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

• 批准号: 8877567
• 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
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8877567
• 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

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）：本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，该系统负责各种外源性化合物和内源性化合物的氧化代谢。该系统的两个关键组件是细胞色素 P450 (P450s) 和 NADPH-细胞色素 P450 氧化还原酶 (CYPOR)。尽管我们对这两个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 个微粒体 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 基因突变会导致多种表型，从胚胎致死、Antley-Bixler 综合征 (ABS) 到轻度类固醇生成性疾病。 与之前获得的CYPOR“封闭形式”结构相比，我们最近获得的CYPOR变体（CYPORTGEE）采用开放构象，能够还原P450，并与其伙伴形成稳定的复合物。我们将在 EPR 和结晶研究中使用 CYPORTGEE，如以下具体目标所述。目标 1：通过定点自旋标记 EPR 方法确定 CYPOR 与其电子转移伙伴结合后的构象变化以及它们之间的相互作用。使用野生型 CYPOR 和 CYPORTGEE 的晶体结构作为指导，对双半胱氨酸突变体（两个黄素结构域各一个）进行自旋标记，并测量自旋-自旋距离。自旋标记的 CYP2B4 和 HO-1 也将与单标记的 CYPOR 一起使用，以测量复合物中两个伙伴之间的自旋-自旋距离。根据 CYPOR 的两个结构域之间以及两个伙伴之间的距离，将构建 CYPOR-2B4/HO-1 复合物的结构。 CYPOR 及其伙伴复合物的模型结构将通过生化表征结合定点诱变进行验证。目标 2：确定 CYPOR 与 CYP2D6、CYP3A4、CYP2B4 和人 HO-1 复合物的晶体结构。我们将使用人类和大鼠 CYPORTGEE 蛋白，它们与合作伙伴形成稳定的复合物。可溶形式的 CYP2B4、CYP3A4 和 CYP2D6 以及可溶形式的 HO-1 将用于与 CYPORTGEE 的共结晶研究。 上述研究的成功完成将影响P450领域，为该领域的核心问题提供答案：CYPOR与其氧化还原伙伴（包括P450s和H2O）之间的识别基础和电子转移机制是什么？这些研究的结果将用于药物开发和有效疗法的设计，分别提供对药物代谢和 HO-1 功能机制的更好理解，并可能用于设计针对 CYPOR 缺陷导致类固醇生成异常的疗法。