Structural Determinants of Functional Cytochrome P450

功能性细胞色素 P450 的结构决定因素

• 批准号: 7487086
• 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
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487086
• 关键词: Affinity Chromatography; Amino Acids; Apoptotic; Area; Biochemical; Biogenesis; Biological Assay; Bleomycin/Doxorubicin/Prednisone; Carcinogens; Catalytic Domain; Characteristics; Compatible; Complex; Cysteine; Cytochrome P450; Degradation Pathway; Dimerization; Disulfide Linkage; Drug Metabolic Detoxication; Endoplasmic Reticulum; Enzymes; Facility Construction Funding Category; Fluorescence; Fluorescence Microscopy; Genetic; Helix (Snails); 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; ear helix; embryonic stem cell; mutant; prevent; programs; protein function; receptor; research study; response; yeast two hybrid system

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生产功能性细胞色素 P450 (P450) 分子的结构基础。 该提案的重点是 a) 靶向由信号锚序列 (SA) 介导的内质网 (ER)，以及 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 直接 ER 保留的辅助蛋白，重点是 BAP31，它已被证明与 P450 相互作用并介导 ER 保留。 其次，检查膜中 N 端信号锚序列的位置和方向（由半胱氨酸可及性决定）是否与 ER 保留功能相关；第三，通过荧光显微镜和出口结构域的免疫富集分析与蛋白质出口标记的共定位，确定 P450 2C2（而不是 P450 2E1）是否被排除在 ER 的蛋白质出口结构域之外；第四，通过检查特定蛋白酶抑制剂对 P450 水平和分布的影响，确定 BAP31 是否通过充当 ER 保留蛋白或靶向 P450 降解来增加 P450 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环区是否是二聚化界面的一部分，如在结晶蛋白中，或者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中，第六，确定催化结构域的膜接触环的疏水性是否 通过引入降低疏水性的突变与 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 介导许多药物和毒素的解毒、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 突变对药物代谢和类固醇生物合成具有显着的临床影响。了解生成功能性 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。