Elucidating the relationship between lipid droplets, oxidative lipid damage, and ferroptosis - PROJECT SUMMARY / ABSTRACT Cells have evolved intricate quality control systems to prevent the accumulation of aberrant or damaged macromolecules, such as proteins and DNA. Lipids are chemically diverse macromolecules that have important functions in membrane biology, energy homeostasis, and signaling. Similar to proteins and DNA, lipids can also be damaged (e.g., oxidized). However, our understanding of the mechanisms that regulate lipid quality control remains at an early stage, representing a key gap in knowledge. The accumulation of oxidatively damaged lipids is a hallmark of ferroptosis, an iron-dependent regulated form of cell death that is an emerging target for the treatment of drug-resistant cancers. Understanding the regulation of lipid peroxidation and ferroptosis provides an exceptional system to uncover the mechanisms that govern lipid quality control and to identify novel therapeutic targets for the treatment of drug-resistant cancers. The prevailing paradigm has focused on the glutathione-dependent peroxidase GPX4, which suppresses ferroptosis by converting lipid peroxides into non- toxic lipid alcohols. Employing CRISPR-based genetic screens, we recently discovered FSP1 as a new ferroptosis resistance factor that acts parallel to GPX4. Mechanistically, FSP1 reduces non-mitochondrial coenzyme Q10, which functions as a lipophilic antioxidant to suppress the propagation of lipid peroxides. Genetic disruption or chemical inhibition of FSP1 sensitizes cancer cells of diverse tissue origins to ferroptosis. FSP1 is present on lipid droplets, the primary lipid storage organelle in cells, but its role on lipid droplets is unknown. The current proposed research aims to exploit lipidomics, imaging, and biochemically reconstituted and cell-based assays of FSP1 function to address several outstanding questions regarding the cellular role of FSP1 and its regulation. In Aim 1, we will test the hypothesis that lipid droplet-localized FSP1 reduces coenzyme Q10 to prevent oxidation to stored neutral lipids such as triacylglycerol. In Aim 2, we will validate and characterize a series of regulators that we identified through genetic screens that govern FSP1 post-translational stability. The completion of these aims will advance our understanding of the mechanisms that mediate cellular lipid quality control, including determining how neutral lipids stored in LDs are protected from oxidative damage and how FSP1 levels are regulated in cancer.
