Regulation of Cytochrome P450 Biosynthesis

描述（由申请人提供）：本申请是对GM 39360“细胞色素P450生物合成的调节”的补充，目的是构建在肝脏中表达用Flag肽标记的组成型雄烷受体（CAR）的转基因小鼠系。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。细胞色素P450是一个超家族的酶，负责通过氧化代谢多种内源性和外源性化合物来激活或失活。激活和失活之间的平衡，可以通过诱导显著改变，决定了摄入的化学品的最终治疗或毒性活性。PB诱导核受体CAR从细胞质易位到细胞核。CAR作为与核受体RXR的异源二聚体，与CYP 2B基因中PB反应单元（PBRU）中的核受体结合位点结合，并组成性诱导基因表达。PB样诱导剂也可以结合CAR并增强其活性。本提案的具体目的是了解CAR/RXR与PBRU结合激活CYP 2B基因的机制，首先表征与PBRU结合的蛋白质及其与CAR的相互作用，然后鉴定和表征与CAR结合的辅助调节蛋白，CYP 2B基因染色质结构在PB作用中的作用，最后是体内方法，以确定体外研究的有效性。该补充剂的主要具体目的是构建转基因小鼠系，其中标记的CAR在肝脏中表达。这种转基因小鼠系将有助于从对照和PB处理的动物的肝脏分离含有CAR的蛋白质复合物，以鉴定CAR相互作用蛋白;通过ChIP分析分析CAR与体内CYP 2B基因的结合;以及从对照和PB处理的动物分离细胞核和细胞质CAR，以分析翻译后修饰在CAR调节中的作用。这些实验将大大增加我们对PB诱导CYPB基因表达机制的了解

DESCRIPTION (provided by applicant): This application is for a supplement to GM39360 "Regulation of cytochrome P450 biosynthesis" for the purpose of constructing a transgenic mouse line expressing constitutive androstane receptor (CAR), tagged with the Flag peptide, in liver. The overall goal of this project is to understand the molecular mechanisms by which phenobarbital (PB) 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. PB induces the translocation from the cytoplasm to the nucleus of the nuclear receptor CAR. CAR, as a heterodimer with the nuclear receptor, RXR, binds to nuclear receptor binding sites in a PB responsive unit (PBRU) in CYP2B genes and constitutively induces gene expression. PB-like inducers may also bind to CAR and enhance its activity. 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, the role of CYP2B gene chromatin structure in PB action, and finally in vivo approaches to establish the validity of the in vitro studies. The major specific aim of the supplement is to construct a transgenic mouse line in which flag-tagged CAR is expressed in the liver. Such a transgenic mouse line will facilitate the isolation from livers of control and PB-treated animals of protein complexes containing CAR to identify CAR interacting proteins; to analyze binding of CAR to the CYP2B genes in vivo by ChIP analysis; and to isolate nuclear and cytoplasmic CAR from control and PB-treated animals to analyze the role of posttranslational modifications in the regulation of CAR. These experiments will greatly increase our understanding of the mechanism of induction of CYPB genes by PB