The terminal steps of cortisol and aldosterone biosynthesis

皮质醇和醛固酮生物合成的最终步骤

基本信息

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
项目类别：
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

项目摘要

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.

人类肾上腺皮质的过量醛固酮和皮质醇产生通常有助于高血压，心力衰竭，肥胖症，葡萄糖肠球和低骨量。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一步。区域 - 这些酶的特定调节维持盐和水平衡或碳氢化代谢和对生理压力的反应。这些酶具有95％的序列相似性，并且都催化了11β- 11-脱氧核心酮和11-脱氧皮质醇的羟基化。相反，P450 11B1的18-羟化酶为与P450 11B2相比，较差，只有P450 11B2具有18-氧化酶活性，可转化18-羟基 - 皮质酮到醛固酮。 P450 11B2具有较高的加工性，因为中间的主要是在醛固酮的额外失误之前解离，但是这种加工性的原因以及是否存在醛固酮生产所需的财产尚不清楚。比较其微观步骤与 P450 11B1的那些提供了一个机会，可以了解醛固酮生产的独特生物化学。我们的长期目标是阐明供认高18--的生化和物理特性羟化酶和中等18氧化酶的活性至P450 11B2，而不是P450 11B1。我们的中心假设是：（1）活性部位从11-脱氧核心酮产生的新生皮质酮的定位 P450 11B2的构象不同，当皮质酮结合为时，复合物的性质初始底物；（2）P450 11B2的加工性反映了缓慢解离率的组合以及与中间体的高耦合效率。因此，此应用程序的目标是表征结合常数，结合和解离速率，稳态态（单个周转）动力学， P450 11B1和P450 11B2的耦合效率与几个基板，并推断出来参数与过程率最相关。作为这些研究的工具，我们将采用改变的突变 P450 11B1和P450 11B2的活动以及具有特定化学特性的替代底物。在AIM 1中，我们将剖析P450 11B1和P450 11B2的18-羟化酶活性，野生型和突变。在AIM 2中，我们将重点关注18-氧化酶活性，并在AIM 2中进行一系列平行的研究。 3，我们将专注于外部因素，特别是膜环境的脂质组成，调节P450 11B2的加工率和后来的两种活动。我们将使用纳米盘生成人工酶 - 膜系统并确认转化为HEK-293或V79细胞的关键结果。工作将利用 Auchus和Waskell实验室已经努力研究细胞色素的专业仪器密歇根大学的P450酶多年来以及最先进的群质谱法。在这种方式，我们将系统地定义P450的反应机理和特性的差异 11B1和P450 11B2。这些研究将为策略提供筛查更好的药物以安全抑制关键步骤的策略。用于治疗人类疾病的醛固酮和皮质醇的产生。