Mitochondrial redox regulation of adrenal steroidogenesis

线粒体氧化还原调节肾上腺类固醇生成

• 批准号: 10381600
• 负责人: Patrick S. Ward
• 金额: $ 17.28万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10381600
• 关键词: Address; Adrenal Glands; Aldosterone; Area; Bioenergetics; Biological; Biological Assay; Biology; Buffers; Cardiovascular Diseases; Cardiovascular system; Cells; Citric Acid Cycle; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; Custom; DNA Sequence Alteration; Data; Doctor of Philosophy; Electron Transport; Endocrinology; Environment; Enzymes; Etiology; Exhibits; Functional disorder; Funding; General Hospitals; Goals; Human; Human Genetics; Hydrocortisone; Hypertension; Impairment; Internal Medicine; Knowledge; Label; Laboratories; Massachusetts; Measurement; Medical; Mentors; Mentorship; Metabolic; Metabolism; Methodology; Methods; Mitochondria; Mitochondrial Proteins; Modeling; NAD(P)+ transhydrogenase; NADH; NADP; NQO1 gene; Output; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathogenicity; Pathway interactions; Physiological; Physiology; Positioning Attribute; Preparation; Production; Proteome; Proteomics; Quinones; Recombinants; Regulation; Research; Resources; Role; Shunt Device; Source; Steroid biosynthesis; Steroids; Stimulus; System; Testing; Tissues; Training; base; career; cofactor; hypertension treatment; inhibitor; instructor; loss of function mutation; novel; programs; stable isotope; tool; tumor metabolism; uptake; ward

PROJECT SUMMARY/ABSTRACT Excess adrenal steroid production is a prevalent but underdiagnosed etiology of hypertension and cardiovascular disease and poorly managed with current medical therapies. Diverse lines of complementary evidence point to dysfunction of adrenal mitochondrial redox pathways as an unexplored pathogenic driver for these conditions. For example, although physiologic stimuli and genetic mutations that increase production of the adrenal steroid aldosterone act through a shared pathway of elevated cytosolic Ca2+, Ca2+ elevations are buffered by mitochondria. Adrenal mitochondria have a unique electron transport chain that uses the redox cofactor NADPH to support several steps in steroidogenesis, and mitochondrial Ca2+ uptake can stimulate production of the steroids aldosterone and cortisol in a manner correlating with increased mitochondrial NADPH. Importantly, human loss of function mutations in nicotinamide nucleotide transhydrogenase (NNT) produce a mitochondrial NADPH deficiency that, in turn, can cause severe cortisol and aldosterone deficiency. While together this suggests a key role for mitochondrial redox biology in adrenal steroid physiology, to date exploration has been limited in this area due to limited knowledge of the basic biology of adrenal mitochondria. Preliminary data presented here highlight a novel purification methodology to produce the first proteomic characterization of adrenal mitochondria, which show notable enrichment for the redox enzymes NNT and NQO1 (NAD(P)H:quinone acceptor oxidoreductase 1). Adrenal mitochondrial preparations are validated with bioenergetic interrogation in a custom-built fluorimeter, and NNT and NQO1-deficient human adrenal cells are generated with CRISPR. With these unique resources, studies are proposed to address the central hypothesis that novel redox pathways of adrenal mitochondria regulate steroidogenesis. Mechanism will be elucidated with bioenergetic studies of isolated mitochondria and permeabilized human adrenal cells, stable isotope tracing of permeabilized cells, targeted LC-MS measurements of steroids and central redox cofactors/metabolites, and activity-based metabolite profiling with recombinant enzymes on fresh adrenal extracts. The studies will launch a new direction in the study of adrenal steroidogenesis. The applicant, Dr. Patrick Ward, Instructor at Massachusetts General Hospital, has delineated a 5 year career plan building upon his Ph.D. in cancer metabolism and his clinical training in internal medicine and endocrinology. Dr. Ward will be mentored by Dr. Vamsi Mootha, a world leader in mitochondrial biology with an exceptional training record, and advised by an outstanding committee with significant expertise in adrenal biology, hypertension, and cardiovascular disease. Dr. Ward’s outstanding mentorship team and institutional environment, coupled with the novel resources he has already generated, uniquely position him to execute his proposed studies and subsequently transition to independence while rapidly ascending as a leader in adrenal metabolism.

项目概要/摘要 肾上腺类固醇产生过多是高血压和高血压的一个普遍但诊断不足的病因。 心血管疾病并且目前的药物治疗效果不佳。多元化线路互补 有证据表明肾上腺线粒体氧化还原途径功能障碍是未探索的致病驱动因素 这些条件。例如，虽然生理刺激和基因突变会增加 肾上腺类固醇醛固酮通过胞质 Ca2+ 升高的共同途径发挥作用，Ca2+ 升高是 由线粒体缓冲。肾上腺线粒体具有独特的电子传递链，利用氧化还原作用 辅因子 NADPH 支持类固醇生成的几个步骤，并且线粒体 Ca2+ 摄取可以刺激 类固醇醛固酮和皮质醇的产生与线粒体增加相关 NADPH。重要的是，人类烟酰胺核苷酸转氢酶（NNT）功能缺失突变 产生线粒体 NADPH 缺乏，进而导致严重的皮质醇和醛固酮缺乏。 迄今为止，这表明线粒体氧化还原生物学在肾上腺类固醇生理学中发挥着关键作用 由于对肾上腺线粒体的基本生物学知识有限，该领域的探索受到限制。 这里提供的初步数据强调了一种新颖的纯化方法来产生第一个蛋白质组 肾上腺线粒体的表征，显示氧化还原酶 NNT 和 NNT 显着富集 NQO1（NAD(P)H：醌受体氧化还原酶 1）。肾上腺线粒体制剂经过验证 在定制的荧光计中进行生物能询问，并且 NNT 和 NQO1 缺陷的人肾上腺细胞 用 CRISPR 生成。借助这些独特的资源，提出研究来解决中心假设 肾上腺线粒体的新氧化还原途径调节类固醇生成。机制将被阐明 通过对分离的线粒体和透化的人肾上腺细胞的生物能研究，稳定同位素 追踪透化细胞、类固醇和中心氧化还原的靶向 LC-MS 测量 使用重组酶对新鲜肾上腺进行辅因子/代谢物和基于活性的代谢物分析 提取物。这些研究将为肾上腺类固醇生成的研究开辟新的方向。申请人，博士。 马萨诸塞州总医院的讲师帕特里克·沃德 (Patrick Ward) 制定了 5 年职业计划 他的博士学位。他在癌症代谢方面的研究以及他在内科和内分泌学方面的临床培训。沃德博士将 接受 Vamsi Mootha 博士的指导，他是线粒体生物学领域的世界领先者，拥有卓越的培训记录， 并由一个在肾上腺生物学、高血压和糖尿病领域拥有丰富专业知识的杰出委员会提供建议 心血管疾病。 Ward博士优秀的导师团队和制度环境，加上 他已经产生的新颖资源使他能够独特地执行他提出的研究和 随后过渡到独立，同时迅速上升为肾上腺代谢领域的领导者。