Structural Determinants of Functional Cytochrome P450

描述(由申请人提供)：这项研究的总体目标是了解生产功能细胞色素P450(P450)分子的结构基础。这一建议的重点是a)信号锚序列(SA)介导的内质网(ER)靶向，以及b)催化结构域与ER膜的相互作用。该提案的具体目的是首先鉴定和鉴定参与P450直接内质网滞留的辅助蛋白，重点是BAP31，它已被证明与P450相互作用并介导内质网滞留。其次，检测半胱氨酸可及性决定的N末端信号锚序列在膜上的位置和方向是否与ER保留功能相关；第三，通过荧光显微镜下与蛋白质退出标记的共定位分析和退出结构域的免疫浓缩，确定P450 2C2而不是P450 2E1是否被排除在ER的蛋白质退出结构域之外；第四，通过检测特定的蛋白酶抑制剂对P450水平和分布的影响，确定BAP31是作为ER保留蛋白发挥作用，还是通过靶向P450降解来增加P450的水平；第五，通过界面序列中取代的半胱氨酸的二硫键确定P450 2C8的F-G环区是否像在结晶蛋白质中那样是二聚界面的一部分，或者通过半胱氨酸可及性分析确定F-G环区是否插入到脂质双层中；第六，通过引入降低疏水性的突变来确定催化结构域的膜接触环的疏水性是否与P450的活性相关。亲和纯化和双杂交方法将用于鉴定与P450相互作用的蛋白质，双分子荧光互补和荧光发射共振转移将用于监测相互作用，siRNA或敲除ES细胞将用于潜在ER保留蛋白的功能分析。P450介导多种药物和毒素的解毒、致癌物和前体药物的激活以及许多内源性化合物的生物发生。人类P450基因突变对药物代谢和类固醇生物发生有显著的临床影响。了解产生功能性P450的遗传和结构基础对于了解这些酶在正常和病理状态下的作用是必要的。

DESCRIPTION (provided by applicant): The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule. The emphasis of this proposal is on a) targeting to the endoplasmic reticulum (ER) mediated by the signal anchor sequence (SA), and b) interaction of the catalytic domain with the ER membrane. The specific aims of the proposal are to first, identify and characterize accessory proteins that are involved in the direct ER retention of P450, with emphasis on BAP31 which has been shown to interact with P450 and mediate ER retention. Second, examine whether the position and orientation of the N-terminal signal anchor sequence in the membrane, determined by cysteine accessibility, correlates with the ER retention function; third, determine if P450 2C2, but not P450 2E1, is excluded from the protein exit domains of the ER by analysis of colocalization with protein exit markers by fluorescence microscopy and by immuno-enrichment of the exit domain; fourth, determine whether BAP31 increases P450 levels by functioning as an ER retention protein or by targeting P450 for degradation by examining effects of specific protease inhibitors on P450 levels and distribution; fifth, determine whether the F-G loop region of P450 2C8 is part of a dimerization interface as in the crystallized protein by disulfide linkage of cysteines substituted in the interface sequences or if the F-G loop region is inserted into the lipid bilayer by cysteine accessibility assays, and sixth, determine if the hydrophobicity of membrane-contacting loops of the catalytic domain correlates with activity of the P450s by introducing mutations that reduce hydrophobicity. Affinity purification and two-hybrid approaches will be used to identify proteins interacting with P450, bimolecular fluorescence complementation and fluorescence emission resonance transfer will be used to monitor interactions, and siRNA or knock-out ES cells will be used for functional analysis of potential ER retention proteins. P450s mediate the detoxification of many drugs and toxins, activation of carcinogens and pro- drugs, and biogenesis of many endogenous compounds. Mutations in human P450s have dramatic clinical effects on drug metabolism and steroid biogenesis. Understanding the genetic and structural basis for generating functional P450s is necessary to understand the role of these enzymes in normal and pathological states.