Regulation of Cytochrome P450 Biosynthesis

描述（由申请人提供）：本申请是GM39360“细胞色素P450生物合成调控”的补充，目的是在肝脏中构建表达组成型雄甾烷受体（CAR）的转基因小鼠系，标记有Flag肽。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形成一个超级家族的酶负责激活或失活的各种内源性和外源性化合物的氧化代谢。激活和失活之间的平衡，可以通过诱导而显著改变，决定了摄入的化学物质的最终治疗活性或毒性活性。PB诱导细胞核受体CAR从细胞质向细胞核的易位。CAR作为具有核受体RXR的异源二聚体，与CYP2B基因中PB响应单元（PBRU）的核受体结合位点结合，并组成性地诱导基因表达。pb样诱导剂也可能与CAR结合并增强其活性。本提案的具体目的是为了了解CAR/RXR结合PBRU激活CYP2B基因的机制，从表征PBRU结合蛋白及其与CAR的相互作用开始，然后鉴定和表征与CAR结合的共调节蛋白，CYP2B基因染色质结构在PB作用中的作用，最后通过体内方法建立体外研究的有效性。补充的主要具体目的是构建一个转基因小鼠系，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处理动物的肝脏中分离出含有CAR的蛋白复合物，以鉴定CAR相互作用蛋白；通过ChIP分析CAR在体内与CYP2B基因的结合情况；并从对照和pb处理的动物中分离细胞核和细胞质CAR，分析翻译后修饰在CAR调控中的作用。这些实验将大大增加我们对PB诱导CYPB基因的机制的认识

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