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.

摘要 多氯联苯和有机磷农药是普遍存在的环境农药 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。它们最好由CYP2B亚家族酶代谢。对经济发展至关重要 这些污染物的解毒是CYP2B酶与其氧化还原作用的相互作用 配对：细胞色素P450氧化还原酶(POR)和细胞色素b5(Cyt B5)。然而，这些 内质网(ER)膜的相互作用仍然知之甚少。 尤其是，细胞色素b5在微粒体p450系统中的作用是个谜。它已经被辩论过了。 几十年来，无论cyt b5作为电子供体或变构效应器影响P450的活性，还是 两者都有。在细胞色素b5在p450活性中的重要作用和 我们对其机制的理解。缺乏理解的部分原因是缺乏3D 天然P450：B5复合体的结构。通过以下方式获得这样的结构是极具挑战性的 常规方法，如X射线结晶学或核磁共振光谱学。单粒子低温电磁波 已成为确定多单元膜结构的首选方法 没有衍射晶的蛋白质复合体。我们假设 天然的CYP2B4-Cytb5复合体的3D结构的确定可以使用 单颗粒冷冻-EM与新的样品制备相结合。我们建议测试一下 假设并开发一种迫切需要的冷冻-EM方法，以达到两个特定的目标。在目标1中，我们将 以新型纳米粒子为载体制备细胞色素P4-细胞色素b5复合体并测定其三维结构 由单粒子低温EM在目标2中实现。两个目标的成功完成不仅将提供 了解Cyt b5在CYP2B4活性中的作用的关键知识，也开发了一种 Cytb5和POR天然络合物结构分析亟需的方法学 与许多其他P450酶结合。此方法的建立和这些方法的可用性 土著建筑群将是开创性的，并提供了一个路线图，以审问 微生物体P450系统解毒环境污染物的机制(S)。