Mitochondrial redox regulation of adrenal steroidogenesis

项目摘要/摘要 肾上腺类固醇分泌过多是高血压和高血压的普遍但未被诊断的病因 心血管疾病，目前的药物治疗效果不佳。互补性不同的系列 有证据表明，肾上腺线粒体氧化还原通路功能障碍是一种未知的致病因素 这些条件。例如，尽管增加细菌产量的生理刺激和基因突变 肾上腺类固醇醛固酮通过共同的细胞内钙升高途径起作用，钙离子升高 由线粒体缓冲。肾上腺线粒体有一条独特的电子传输链，它使用氧化还原 辅因子NADPH支持类固醇合成的几个步骤，线粒体钙摄取可以刺激 与线粒体增加相关的类固醇、醛固酮和皮质醇的产生 NADPH。重要的是，人类烟酰胺核苷酸转氢酶(NNT)功能突变 产生线粒体NADPH缺乏症，进而导致严重的皮质醇和醛固酮缺乏症。 综上所述，这表明到目前为止线粒体氧化还原生物学在肾上腺类固醇生理中起着关键作用。 由于对肾上腺线粒体的基本生物学知识有限，在这一领域的探索一直受到限制。 这里给出的初步数据强调了一种新的纯化方法来生产第一个蛋白质组 肾上腺线粒体的特征，显示氧化还原酶NNT和NNT显著丰富 NQO1(NAD(P)H：苯二酚受体氧化还原酶1)。肾上腺线粒体制剂是有效的 在定制的荧光仪中进行生物能量检测，缺乏NNT和NQO1的人肾上腺细胞 由CRISPR生成。有了这些独特的资源，提出了解决中心假设的研究 肾上腺线粒体新的氧化还原途径调节类固醇的生成。机理将被阐明 通过对分离的线粒体和通透性的人肾上腺细胞的生物能量学研究，稳定同位素 通透性细胞的示踪，类固醇和中心氧化还原的靶向LC-MS测量 利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 萃取物。这些研究将为肾上腺类固醇激素生成的研究开辟新的方向。申请者是Dr。 马萨诸塞州综合医院的讲师帕特里克·沃德勾勒出了一份建立在 他在癌症新陈代谢方面的博士学位，以及在内科和内分泌学方面的临床训练。沃德医生会 由Vamsi Mootha博士指导，Vamsi Mootha博士是线粒体生物学的世界领导者，有着非凡的训练记录， 并由一个在肾上腺生物学、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 他已经产生的小说资源，使他处于独特的地位，可以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新陈代谢的领导者。

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.