Regulation of Cytochrome P450 Biosynthesis

描述（来自申请人摘要的逐字描述）：本申请的总体目标 项目是了解苯巴比妥的分子机制， 诱导细胞色素P450基因（CYP 450）表达。细胞色素P450形成一个超级 一个负责通过氧化作用激活或失活的酶家族 各种内源性和外源性化合物的代谢。余额 在激活和失活之间，这可以通过 诱导，决定最终的治疗或毒性活性的摄入 化工有限 一种苯巴比妥（PB）作用模型，其中PB诱导从 细胞质到细胞核的核受体，组成型雄甾烷 最近，研究人员开发了一种新的CAR受体。CAR，作为异二聚体， 核受体RXR与PB中的核受体结合位点结合 在-2.3Kb处的PBRU反应单位（PBRU）激活肝CYP 2B基因表达。 PBRU中的其他元素有助于诱导，包括NF-1， 表明蛋白质复合物介导基因的激活。的 CYP 2B PBRU和近端启动子的染色质结构改变， PB治疗导致额外的蛋白质结合到肝组织， PBRU。 这一建议的具体目的是为了了解这一机制 CAR/RXR与PBRU的结合激活CYP 2B基因， 表征与PBRU结合的蛋白质及其与 CAR，然后鉴定和表征共调节蛋白结合 到CAR，最后是体内方法，以确定在体内的有效性。 体外研究。蛋白质与PBRU序列的结合将通过在 体外足迹法和凝胶迁移试验，以评估结合是否 合作、对抗或独立。染色质结构的影响 对蛋白质结合的影响将通过体外组装 染色质和足迹分析。潜在的协同调节因子， CAR将通过GST-唐斯和酵母双杂交分析鉴定。的 DNA结合蛋白和潜在的辅助调节因子的功能意义 将通过培养细胞的瞬时和稳定转染来确定， 体外转录结合PBRU的诱变和共表达 与CAR/RXR相关的因素。组蛋白修饰在活化细胞中的作用 将研究CYP 2B基因。在体外的体内意义 实验将通过测定鉴定的化合物的体内结合来评估。 通过染色质免疫沉淀技术检测PBRU因子， 在体内通过瞬时和稳定转染肝细胞在功能上 尾静脉注射载体DNA。这些研究将确定 负责PB激活CYP 2B的蛋白质复合物的功能 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.