub-families, in which the CYP11 controls the first and rate-limiting step of steroid biosynthesis. Nuclear receptor SF-1 plays amajor role in determining the cell-specific expression of P450 steroidogenic enzymes. Previously we have shown that CYP1C1 mRNA is extensively expressed in the vascular cells after hatching. This temporal expression is attenuated after 4 dpf stage. Nevertheless, TCDD treatment greatly enhances CYP1C1 transcription in an AHR2-dependent manner. This year our study focused on the regulatory mechanism of CYP3A65 in vivo. We have constructed a CYP3A65-EGFP transgenic line which contains an insert fragment with 2.8 kb of CYP3A65 upstream sequence and a complete ORF of EGFP gene. There are 6 XRE, 21 CdxA-binding site and 9 HNF-1/3 recognition sites in this 2.8 Kb upstream region. The F2 exhibits a strong EGFP fluorescence at 3 dpf stage in gastrointestinal tract and the expression is greatly enhanced by TCDD treatment. AHR2 knockdown eliminate CYP3A65 transcription. This line will be very useful in xenobiotic screening test in the future. Currently we are examining the function of multiple XRE modules on CYP3A65 regulation. We hypothesize that AHR::ARNT pathway plays critical role in fish CYP3A activity. The evolution of vertebrate CYP3A genes will also be studied by bioinformatics methods next year.

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Xenobiotic-Metabolism and Steroidogenic Enzyme Cytochrome P450 and Their Regulation---Bioinformatic and Functional Genomic Approach (II)

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