Project Summary The goal of this project is to define the metabolic and molecular basis of fatty liver disease (FLD), a burgeoning health problem with few therapeutic options. Fatty liver disease has been a major focus of our laboratory since 2004, when we undertook the first survey of hepatic triglyceride (TG) content (HTGC) in a population-based sample of different ancestries, the Dallas Heart Study (DHS). Hepatic steatosis was found to be strongly influenced by ancestry (Hispanics>European>African) and adiposity, but varied widely even among individuals who were matched for ancestry and body mass index (BMI). We used human genetics to identify the first and most clinically impactful genetic risk factor variant for FLD: PNPLA3(148M). This variant confers susceptibility to the full spectrum of both alcoholic and nonalcoholic FLD. In the same study in which we identified PNPLA3(148M), we also identified another variant in PNPLA3, S453I, that is associated with reduced HTGC; this variant is present almost exclusively in individuals of African descent, the group with the lowest prevalence of FLD. These two variants together account for ~70% of ancestry-related differences in HTGC. Despite having made significant progress elucidating the pathogenic mechanism of the 148M variant, and having performed successful proof-of-concept studies in mice of potential therapeutic avenues to combat the effects of 148M, important questions regarding the pathobiology of the variant and how it is related to FLD remain unanswered or disputed. Accordingly, we will focus this application on three critical questions: 1) How does PNPLA3(148M) evade ubiquitylation and degradation? 2) How does the 148M variant impair TG hydrolysis? and 3) How does PNPLA3-S453I lower hepatic TG content and protect against FLD? Each of these questions constitutes a Specific Aim. We will take advantage of cutting edge technologies to overcome important limitations in prior methods used by us and others to address these questions. In AIM 1 we will use a CRISPR/Cas9 inactivation screen to identify the E3-ligase that ubiquitylates PNPLA3. In AIM 2 we will use a highly tunable system to control protein expression at the level of translation, and a sensitive, luciferase reconstitution assay to biochemically define the interactions among PNPLA3, ATGL, and ABHD5 at physiologically relevant concentrations in cells. In AIM 3 we will develop the first mouse model of PNPLA3(S453I) to determine how the variant lowers HTGC. Since the region of PNPLA3 spanning residue 453 is not present in mice, we will replace the mouse gene with a human mini-gene containing the S453I variant using CRISPR-Cas9 technology. These mice will be used to determine how this missense variant results in lower hepatic TG levels. These studies, when taken together, hold the promise of revealing new pathways and processes that can be therapeutically manipulated for the prevention and treatment of PNPLA3-related FLD.