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Regulation of P450 Activity by Cytochrome P450 Oxidoreductase

细胞色素 P450 氧化还原酶对 P450 活性的调节

基本信息

批准号：
9091550
负责人：
JUNG JA P. KIM
金额：
$ 29.07万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9091550
关键词：
Adopted Affinity Amino Acids Antley-Bixler syndrome Binding Biochemical Biological Assay Bioluminescence C-terminal CYP2B4 gene CYP2B6 gene CYP2D6 gene CYP3A4 gene Cell Respiration Cells Chimeric Proteins Cholates Collaborations Complex Crystallization Cysteine Cytochrome P450 Data Detergents Development Disease Drug Design Electron Transport Electrons Embryo Energy Transfer Enzymes Family Flavin Mononucleotide Flavins Goals Health Heme Homeostasis Human Label Length Ligands Lipids Measurement Measures Methods Modeling Molecular Molecular Conformation Mutation NADP NADPH-Ferrihemoprotein Reductase Oryctolagus cuniculus Oxidation-Reduction Oxidoreductase Oxygenases POR gene Patients Pharmaceutical Preparations Phenotype Physiologic pulse Probability Process Protein Isoforms Proteins Rattus Regulation Robotics Site Site-Directed Mutagenesis Solvents Spectrum Analysis Spin Labels Steroid biosynthesis Structure System Testing Therapeutic Variant Xenobiotics analog base biophysical properties design drug development drug metabolism effective therapy heme oxygenase-1 heme oxygenase-2 improved inhibitor/antagonist mutant reconstitution steroid hormone

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the structural and molecular basis for the mechanisms of the microsomal monooxygenation system that is responsible for the oxidative metabolism of various xenobiotics and endogenous compounds. The two key components of this system are cytochromes P450 (P450s) and NADPH-cytochrome P450 oxidoreductase (CYPOR). Although we know much about the mechanisms and structures of each of these two partners individually, their interactions with each other are poorly understood. In humans, the single CYPOR protein is capable of interacting with ~50 microsomal P450s, as well as the non-P450 enzymes, heme oxygenases (HO-1 and HO-2). Furthermore, mutations of the POR gene, encoding CYPOR, result in a wide range of phenotypes, from embryonic lethality, Antley-Bixler Syndrome (ABS) to mild steroidogenic disorders. In contrast to the previously obtained "closed form" structure of CYPOR, our recently obtained variant form of CYPOR (CYPORTGEE) adopts an open conformation that is capable of reducing P450s, and forms stable complexes with its partners. We will use CYPORTGEE in our EPR and crystallization studies as outlined in the following specific aims. Aim 1: To determine conformational changes of CYPOR upon binding to its electron transfer partners and the interactions between them by site-directed spin-labeling EPR methods. Using the crystal structures of wild type CYPOR and CYPORTGEE as guides, double cysteine mutants (one in each of the two flavin domains) will be spin labeled, and the spin-spin distances will be measured. Spin-labeled CYP2B4 and HO-1 will also be used with single labeled CYPOR to measure the spin- spin distances between the two partners in the complex. From the distances between the two domains of CYPOR and between the two partners, structures of the complexes of CYPOR-2B4/HO-1 will be constructed. The resulting model structures of complexes of CYPOR and its partners will be validated by biochemical characterization combined with site-specific mutagenesis. Aim 2: To determine the crystal structures of complexes of CYPOR with CYP2D6, CYP3A4, CYP2B4, and human HO-1. We will use both human and rat CYPORTGEE proteins that form stable complexes with the partners. Soluble forms of CYP2B4, CYP3A4, and CYP2D6 and the soluble form of HO-1 will be used in co-crystallization studies with CYPORTGEE. The successful completion of the above studies will impact the P450 field by providing the answer to the central question in the field: What is th basis for recognition and the mechanism of electron transfer between CYPOR and its redox partners, including P450s and HO? The results from these studies will be used for drug development and designing of effective therapies by providing a better understanding of drug metabolism and the mechanism of HO-1 functions, respectively, and possibly to the design of therapeutics for CYPOR deficiencies resulting in abnormal steroidogenesis.

描述（由申请方提供）：本提案的总体目标是了解微粒体单氧合系统机制的结构和分子基础，该系统负责各种外源性物质和内源性化合物的氧化代谢。该系统的两个关键组分是细胞色素P450（P450 s）和NADPH-细胞色素P450氧化还原酶（CYPOR）。尽管我们对这两种伙伴各自的机制和结构了解很多，但对它们之间的相互作用却知之甚少。在人类中，单个CYPOR蛋白能够与约50种微粒体P450以及非P450酶、血红素加氧酶（HO-1和HO-2）相互作用。此外，编码CYPOR的POR基因的突变导致广泛的表型，从胚胎致死性、Antley-Bixler综合征（ABS）到轻度类固醇生成障碍。与先前获得的CYPOR的“闭合形式”结构相反，我们最近获得的CYPOR的变体形式（CYPORTGEE）采用能够还原P450的开放构象，并与其伴侣形成稳定的复合物。我们将在EPR和结晶研究中使用CYPORTGEE，具体目标如下。目标1：用自旋标记EPR方法研究CYPOR与其电子转移配偶体结合后的构象变化及电子转移配偶体之间的相互作用。使用野生型CYPOR和CYPORTGEE的晶体结构作为指导，将自旋标记双半胱氨酸突变体（两个黄素结构域中的每一个中一个），并测量自旋-自旋距离。自旋标记的CYP 2B 4和HO-1也将与单标记的CYPOR一起使用，以测量复合物中两个配偶体之间的自旋-自旋距离。根据CYPOR两个结构域之间的距离以及两个配偶体之间的距离，构建CYPOR-2B 4/HO-1复合物的结构。CYPOR及其伴侣的复合物的模型结构将通过结合位点特异性诱变的生物化学表征来验证。目的2：确定CYPOR与CYP 2D 6、CYP 3A 4、CYP 2B 4和人HO-1复合物的晶体结构。我们将使用与伴侣形成稳定复合物的人类和大鼠CYPORTGEE蛋白。CYP 2B 4、CYP 3A 4和CYP 2D 6的可溶性形式以及HO-1的可溶性形式将用于与CYPORTGEE的共结晶研究。上述研究的成功完成将通过回答该领域的中心问题来影响P450领域：CYPOR与其氧化还原伙伴（包括P450和HO）之间的识别基础和电子转移机制是什么？这些研究的结果将分别通过提供对药物代谢和HO-1功能机制的更好理解而用于药物开发和有效疗法的设计，并且可能用于导致异常类固醇生成的CYPOR缺陷的治疗剂的设计。