Molecular basis of mitochondrial calcium extrusion - a key target in ischemic heart disease - PROJECT SUMMARY/ABSTRACT Ischemic heart disease remains the leading cause of death in the United States. Treating its associated pathologies is complicated by ischemia-reperfusion (I/R) injury, which further damages affected tissue due, in part, to mishandling of mitochondrial Ca2+. While physiological mitochondrial Ca2+ is an important component of cellular metabolism and signaling, aberrant Ca2+ accumulation during I/R injury is a key contributor to apoptotic and necrotic cell death. Targeting the molecular agents mediating mitochondrial Ca2+ extrusion thus poses a promising therapeutic avenue against I/R injury. However, the underlying agents and mechanisms facilitating mitochondrial Ca2+ extrusion remain poorly characterized. Here, TMEM65 is an emerging target implicated in the process of mitochondrial sodium-dependent calcium extrusion (mNCX) as its overexpression was recently reported to enhance cellular mNCX activity. However, the molecular basis of this important function remains to be elucidated. The proposed project will investigate the molecular mechanisms bridging TMEM65 to mNCX. The Research Training Plan proposes an innovative, multidisciplinary approach incorporating reductive biochemistry (Aim 1), structural studies (Aim 2.1), and cell biology (Aim 2.2) to investigate the role of TMEM65 in Na+-coupled Ca2+ transport and its relevance to cellular Ca2+ signaling. In Aim 1, the Applicant will functionally interrogate and quantitatively analyze the activity of purified TMEM65 molecules in a chemically defined, liposomal environment to unambiguously characterize the function of TMEM65. In Aim 2, the Applicant will determine the molecular- basis for TMEM65 function and modulation at the atomic level via cryogenic electron microscopic studies and perform structure-function analyses. Completion of the proposed aims will yield critical insights on mitochondrial Ca2+ signaling and cellular energetics. The Applicant has suitable prior training in protein biochemistry and strong preliminary data to support the feasibility of his proposed work. Additionally, the Applicant will receive further training in membrane protein biochemistry, functional assays, and structural analysis as well as career development skills in scientific communication, project management, and mentorship. The Project Sponsor and surrounding Training Environment are well-suited to provide the guidance and resources necessary for completing the proposed scientific and training aims, which will additionally prepare the Applicant for a career as an independent research scientist. Ultimately, the research proposed here will improve our understanding of cellular physiology and, by furthering our understanding of mitochondrial Ca2+ extrusion, effectively provide a new avenue for the development of therapeutics against I/R injury.
