Lipid homeostasis is crucially important for cells, and disruptions in this balance can lead to diseases of lipid overload, such as obesity-related disorders. Lipid homeostasis is mainly coordinated in the endoplasmic reticulum (ER), the largest membrane system in cells. The ER is a well-recognized location for synthesis of membrane and secreted proteins, processes that are safeguarded by the “unfolded protein response” (UPR). Recent studies suggest that a complementary and interrelated pathway, which we term the “lipotoxic protective response” (LPR), acts in parallel to maintain ER lipid homeostasis. In contrast to the UPR, however, knowledge of the molecular aspects of the LPR remain rudimentary. Here we describe studies to overcome this knowledge gap through investigations through our recent identification of the mammalian FIT2 protein as a key guardian of ER lipid homeostasis. FIT2 has been a mysterious ER protein, of crucial importance for cell health and function, that was implicated in ER homeostasis and lipid metabolism but lacked an identified function. After years of effort and a biochemical tour de force, we have discovered that FIT2 is an acyl-CoA diphosphatase enzyme that catabolizes fatty acyl-CoAs, the activated forms of fatty acids, in the ER. Our preliminary data indicate that this activity may be localized on the luminal leaflet and is crucially important for cell health and viability. Absence of FIT2 triggers ER stress and, in a mouse model we have generated, liver injury. Our findings break new ground and raise many important questions about FIT2 and the LPR. Here we propose to answer these questions by using an interdisciplinary approach. Aim 1 will determine mechanisms of the LPR at the molecular level by deciphering the biochemical mechanism for FIT2’s catabolism of fatty acyl-CoA in the ER. We will combine biochemical and structural biology approaches to answer: What is the enzyme’s catalytic mechanism? Is FIT2 active towards ER luminal substrates? Is its activity regulated by lipid metabolites (e.g., diacylglycerol, DAG)? What is the fate of the metabolites generated by FIT2 activity? Aim 2 will determine mechanisms of the LPR at the cellular level by elucidating how FIT2 and the LPR are integrated with other ER stress protection pathways. Specifically, we will address: How does FIT2 mechanistically trigger the UPR? Does the mammalian UPR pathway depend on FIT2 activity? Can the interdependency of the FIT2/LPR and the UPR be exploited for therapeutic purposes in cancer, for example those with high demands on ER function, such hepatocellular carcinoma and multiple myeloma? Aim 3 will determine mechanisms of the LPR at the physiological level by determining how FIT2 maintains lipid homeostasis in mammalian liver. We have generated mice lacking FIT2 in hepatocytes, and preliminary studies indicate these mice have ER stress, increases in hepatocyte lipid storage, defects in lipid secretion, and signs of liver disease. We will...