PROJECT SUMMARY Alcohol-related liver disease (ALD) and nonalcoholic steatohepatitis (NASH) are two reasons Americans mostly require a liver transplant. Furthermore, aldehyde dehydrogenase gene 2 (ALDH2) deficiency, which impairs alcohol and lipid metabolism, affects ~560 million people globally. Our earlier studies show that a pathway from TGF-β to βII-spectrin (encodes the disordered protein βII-spectrin) via SMAD3 (a TGF-β -activated transcription factor) is essential for proper development of the liver and preventing many liver pathologies. βII-spectrin-deficient mice are highly susceptible to alcohol-induced liver injury, and complete knockout of this gene results in embryonic death with features like fetal alcohol syndrome. Compromised TGF-β signaling through SMAD3 and βII-spectrin dysregulates ALDH1A and ALDH2 genes and disrupts DNA repair, contributing to oncogenesis from elongated telomeres and genome instability. To explore the roles of βII-spectrin and the TGF-β pathway in aldehyde-related and alcohol-related liver injury, we generated Aldh2-/-Sptbn1+/- (ASKO) double-knockout mice, which become obese and develop NASH and characteristics of metabolic syndrome (MetS). Their livers have altered lipid profiles, increased reactive aldehydes, and increased lipid synthesis. Treating Aldh2-/- mice with siRNA targeting βII-spectrin reduces diet- induced lipid accumulation and improves glucose handling. We hypothesize that hepatocyte stress from high lipid levels, reactive aldehydes, such as 4-hydroxynonenol (4-HNE), or inflammation activates caspases that cleave βII-spectrin, which, in turn, promote lipid synthesis by interacting with SREBP1 (sterol regulatory element binding protein1). This process also diverts βII-spectrin from TGF-β-SMAD3 signaling, activating fibrogenic and oncogenic pathways instead. We will explore how cleavage of βII-spectrin and aldehyde-modification of βII- spectrin affect the interactions between βII-spectrin, SMAD3, and SREBP1 by addressing the following specific aims: Aim 1. Determine the hepatocyte-specific roles of βII-spectrin by studying Aldh2-/- mice crossed with liver-specific Sptbn1 knockout mice. Aim 2. Define molecular mechanisms by which the interaction between βII-spectrin and SREBP1 promotes lipogenesis. Aim 3. Assess the translational relevance of SPTBN1 and ALDH2 in human obesity and NASH, and if pathological effects are reduced by siRNA targeting βII-spectrin. The outlined studies are aimed at improving the understanding of diet-induced fatty liver disease in the context of ALDH2 deficiency. The outcomes promise to expose the many intricacies of liver diseases and inform the development of novel intervention strategies for liver-associated diseases, such as MetS, ALD, NAFLD, and NASH.