Mitochondrial redox regulation of adrenal steroidogenesis

项目总结/摘要 肾上腺类固醇分泌过多是一种普遍但未被诊断的高血压病因， 心血管疾病和目前的药物治疗管理不善。不同的互补线 有证据表明，肾上腺线粒体氧化还原途径功能障碍是一种未探索的致病驱动因素， 了以下条件例如，虽然生理刺激和基因突变增加了 肾上腺甾体醛固酮通过升高胞质Ca 2+的共同途径起作用，Ca 2+升高是 由线粒体缓冲。肾上腺线粒体有一个独特的电子传递链， 辅因子NADPH支持类固醇生成的几个步骤，线粒体Ca 2+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，人的烟酰胺核苷酸转氢酶（NNT）功能缺失突变 产生线粒体NADPH缺乏，这反过来又会导致严重的皮质醇和醛固酮缺乏。 虽然这表明线粒体氧化还原生物学在肾上腺类固醇生理学中起着关键作用，但迄今为止， 由于肾上腺线粒体的基础生物学知识有限，在该领域的探索受到限制。 这里提出的初步数据强调了一种新的纯化方法，以产生第一个蛋白质组 肾上腺线粒体的特征，显示氧化还原酶NNT和 NQO 1（NAD（P）H：醌受体氧化还原酶1）。肾上腺线粒体制备物经验证， 生物能量询问在一个定制的荧光计，和NNT和NQO 1缺陷的人肾上腺细胞， 用CRISPR产生。有了这些独特的资源，研究提出了解决中心假设 肾上腺线粒体的新氧化还原途径调节类固醇生成。将阐明机制 通过对分离的线粒体和透化的人肾上腺细胞的生物能量学研究，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 辅因子/代谢物，以及重组酶对新鲜肾上腺的基于活性的代谢物分析 萃取物这些研究将为肾上腺类固醇合成的研究开辟一个新的方向。申请人博士 马萨诸塞州总医院的讲师帕特里克沃德描绘了一个5年的职业计划， 博士学位在癌症代谢和他的临床培训内科和内分泌学。沃德医生会 由Vamsi Mootha博士指导，他是线粒体生物学领域的世界领导者，拥有出色的培训记录， 并由一个杰出的委员会提供咨询，该委员会在肾上腺生物学、高血压和 心血管疾病沃德博士杰出的导师团队和机构环境，加上 他已经产生的新资源，使他能够独特地执行他提出的研究， 随后过渡到独立，同时迅速上升为肾上腺代谢的领导者。

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.