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（由申请人提供）：该提案的总体目标是了解微粒体单氧化系统机制的结构和分子基础，该系统负责各种异种生物和内源性化合物的氧化代谢。该系统的两个关键组成部分是细胞色素P450（P450）和NADPH-CYTOCHROME P450氧化还原酶（CYPOR）。尽管我们对这两个伙伴的每个伙伴的机制和结构都了解很多，但他们彼此之间的相互作用知之甚少。在人类中，单个CYPOR蛋白能够与〜50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基因的突变导致了各种表型，从胚胎致死性，安特利 - 蛋白综合征（ABS）到轻度类固醇生成疾病。与先前获得的CYPOR的“封闭形式”结构相反，我们最近获得的Cypor（Cyportgee）的变体形式采用了一个开放的构象，能够减少P450，并与其合作伙伴形成稳定的复合物。如以下特定目的，我们将在EPR和结晶研究中使用cyportgee。目标1：确定CYPOR与其电子转移伙伴结合后的构象变化，以及通过位置定向自旋标记的EPR方法之间的相互作用。将使用野生型Cypor和Cyportgee的晶体结构作为指南，将旋转标记双半胱氨酸突变体（两个黄素结构域中的每个），并将测量自旋旋转距离。自旋标记的CYP2B4和HO-1也将与单个标记的CYPOR一起使用，以测量复合物中两个伴侣之间的自旋距离。从Cypor的两个域之间以及两个伙伴之间的距离，将构建CYPOR-2B4/HO-1的复合物的结构。 CYPOR及其伴侣复合物的所得模型结构将通过生化表征与位点特异性诱变相结合来验证。目标2：确定CYPOR与CYP2D6，CYP3A4，CYP2B4和人HO-1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。 CYP2B4，CYP3A4和CYP2D6的可溶形式以及HO-1的可溶形式将用于与Cyportgee的共结晶研究。上述研究的成功完成将通过提供该领域的中心问题的答案来影响P450领域：识别的基础和电子转移机理是什么基础，以及包括P450和HO在内的CYPOR及其氧化还原伙伴之间的电子转移机制是什么？这些研究的结果将通过更好地理解药物代谢和HO-1功能的机制，并可能用于针对CYPOR缺乏症的治疗剂，从而用于药物开发和设计有效疗法。