Project summary Obesity is a major risk factor for NAFLD/NASH. In obesity, energy accumulation causes metabolic dysfunction and endoplasmic reticulum (ER) and oxidative stress, leading to tissue inflammation and injury, which are hallmarks of NASH. The DNA damage response and unfolded protein response are vital to maintain the integrity of cellular genome and proteome. The ER maintains proteostasis via ER-associated protein degradation and unfolded protein response. Metabolic dysfunction alters ER-associated protein degradation and unfolded protein response, leading to ER stress, caspase activation and cell death. Moreover, increased oxidative stress enhances DNA damage-induced cell death. Defining how these pathways integrate to dictate cell metabolism and survival is the long-term goal of The Chakraborty lab. The objective of this proposal is to decipher the role of the E3 ubiquitin ligase Ube4A in the obesogenic IP6K1 protein-mediated hepatocyte metabolic dysfunction and DNA damage response and unfolded protein response mediated hepatocyte survival and NAFLD/NASH. The overarching hypothesis is that Ube4A maintains metabolic homeostasis and protects hepatocytes from stress-induced death, delaying the development and progression of obesity and NAFLD/NASH. The rationale is that determining the role of Ube4A in NAFLD/NASH and the mechanisms by which it regulates hepatocyte metabolism and survival will provide new therapeutic opportunities. Our specific Aims will test the following hypotheses: (Aim 1) Test the impact of whole-body- and hepatocyte-Ube4A deletion on metabolic dysfunction, liver injury, and NAFLD/NASH in mice; (Aim 2) Determine mechanisms of Ube4A-mediated IP6K1 inhibition and its impact on metabolic dysfunction and NAFLD/NASH in mice; (Aim 3) Decipher the mechanisms by which Ube4A regulates hepatocyte survival. The contribution is significant and transformative because it is expected to unravel the role of a novel pathway that regulates obesity, insulin resistance and hepatic steatosis and distinguish the hepatocyte-specific impact of this pathway on NAFLD/NASH. Moreover, it is the first step to defining the mechanisms of how Ube4A regulates hepatocyte metabolism and survival and how the obesogenic protein IP6K1 is modulated in vivo. These exciting findings could lead to development of new therapeutic approaches to treat obesity and NAFLD/NASH. The proposed research is innovative as it will utilize exciting new tools to unravel a novel pathway that regulates cell metabolism and survival, which is therapeutically relevant and has broad implications for many diseases.