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Structural Determinants of Functional Cytochrome P450

功能性细胞色素 P450 的结构决定因素

基本信息

批准号：
7684235
负责人：
Byron W Kemper
金额：
$ 26.74万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-01-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7684235
关键词：
Affinity Chromatography Amino Acids Apoptotic Area Biochemical Biogenesis Biological Assay Carcinogens Catalytic Domain Characteristics Complex Cysteine Cytochrome P450 Degradation Pathway Dimerization Disulfide Linkage Drug Metabolic Detoxication Endoplasmic Reticulum Enzymes Fluorescence Fluorescence Microscopy Genetic Hemeproteins Human Hydrophobicity Knock-out Lipid Bilayers Mediating Membrane Modeling Molecular Chaperones Monitor Mutation N-terminal Oxidoreductase Pharmaceutical Preparations Positioning Attribute Prodrugs Production Protease Inhibitor Proteins Quality Control Regulation Research Research Personnel Retrieval Role Scanning Signal Transduction Site Small Interfering RNA Steroids Structure Theoretical model Toxin Transmembrane Domain Transport Vesicles base clinical effect cytochrome P-450 2C2 dimer drug metabolism embryonic stem cell mutant prevent programs protein function receptor research study response yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：本研究的总体目标是了解功能性细胞色素P450（P450）分子产生的结构基础。该建议的重点是a）通过信号锚序列（SA）介导的靶向内质网（ER），和B）催化结构域与ER膜的相互作用。该提案的具体目标是首先鉴定和表征参与P450的直接ER保留的辅助蛋白，重点是已显示与P450相互作用并介导ER保留的BAP 31。第二，检查膜中N末端信号锚序列的位置和方向（由半胱氨酸可及性确定）是否与ER保留功能相关;第三，通过荧光显微镜分析与蛋白质出口标志物的共定位和出口结构域的免疫富集，确定P450 2C 2是否被排除在ER的蛋白质出口结构域之外，而不是P450 2 E1;第四，通过检查特异性蛋白酶抑制剂对P450水平和分布的影响，确定BAP 31是否通过充当ER滞留蛋白或通过靶向P450降解来增加P450水平;第五，确定F-P450 2C 8的G环区是二聚化界面的一部分，如在结晶蛋白质中，通过界面序列中取代的半胱氨酸的二硫键，或通过半胱氨酸可及性测定确定F-G环区是否插入脂质双层，以及第六，通过引入降低疏水性的突变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滞留蛋白的功能分析。 P450介导许多药物和毒素的解毒、致癌物和前药的活化以及许多内源性化合物的生物合成。人类P450突变对药物代谢和类固醇生物合成具有显著的临床影响。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。