Project Summary: The overall goal of our research program is to develop chemical tools and an in vivo pipeline to delineate mechanisms of ferroptosis regulation by endogenous oxidized polyunsaturated fatty acids (PUFAs). Ferroptosis is a recently discovered iron-dependent non-apoptotic programmed cell death that is characterized by an increase in membrane lipid peroxidation. Like other programmed cell death, ferroptosis has a broad impact on cell and embryo development, cell senescence, tissue homeostasis, and many diseases. Several key proteins and compounds that regulate ferroptosis have been identified, but the underlying mechanism of ferroptosis and its endogenous mediators are poorly understood. This proposal is based on our research centered around PUFA oxidative metabolism and health. Our research focuses on generating novel chemical tools to investigate the mechanism of PUFA metabolites on human health. Specifically, we have developed multiple synthetic methods to prepare cytochrome P450 (CYP) PUFA metabolites. We also designed and synthesized the corresponding PUFA novel mimics. We applied these synthesized metabolites and mimics to identify many new biological effects of PUFA CYP metabolites. To investigate the mechanism behind the biological effects of CYP PUFA metabolites, we established C. elegans as a novel biological model in our lab due to the vast genetic tools available and the adaptability for high-throughput screening. All of these features greatly facilitate mechanistic studies. Using C. elegans, we found that endogenous PUFAs and the corresponding CYP metabolites induce ferroptosis only in the germline and neurons. Based on these findings, our central hypothesis for this proposal is that PUFA metabolites are endogenous lipid mediators of ferroptosis. To significantly advance the understanding of this novel programmed cell death, this R35 will address two questions: 1) What are the endogenous signaling molecules of ferroptosis? And 2) How is ferroptosis regulated in a cell type-specific manner? In the next five years, we will use PUFA metabolites, metabolite mimics, and metabolic inhibitors that we will synthesize in combination with a novel model organism (C. elegans) and their vast genetic tools to answer our scientific questions by 1) identifying endogenous PUFA metabolites that trigger or inhibit ferroptosis and 2) developing a novel in vivo platform to delineate cell-specific ferroptosis regulatory pathways. Understanding how oxidized PUFA metabolites trigger ferroptosis in specific cell types will have implications for how ferroptosis may affect human health. The outcomes from this proposal will build a strong foundation for our future research on multiple levels. The identified lipid mediators and protein targets will be further tested in mammalian models for research translation and mechanistic studies. The discovery of lipid mediators and protein targets also allows us to develop drug candidates to treat diseases rel...