The terminal steps of cortisol and aldosterone biosynthesis

皮质醇和醛固酮生物合成的最终步骤

• 批准号: 10409567
• 负责人: RICHARD J. AUCHUS
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10409567
• 关键词: Active Sites; Adrenal Cortex; Adrenal Glands; Adverse effects; Affinity; Age; Aldosterone; Anabolism; Androgens; Binding; Biochemical; Biochemistry; CYP11B2 gene; Cell Line; Cells; Cessation of life; Chromosome 8; Complex; Consumption; Corticosterone; Cortodoxone; Coupling; Cytochrome P450; Data; Deoxycorticosterone; Diabetes Mellitus; Disease; Dissociation; Drug Design; Drug Targeting; Dyslipidemias; Environment; Enzymes; Equilibrium; Genes; Geometry; Glucocorticoids; Glucose Intolerance; Goals; Heart failure; Homeostasis; Human; Hydrocortisone; Hydroxylation; Hypertension; Hypotension; Impairment; Intrinsic factor; Kinetics; Laboratories; Lead; Lipids; Medical; Medical History; Membrane; Membrane Lipids; Metabolism; Michigan; Microscopic; Mineralocorticoids; Mitochondria; Mixed Function Oxygenases; Molecular Conformation; Morbidity - disease rate; Mutagenesis; Mutation; NADP; Nature; Obesity; Operative Surgical Procedures; Organ; Oxidases; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; Physiological; Point Mutation; Positioning Attribute; Production; Property; Reaction; Regulation; Reproducibility; Research; Series; Sodium Chloride; Steroids; Stress; System; Testing; Therapeutic Index; Universities; Veterans; Water; Work; Zona Fasciculata; Zona Glomerulosa; Zona Reticularis; antagonist; bone mass; carbohydrate metabolism; chemical property; comorbidity; enzyme activity; enzyme structure; enzyme substrate; experimental study; human disease; hypertension treatment; in vivo; inhibitor therapy; insight; instrumentation; nanodisk; novel; oxidation; physical property; rational design; receptor; response; side effect; single molecule; small molecule; steroid hormone; surgical risk; tandem mass spectrometry; tool

Excess aldosterone and cortisol production from the human adrenal cortex commonly contributes to hypertension, heart failure, obesity, glucose intolerance, and low bone mass. Cytochromes P450 11B2 and P450 11B1 catalyze the final steps in the biosynthesis of aldosterone and cortisol, respectively. The zone- specific regulation of these enzymes maintains salt and water balance or carbohydrate metabolism and response to physiologic stress. The enzymes share 95% sequence similarity, and both catalyze the 11β- hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol. In contrast, the 18-hydroxylase of P450 11B1 is poor compared to P450 11B2, and only P450 11B2 has 18-oxidase activity, which converts 18-hydroxy- corticosterone to aldosterone. P450 11B2 has high processivity, in that the intermediates predominantly do not dissociate before additional turnovers to aldosterone, but the reasons for this processivity and whether this property is required for aldosterone production are not known. A comparison of its microscopic steps with those of P450 11B1 offers an opportunity to understand the unique biochemistry of aldosterone production. Our long-term goal is to elucidate the biochemical and physical properties that confers high 18- hydroxylase and moderate 18-oxidase activities to P450 11B2 but not P450 11B1. Our central hypotheses are that: (1) the positioning of nascent corticosterone generated from 11-deoxycorticosterone in the active site and the conformation of P450 11B2 is different that the nature of the complex when corticosterone binds as initial substrate; and (2) that the processivity of P450 11B2 reflects a combination of slow dissociation rates and high coupling efficiency with the intermediates. Consequently, the objectives of this application are to characterize the binding constants, binding and dissociation rates, pre-steady state (single turnover) kinetics, and coupling efficiencies for P450 11B1 and P450 11B2 with several substrates and to deduce which parameters correlate best with processivity. As tools for these studies, we will employ mutations that alter the activities for P450 11B1 and P450 11B2 and alternate substrates that have specific chemical properties. In Aim 1, we will dissect the 18-hydroxylase activity of P450 11B1 and P450 11B2, wild-type and mutations. In Aim 2, we will focus on the 18-oxidase activity with a parallel series of studies as in Aim 2. In Aim 3, we will focus on extrinsic factors, specifically the lipid composition of the membrane environment, in regulating the processivity and latter two activities of P450 11B2. We will use nanodiscs to generate artificial enzyme-membrane systems and confirm key results in transfected HEK-293 or V79 cells. The work will utilize the specialized instrumentation that the Auchus and Waskell laboratories have employed to study cytochrome P450 enzymes for many years and state-of-the art tandem mass spectrometry at the University of Michigan. In this manner, we will systematically define the differences in the reaction mechanisms and properties for P450 11B1 and P450 11B2. These studies will inform strategies to screen for better drugs to safely inhibit key steps of aldosterone and cortisol production for the treatment of human diseases.

人类肾上腺皮质产生过量的醛固酮和皮质醇通常会导致 高血压、心力衰竭、肥胖、葡萄糖不耐症和低骨量。细胞色素 P450 11B2 和 P450 11B1 分别催化醛固酮和皮质醇生物合成的最后步骤。该区域- 这些酶的特定调节可维持盐和水平衡或碳水化合物代谢， 对生理应激的反应。这两种酶具有 95% 的序列相似性，并且都催化 11β- 11-脱氧皮质酮和11-脱氧皮质醇的羟基化。相反，P450 11B1 的 18-羟化酶是 与 P450 11B2 相比较差，并且只有 P450 11B2 具有 18-氧化酶活性，可将 18-羟基- 皮质酮转变为醛固酮。 P450 11B2 具有高持续合成能力，因为中间体主要不 在额外转化为醛固酮之前解离，但这种持续性的原因以及这是否 醛固酮生产所需的特性尚不清楚。其微观步骤与 P450 11B1 的那些提供了了解醛固酮产生的独特生物化学的机会。 我们的长期目标是阐明赋予高 18- 的生化和物理特性 羟化酶和中等 18-氧化酶活性对 P450 11B2 具有作用，但对 P450 11B1 没有作用。我们的中心假设 是：（1）11-脱氧皮质酮产生的初生皮质酮在活性位点的定位 P450 11B2 的构象与皮质酮结合时复合物的性质不同 初始底物； (2) P450 11B2 的持续合成能力反映了缓慢解离速率的组合 与中间体的偶联效率高。因此，该应用程序的目标是 表征结合常数、结合和解离速率、前稳态（单次转换）动力学， 以及 P450 11B1 和 P450 11B2 与多种基材的耦合效率，并推断出 参数与持续加工能力最相关。作为这些研究的工具，我们将利用突变来改变 P450 11B1 和 P450 11B2 以及具有特定化学性质的替代底物的活性。 在目标 1 中，我们将剖析 P450 11B1 和 P450 11B2、野生型和 突变。在目标 2 中，我们将重点关注 18-氧化酶活性，并与目标 2 一样进行一系列平行研究。 3、我们将重点关注外在因素，特别是膜环境的脂质成分， 调节 P450 11B2 的持续合成能力和后两项活性。我们将使用纳米圆盘来生成人工 酶膜系统并确认转染 HEK-293 或 V79 细胞的关键结果。该工作将利用 奥克斯和瓦斯克尔实验室用于研究细胞色素的专用仪器 密歇根大学多年来一直致力于 P450 酶研究和最先进的串联质谱分析。在 通过这种方式，我们将系统地定义P450的反应机制和性质的差异 11B1 和 P450 11B2。这些研究将为筛选更好的药物以安全地抑制关键步骤提供策略 用于治疗人类疾病的醛固酮和皮质醇生产。