Mammalian cells require fatty acids to continuously synthesize cellular membranes and generate energy. At the cellular level, these fatty acids are either taken up or synthesized de novo from other nutrients and incorporated into glycerolipids as major constituents of membrane phospholipids and triacylglycerols. Balancing glycerolipid synthesis with fatty acid availability must involve strict regulatory mechanisms. While there has been substantial progress to identify transcription factors involved in lipid metabolism, there are still several gaps in our understanding of allosteric mechanisms that regulate glycerolipid synthesis and storage. Addressing this through a systematic analysis of regulatory or enzymatic components of lipid synthesis and storage can provide significant insights in the field of metabolic disorders. Our long-term goal is to elucidate these regulatory components and to understand their roles in normal and disease physiology. We previously devised a CRISPR-based genetic screening strategy utilizing a toxic saturated fatty acid, palmitate, and systematically defined key metabolic enzymes and regulators of the glycerolipid synthesis pathway. We discovered calcineurin B homologous protein 1 (CHP1) as an essential regulatory protein of glycerolipid synthesis and storage. Through a myristoyl modification, CHP1 binds to and activates an endoplasmic reticulum GPAT (GPAT4), the first committed enzyme for the de novo synthesis of triacylglycerols and membrane lipids. Our preliminary data, which form the premise of our application, point to an unexpected mode of glycerolipid synthesis and storage regulation by CHP1. Given the conserved and critical role of CHP1 in glycerolipid synthesis, a chemical designed to impair the CHP1-GPAT4 complex could be used to treat metabolic disorders associated with dysfunctional lipid accumulation. However, the lack of the precise regulatory mechanisms and structural information of the CHP1-GPAT complex precludes sufficient mechanistic understanding to guide drug design. In this proposal, building on our previous data, I aim to test the hypothesis that CHP1 regulates ER GPAT function and may be used as a therapeutic target for metabolic disorders with dysfunctional lipid accumulation. To address this, we will first identify the precise mechanism by which CHP1 activates GPAT4 through structural and biochemical studies (Aim1). We will then determine whether upstream metabolic cues regulate the CHP1-GPAT4 complex in mammalian cells (Aim 2). Finally, we will test the therapeutic potential of targeting CHP1 in murine models of hepatic steatosis (Aim 3). Our proposal is highly innovative because we aim to identify new regulatory mechanisms for lipid storage and synthesis that could potentially be drug targets for disorders associated with dysfunctional lipid accumulation. Finally, this endeavor represents the first attempt to apply structural biology and genetics to the GPAT family of enzymes and will bring ...