Molecular Mechanisms of TRIB1 Regulation of Hepatic Metabolism

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. This constellation of genetic associations is unique in the genome, and suggests that TRIB1 is a critical regulator of human metabolic health. Many of these traits are governed by hepatic metabolism, and knockout and overexpression studies in mouse livers have demonstrated that Trib1 is a critical regulator of hepatic lipid metabolism. 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. Prior work in myeloid cells has established a model of TRIB1 function wherein it promotes the ubiquitination and degradation of the transcription factor C/EBPα, but the mechanisms governing this function have not been explored in hepatocytes. Additionally, while previous work demonstrated that hepatic overexpression of Trib1 reduces plasma lipids in mice, this therapeutic paradigm has not been tested in preclinical models of cardiometabolic disease. Finally, the direction of effect of TRIB1 in humans remains unknown, hampering the translational and therapeutic potential of this gene and related pathways. We present here preliminary data demonstrating that hepatic TRIB1 does promote COP1-dependent ubiquitination of the transcription factor C/EBPα, and this process requires a novel interaction with a different pseudokinase, STK40. Additionally, we have established multiple in vivo animal models to test the therapeutic potential of increased hepatic Trib1 activity in animal models of metabolic disease. Finally, we have 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 experimental plan aims to address the above outlined knowledge gaps through accomplishment of 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. Completion of these aims will not only elucidate novel biology surrounding E3 ligases, pseudokinases, and the therapeutic potential of TRIB1, but 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 TRIB1 with the hope that this can inform therapeutic strategies targeting dyslipidemia and resultant CAD, steatosis and NAFLD, and metabolic syndrome in humans.