Our multi-institutional team will investigate ZMPSTE24 inhibition by HIV-protease inhibitors and the resulting accumulation of farnesyl-prelamin A as a mechanism for metabolic syndrome and atherosclerotic cardiovascular disease in HIV-PI-treated patients. This grant proposal is submitted in response to a Request for Applications (RFA-HL-14-024) on mechanisms of heart disease in HIV patients. Our team was enthusiastic about responding to this RFA because we have collective expertise in cardiology, HIV medicine, lipid metabolism, atherogenesis, and nuclear lamins. Also, this RFA provides an opportunity for us to pursue one of our own discoveries-that commonly used HIV-PIs (e.g., atazanavir, ritonavir, lopinavir) inhibit ZMPSTE24 and lead to an accumulation of farnesyl-prelamin A in cells and tissues. This discovery is noteworthy because genetic syndromes associated with an accumulation of prelamin A (e.g., Hutchinson-Gilford progeria syndrome) lead to severe atherosclerotic heart disease. We fully appreciate that heart disease in HIV patients is multifactorial and influenced by several classes of drugs. No single research group could possibly follow-up on all of the potential mechanisms. Our team has decided to focus and to dig deeper into the connection between HIV-PIs, reduced ZMPSTE24 activity, farnesyl-prelamin A accumulation, and heart disease. Over the past few years, several stumbling blocks have slowed progress in understanding the connection between HIV-PIs, ZMPSTE24 inhibition, and heart disease. First, a paucity of specific prelamin A antibodies has made it difficult to perform rigorous research in animal models or with the cells and tissues of HIV patients. Our laboratory's recent development of a panel of monoclonal antibodies against prelamin A will be very helpful in overcoming that obstacle. Second, the link between prelamin A accumulation and atherogenesis is poorly understood. The cells in the arterial wall that are susceptible to prelamin A toxicity-and the link to atherogenesis-are not understood. To overcome that obstacle, we have created a conditional knockout allele for Zmpste24. Third, the mechanisms by which HIV-PIs inhibit ZMPSTE24 are not known. However, overcoming that obstacle is now quite feasible because the structure for human ZMPSTE24, alone and complexed to a prelamin A peptide substrate, was recently solved by our collaborator, Dr. Elisabeth Carpenter. We have three Specific Aims. The first is to investigate the impact of HIV-PIs on prelamin A processing in cultured cell lines, in mouse models, and in the cells and tissues of HIV patients. The second is to determine, with tissue-specific Zmpste24 knockout mice, how prelamin A accumulation affects different cell types of the arterial wall and leads to atherogenesis. The third is to investigate, with Dr. Carpenter, mechanisms for ZMPSTE24 catalysis and how HIV-PIs block ZMPSTE24 activity. We are excited by all three Aims and are poised-with all of the expertise, reagents, techniques, and expert collaborators-to carry out our proposed studies. We expect that our studies will yield fresh insights into mechanisms of heart disease in HIV patients.