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 sub...