The synthesis and trafficking of phosphatidylserine in cellular cholesterol homeostasis. - ABSTRACT Significance: Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in mammalian cells. Besides being a key structural component of cellular membranes, PS also regulates multiple cellular processes, such as cell signaling, mitochondrial function, the synthesis of phosphatidylethanolamine, programmed cell death, blood clotting, viral infection and cancer immunotherapy. Recently, PS has been shown to govern the transport of low-density lipoprotein (LDL)-derived cholesterol and also to contribute to the repair of damaged lysosomes and plasma membrane. However, our understanding of its synthesis via PSS1/2 (PS synthase 1/2), its flip-flop via TMEM41B/VMP1 and its transport via ORP5/8, remain very limited, and much less is known about the regulation of these processes. Builidng on exciting preliminary findings, this project will determine the structure of PSS1/2 and investigate how cholesterol may regulate the synthesis and distribution of cellular PS. Results from our proposed work will provide fundamental new insights into how two major lipid species, PS and cholesterol, may impact each other's cellular homeostasis. Innovation: Our work will establish a new cholesterol sensing mechanism to control cellular and systemic cholesterol homeostasis, and provide major conceptual innovation on the interplay between two of the most important lipid species in cell biology: PS and cholesterol. Approach: Aim 1 will determine the structures of PSS1/2 and their regulation by PS and cholesterol. Novel in vitro assays will be developed. Aim 2 will determine the role of TMEM41B/VMP1 in cholesterol transport and their regulation by cholesterol. TMEM41B and VMP1 are key phospholipid scramblases of the ER and regulate the level of cholesterol and PS in the plasma membrane. Aim 3 will determine how cholesterol may regulate the targeting of ORP5/8 and their interaction with cholesterol carriers: the GRAMD1 proteins. Impact: If successfully completed, the proposed work will establish a novel control mechanism for maintaining cellular cholesterol homeostasis and will provide new insights into how the synthesis and distribution of a major phospholipid, PS, is regulated.
