Project Summary Alcohol-associated liver disease (ALD) is a complex disorder and developed as an adverse consequence of excessive alcohol use. It is a major public health problem among veterans and a leading cause of chronic liver diseases and liver transplantation in the US. Mitochondria-associated ER membranes (MAM) are the site where mitochondria and endoplasmic reticulum (ER) form contact points; it is a signaling hub enriched with a wide range of proteins. One of its crucial functions is to regulate Ca2+ transport from the ER to mitochondria to support mitochondrial metabolism. In the MAM, Ca2+ transfer is regulated by a formation of MAM Ca2+- channeling complex, consisting of the chaperone glucose-regulated protein 75 (GRP75). GRP75 acts as a tether between ER and mitochondria by physically binding with ER membrane Ca2+ efflux channel, Inositol-1- tri-phosphate receptor 1 (IP3R1), and outer mitochondrial membrane protein, Voltage-dependent anion channel 1 (VDAC1). The role of MAM in ALD pathogenesis has not been explored. We found that hepatic pyruvate dehydrogenase kinase 4 (PDK4), one of the MAM-associated genes, was significantly upregulated in ethanol-fed mice and patients with ALD. Our overarching goal is to further understand the mechanistic action of the PDK4 and the MAM axis in mediating alcohol-induced liver injury. In the first aim, we will determine the effect of hepatic PDK4 and MAM Ca2+-channeling complex formation in alcohol-associated liver disease. Subsequently, in the second aim, we proposed a mechanistic study to determine the molecular mechanism of PDK4 on MAM Ca2+-channeling complex proteins in the pathogenesis of alcohol-associated liver disease. This is based on our novel preliminary data that PDK4 modulates the MCC complex via the phosphorylation of GRP75. In the last aim, we proposed several translational experiments to determine if PDK4 deficiency ameliorates ethanol-induced liver injury via suppression of the MAM formation. Taken together, we have developed a mechanistic proposal to determine the role of PDK4 and MAM in ALD pathogenesis. Our proposed experiments also open an avenue for the future therapy of ALD by employing PDK4 as its target; illustrating the clinical applicability of our project.