PROJECT SUMMARY The 8q24 genomic locus, containing the gene Tribbles pseudokinase 1 (TRIB1) has been repeatedly linked via human genome-wide association study with multiple cardiometabolic parameters. This includes plasma total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, coronary artery disease (CAD), circulating liver enzymes and non-alcoholic fatty liver disease (NAFLD), circulating adiponectin, and HbA1c. Despite the genetic evidence supporting a role for this gene in human disease and early observations underscoring the importance of hepatic Trib1, mechanistic studies of this gene have lagged. In the parent application, we presented preliminary data demonstrating that hepatic TRIB1 promotes COP1-dependent ubiquitination of the transcription factor C/EBPα, and this process requires a novel interaction with a different pseudokinase, STK40. Additionally, we established multiple in vivo animal models to test the therapeutic potential of increased hepatic Trib1 activity in animal models of metabolic disease. Finally, we utilized whole exome sequencing in a highly novel consanguineous population to identify predicted loss-of-function (pLoF) variants in TRIB1 that can help determine its function in humans. This parent award experimental plan aims to address the following specific aims: 1) To determine how hepatic TRIB1-mediated ubiquitination of target proteins is regulated by STK40; 2) To determine the therapeutic benefit of Trib1 overexpression in animal models of CAD and NAFLD, and the requirement for C/EBPα for such benefits; and 3) To determine the direction of effect and therapeutic potential for TRIB1 through the identification and metabolic phenotyping of subjects harboring TRIB1 pLoF variants. This diversity supplement expands the scope of aim 3 from the parent award by investigating pLoF variants identified in CEBPA. As this is the major downstream target of TRIB1, the identification of pLoF C/EBPα proteins can help elucidate the function of the TRIB1-C/EBPα hepatic regulatory axis in human physiology and disease. During this diversity supplement, the candidate will test the functional effects of pLoF CEBPA variants via the following in vitro and in vivo techniques: 1) generate recombinant mutant CEBPA cDNA constructs containing rare pLoF mutations and test their ability to activate a custom C/EBPα-responsive luciferase reporter, and 2) generate AAV plasmid constructs of mutant CEBPA to test the ability of these to induce steatosis in wild-type mice and rescue the phenotype of mice with hepatic Cebpa deletion. The completion of these studies will add to our understanding of the role of the TRIB1-C/EBPα regulatory axis in human hepatic metabolism and help elucidate the therapeutic potential of TRIB1. These studies will also further our understanding of the mechanistic underpinnings of the myriad genetic associations with the TRIB1 gene in humans. Our ultimate goal is an in depth understanding of the functions of hepatic TR...