Regulation of Cytochrome P450 Biosynthesis

描述(申请人提供)：本项目的总体目标是了解苯巴比妥(PB)诱导细胞色素P450基因(CYP)表达的分子机制。P450催化多种内源和外源化合物的激活或失活。在临床上，P450的诱导是许多药物相互作用的基础，而失活和激活之间的平衡受诱导的影响，决定了摄入化合物的最终治疗或毒性效果。PB处理可诱导组成性雄烷受体(CAR)的核移位。CAR作为RXR的异源二聚体与PB反应增强子(PBRU)中的三个核受体(NR)结合，并招募p160共激活子和其他未知蛋白，导致染色质结构和基因激活的变化。其具体目的是确定CAR招募到PBRU的调控复合体，确定与CYP基因激活相关的PBRU染色质结构的变化，并确定CAR的核质穿梭所需的输入和输出信号。为了便于从小鼠肝脏中分离CAR及其蛋白复合体，我们将构建一个表达标记CAR的转基因小鼠。经PB处理的动物的核CAR蛋白复合体将通过FLAG免疫亲和分离而得到丰富，并将通过质谱仪鉴定复合体中的蛋白质。三个p160共激活子和三个NR结合位点的相对重要性将被确定。PB处理后招募到PBRU的蛋白质将通过染色质免疫沉淀分析进行鉴定，染色质结构将通过对核酸酶和羟基自由基切割的敏感性来探索。在PBRU或CAR复合体中检测到的蛋白质的功能意义将通过在培养细胞和活体肝细胞中的瞬时转基因来确定。通过检测CAR片段的嵌合体和荧光蛋白在体内肝细胞中的分布，鉴定CAR的核进出口信号和受体。这些研究强调体内的方法，应该提供对PBRU调控复合体的描述，这将为PB诱导CYP基因的机制提供洞察力，并确定核质穿梭的运输信号，这是PB处理调控的一个关键过程。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the molecular mechanisms by which phenobarbital (PB) induces cytochrome P450 gene (CYP) expression. P450s catalyze the activation or inactivation of a wide variety of endogenous and exogenous compounds. Clinically, induction of P450s underlies many drug interactions, and the balance between inactivation and activation, which is influenced by induction, determines the ultimate therapeutic or toxic effect of ingested compounds. PB treatment induces the nuclear translocation of the constitutive androstane receptor (CAR). CAR binds to three nuclear receptor (NR) binding sites in a PB responsive enhancer (PBRU) as a heterodimer with RXR and recruits p160 coactivators and other unknown proteins resulting in changes in chromatin structure and gene activation. The specific aims are to identify the regulatory complex recruited by CAR to the PBRU, to determine changes in chromatin structure at the PBRU correlated with activation of the CYP genes, and to determine the import and export signals of CAR necessary for its nucleocytoplasmic shuttling. To facilitate the isolation of CAR and its protein complexes from mouse liver, a transgenic mouse expressing flag-tagged CAR will be constructed. Nuclear CAR protein complexes in PB-treated animals will be enriched by flag immunoaffinity isolation and proteins in the complex will be identified by mass spectrometry. The relative importance of the three p160 coactivators and the three NR binding sites will be determined. Proteins recruited to the PBRU after PB treatment will be identified by chromatin immunoprecipitation assays and chromatin structure will be probed by sensitivity to cleavage by nucleases and hydroxyl radicals. The functional significance of proteins detected at the PBRU or in CAR complexes will be determined by transient transfections in cultured cells and in hepatocytes in vivo. Nuclear import and export signals and receptors for CAR will be identified by examining the distribution of chimera of CAR fragments and fluorescent proteins in hepatocytes transfected in vivo. These studies, emphasizing in vivo approaches, should provide a description of the regulatory complex at the PBRU, which will provide insight into the mechanism of PB induction of CYP genes, and determine transport signals for nucleocytoplasmic shuttling, which is a key process regulated by PB treatment.