Structural basis for detoxification of environmental pollutants by native complexes of CYP2B family with its redox partners

CYP2B家族天然复合物及其氧化还原伙伴对环境污染物解毒的结构基础

• 批准号: 9979011
• 负责人: Min Su
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979011
• 关键词: Affect; Apolipoproteins; Architecture; Aryl Hydrocarbon Hydroxylases; CYP2B4 gene; Catalysis; Cell Respiration; Complex; Computer software; Cryoelectron Microscopy; Crystallization; Cytochrome P450; Cytochromes b5; Data Set; Detoxification Process; Drug Metabolic Detoxication; Due Process; Endoplasmic Reticulum; Environment; Environmental Pollutants; Enzymes; Exhibits; Family; Genetic Polymorphism; Health; Human; Ice; Image; Knowledge; Length; Lipids; Lipoproteins; Malignant Neoplasms; Maps; Membrane; Membrane Proteins; Methodology; Methods; Molecular; Molecular Conformation; NMR Spectroscopy; Negative Staining; Organophosphates; Oxidation-Reduction; Oxidoreductase; Pathogenesis; Pesticides; Phospholipids; Play; Polychlorinated Biphenyls; Preparation; Process; Proteins; Role; SAP-A Protein; Sampling; Structural Models; Structure; System; Testing; Time; Toxic effect; X-Ray Crystallography; base; bioaccumulation; contrast imaging; electron donor; flexibility; human tissue; improved; insight; nanodisk; nanometer resolution; nanoparticle; neurotoxicity; novel; particle; pollutant; pollutant interaction; polybrominated diphenyl ether; protein aggregation; protein complex; reconstitution; reconstruction; stoichiometry; three dimensional structure

ABSTRACT Polychlorinated biphenyls and organophosphorus pesticides are ubiquitous environmental pollutants. Bioaccumulation of these pollutants in human tissues is associated with cancer and neurotoxicity. They are preferably metabolized by CYP2B subfamily enzymes. Essential to the detoxification of these pollutants are the interactions of CYP2B enzymes with their redox partners, cytochrome P450 oxidoreductase (POR) and cytochrome b5 (cyt b5). However, these interactions in the context of endoplasmic reticulum (ER) membrane remain poorly understood. In particular, the role of cyt b5 in the microsomal P450 system is enigmatic. It has been debated for decades whether cyt b5 affects P450 activity as an electron donor or allosteric effector or both. There is a clear knowledge gap between the important role of cyt b5 in P450 activity and our understanding of its mechanism. Lack of understanding is in part due to the absence of 3D structure of a native P450:b5 complex. It is highly challenging to obtain such a structure by conventional methods like X-ray crystallography or NMR spectroscopy. Single-particle cryo-EM has emerged as the method of choice for structural determination of multi-unit membrane protein complexes where diffracting crystals are not available. We hypothesize that determination of the 3D structure of a native CYP2B4-cyt b5 complex can be achieved using single-particle cryo-EM in conjunction with novel sample preparations. We propose to test the hypothesis and develop a much-needed cryo-EM method in two specific aims. In Aim 1, we will prepare the CYP2B4-cyt b5 complex in novel nanoparticles and then determine its 3D structure by single-particle cryo-EM in Aim 2. Successful completion of the two aims will not only provide critical knowledge for understanding the role of cyt b5 in CYP2B4 activity, but also develop a much-needed methodology for structural analysis of the native complexes of cyt b5 and POR with many other P450 enzymes. Establishment of this methodology and availability of these native complexes will be groundbreaking and provide a roadmap to interrogate the mechanism(s) by which the microsomal P450 system detoxifies environmental pollutants.

抽象的 多氯联苯和有机磷农药是无处不在的环境 污染物。这些污染物在人体组织中的生物积累与癌症和 神经毒性。最好由CYP2B亚家族酶代谢。对 这些污染物的排毒是CYP2B酶与氧化还原的相互作用 合作伙伴，细胞色素P450氧化还原酶（POR）和细胞色素B5（Cyt B5）。但是，这些 在内质网（ER）膜的背景下的相互作用仍然鲜为人知。 特别是，Cyt B5在微粒体P450系统中的作用是神秘的。它已经辩论 几十年来，CYT B5是否影响P450活性作为电子供体还是变构效应器或 两个都。 Cyt B5在P450活动中的重要作用与 我们对其机制的理解。缺乏理解部分是由于缺乏3D 天然P450：B5复合物的结构。通过 常规方法，例如X射线晶体学或NMR光谱法。单粒子冷冻EM 已成为多单位膜结构测定的首选方法 没有衍射晶体的蛋白质复合物。我们假设这一点 可以使用天然CYP2B4-CYT B5复合物的3D结构的测定 单粒子冷冻EM与新型样品制剂结合使用。我们建议测试 假设并在两个具体目标中开发了急需的冷冻EM方法。在AIM 1中，我们将 在新型纳米颗粒中准备CYP2B4-CYT B5复合物，然后确定其3D结构 通过AIM 2中的单粒子冷冻EM。成功完成两个目标不仅会提供 了解Cyt B5在CYP2B4活动中的作用的批判知识，但也会发展 急需的方法，用于对Cyt B5和POR的天然复合物进行结构分析 与许多其他P450酶。建立这种方法和这些方法的可用性 本地建筑群将是开创性的，并提供了询问的路线图 微粒体P450系统对环境污染物解毒的机制。