Regulation of Cytochrome P450 Biosynthesis

描述(逐字摘自申请者摘要)：总体目标 项目是了解苯巴比妥的分子机制 诱导细胞色素P450基因(CYP)表达。细胞色素P450形成一个超级 负责氧化激活或失活的一类酶 多种内源和外源化合物的代谢。余额 在激活和失活之间，可以通过以下方式显著改变 诱导，决定了摄入的最终治疗或毒性活性 化学药品。 苯巴比妥(PB)的作用模型，其中PB诱导细胞易位 核受体的胞质至核，构成雄烷 受体(CAR)是最近发展起来的。Car作为异源二聚体与 核受体RXR与外周血中的核受体结合部位结合 反应单位(PBRU)在-2.3 kb处激活肝脏细胞色素P450 2 B基因的表达。 PBRU中的其他成分也参与了诱导，包括核因子-1， 这表明一种蛋白质复合体介导了基因的激活。这个 CYP2B PBRU和近端启动子的染色质结构在 肝组织，PB治疗导致额外的蛋白质结合到 PBRU。 这项建议的具体目的是为了了解这一机制 通过与PBRU结合的CAR/RXR激活CYP2B基因 与PBRU结合的蛋白质特性及其与PBRU的相互作用 Car，然后鉴定并鉴定与协同调节蛋白结合的蛋白 到CAR，最后在体内建立In的有效性的方法 体外研究。蛋白质与PBRU序列的结合将通过In 体外足迹和凝胶移位分析以评估结合是否 合作的、对立的或独立的染色质结构的影响 将通过体外组装来研究蛋白质的结合 染色质和足迹分析。与之互动的潜在共同监管机构 Car将通过GST下拉和酵母双杂交分析进行鉴定。这个 DNA结合蛋白及其潜在共调控因子的功能意义 将通过瞬时和稳定的细胞转染法确定 PBRU的体外转录与诱变及其共表达 影响CAR/RXR的因素。组蛋白修饰在细胞活化中的作用 将对CYP2B基因进行研究。体外试验的体内意义 实验将通过测定确定的在体内的结合来进行评估 染色质免疫沉淀技术对PBRU影响因素的研究 通过瞬时和稳定的体内肝细胞转染法实现功能 尾静脉注射载体DNA。这些研究将建立自然和 参与细胞色素P450酶B活化的蛋白质复合体的功能 基因。

DESCRIPTION (Verbatim from the Applicant's Abstract): The overall goal of this project is to understand the molecular mechanisms by which phenobarbital induces cytochrome P450 gene (CYP) expression. Cytochromes P450 form a super family of enzymes responsible for activation or inactivation by oxidative metabolism of a wide variety of endogenous and exogenous compounds. The balance between activation and inactivation, which can be dramatically altered by induction, determines the ultimate therapeutic or toxic activity of an ingested chemical. A model of phenobarbital (PB) action in which PB induces the translocation from the cytoplasm to the nucleus of the nuclear receptor, constitutive androstane receptor (CAR) has been recently developed. CAR, as a heterodimer with the nuclear receptor, RXR, binds to nuclear receptor binding sites in a PB responsive unit (PBRU) at -2.3 Kb to activate hepatic CYP2B gene expression. Other elements in the PBRU contribute to the induction, including NF-1, indicating that a complex of proteins mediates the activation of the gene. The chromatin structure of the CYP2B PBRU and the proximal promoter is altered in hepatic tissue, and PB treatment results in additional protein binding to the PBRU. The specific aims of this proposal are directed at understanding the mechanism by which CAR/RXR binding to the PBRU activates CYP2B genes beginning with characterization of the proteins binding to the PBRU and their interaction with CAR, then identification and characterization of co-regulator proteins binding to CAR, and finally in vivo approaches to establish the validity of the in vitro studies. The binding of proteins to PBRU sequences will be studied by in vitro footprinting and gel shift assays to assess whether the binding is cooperative, antagonistic, or independent. The influence of chromatin structure on the binding of the proteins will be studied by in vitro assembly of chromatin and footprinting analysis. Potential co-regulators that interact with CAR will be identified by GST-pull downs and yeast two-hybrid analysis. The functional significance of the DNA binding proteins and potential co-regulators will be determined by transient and stable transfections of cultured cells in vitro transcription combined with mutagenesis of the PBRU and co-expression of the factors with CAR/RXR. The role of histone modification in activation of the CYP2B genes will be studied. The in vivo significance of the in vitro experiments will be assessed by determining the binding in vivo of identified factors to the PBRU by the chromatin immunoprecipitation technique and functionally by transient and stable transfections of hepatocytes in vivo by tail vein injection of vector DNA. These studies will establish the nature and function of the complex of proteins responsible for PB activation of CYP2B genes.