STUCTURE FUNCTION AND REGULATION OF CYTOCHROME P450 ENZYMES AND EPOXIDE HYDROLASE

细胞色素P450酶和环氧化物水解酶的结构、功能和调控

• 批准号: 5206980
• 负责人: CHARLES B KASPER
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5206980
• 关键词: NAD(P)H oxidoreductase; active sites; chemical carcinogen; chemical carcinogenesis; corticosteroid receptors; cytochrome P450; dexamethasone; enzyme induction /repression; enzyme model; enzyme structure; epoxide hydrolase; gene expression; gene induction /repression; genetic promoter element; laboratory rat; molecular oncology; nucleic acid sequence; oxides; phenobarbital; protein sequence; protein structure function; site directed mutagenesis; stilbenes

This proposal is an extension of our ongoing studies dealing with the structure, function, and regulation of NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450, and epoxide hydrolase. The application is divided into three major parts, one for each of the enzymes listed above. The first deals with (1) the basal regulation of the oxidoreductase gene, (2) the mechanism of phenobarbital transcriptional activation, (3) the molecular basis for trans-stilbene oxide induction, and (4) the structure-function relationship of the oxidoreductase enzyme. The promoter region of the oxidoreductase gene is of particular interest since it lacks both TATA and CCAAT sequence motifs but instead contains multiple consensus sequences for Sp-1. In this respect, it differs from other genes encoding for members of the mixed-function oxidase system as well as the UDP-glucuronosyltransferase and the glutathione S-transferases. Our goal is to identify and characterize those factors and sequences important in the regulation of the oxidoreductase gene and also to elucidate the biochemical pathways for xenobiotic induction. Furthermore, in the absence of the crystallographic structure of the oxidoreductase protein, we will use computer modeling and site-directed mutagenesis to analyze the structure-function relationships of the enzyme. Our immediate goal is to identify amino acids that are functionally important for biological activity. Five domains are targeted for characterization and include the binding sites for the flavins (FAD and FMN), cytochrome P-450, NADPH, and the amino terminal anchor required for membrane integration. The second part of the proposal examines the basal regulation of the epoxide hydrolase gene along with the biochemical events responsible for the glucocorticoid receptor-dependent dexamethasone repression of the gene. Down-regulation appears to be linked to a proximal AP-1 site and may involve inhibition of AP-1 activity by the glucocorticoid receptor. The mechanisms by which various compounds (phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene, and diethylnitrosamine) induce epoxide hydrolase also will be studied using promoter deletion mutants. It will be of particular interest to compare the mechanism of phenobarbital induction for genes lacking (oxidoreductase) and possessing (epoxide hydrolase and P-450 PCN-3) TATA and CCAAT regulatory sequences. In addition, the active site of epoxide hydrolase will be characterized using group specific chemical modifications in the presence and absence of an inhibitor followed by site-directed mutagenesis of the protected amino acid to establish biological relevancy. The third section of this application poses questions about the gene organization and regulation of the steroid-inducible P-450s (P-450III gene family). Emphasis will be placed on understanding the basis for the differential regulation of the P-450 PCN 2 and 3 genes.

该提案是我们正在进行的研究的延伸，涉及 NADPH-细胞色素 P-450 的结构、功能和调节 氧化还原酶、细胞色素 P-450 和环氧化物水解酶。 这 应用程序分为三个主要部分，每个部分一个 上面列出的酶。 第一个涉及（1）基本监管 氧化还原酶基因，（2）苯巴比妥的作用机制 转录激活，（3）反式二苯乙烯的分子基础 氧化物诱导，以及（4）结构-功能关系 氧化还原酶。 氧化还原酶基因的启动子区 特别令人感兴趣，因为它缺少 TATA 和 CCAAT 序列 基序，但包含 Sp-1 的多个共有序列。 在 在这方面，它不同于编码成员的其他基因 混合功能氧化酶系统以及UDP-葡萄糖醛酸基转移酶 和谷胱甘肽S-转移酶。 我们的目标是识别并 描述那些在调节中重要的因素和序列 氧化还原酶基因并阐明生化途径 用于外源物诱导。 此外，在没有 氧化还原酶蛋白的晶体结构，我们将使用 计算机建模和定点突变来分析 酶的结构与功能的关系。 我们的近期目标是 鉴定对生物功能重要的氨基酸 活动。 五个领域是表征的目标，包括 黄素（FAD 和 FMN）、细胞色素 P-450 的结合位点， NADPH，以及膜整合所需的氨基末端锚定。 该提案的第二部分审查了 环氧化物水解酶基因以及负责的生化事件 糖皮质激素受体依赖性地塞米松抑制 基因。 下调似乎与近端 AP-1 位点有关 可能涉及糖皮质激素受体对 AP-1 活性的抑制。 各种化合物（苯巴比妥、反式二苯乙烯）的作用机制 氧化物、2-乙酰氨基芴和二乙基亚硝胺）诱导环氧化物 水解酶也将使用启动子缺失突变体进行研究。 它将 特别感兴趣的是比较苯巴比妥的机制 诱导缺乏（氧化还原酶）和拥有（环氧化物）的基因 水解酶和 P-450 PCN-3) TATA 和 CCAAT 调节序列。 在 此外，环氧化物水解酶的活性位点将被表征 在存在和不存在的情况下使用组特定的化学修饰 抑制剂的作用，然后对受保护的区域进行定点诱变 氨基酸以建立生物学相关性。 本申请的第三部分提出了有关基因的问题 类固醇诱导型 P-450（P-450III 基因家族）。 重点将放在理解基础上 P-450 PCN 2 和 3 基因的差异调节。