Molecular Mechanisms of TRIB1 Regulation of Hepatic Metabolism

项目摘要 含有Tribbles假激酶1（TRIB 1）基因的8 q24基因组位点通过以下方式重复连接： 人类全基因组与多个心脏代谢参数关联研究。这包括血浆总量 胆固醇、LDL胆固醇、HDL胆固醇、甘油三酯、冠状动脉疾病（CAD）、循环肝 酶和非酒精性脂肪肝（NAFLD），循环脂联素和HbA 1c。这个星座 在基因组中是独一无二的，这表明TRIB 1是人类基因组的关键调节因子。 代谢健康这些性状中的许多受肝脏代谢、基因敲除和过表达控制 在小鼠肝脏中的研究已经证明Trib 1是肝脏脂质代谢的关键调节剂。尽管 支持这种基因在人类疾病中的作用的遗传学证据和早期观察强调了 肝脏Trib 1的重要性，该基因的机制研究已经滞后。先前在骨髓细胞方面的工作 建立了TRIB 1功能的模型，其中它促进转录的泛素化和降解， 因子C/EBPα，但尚未在肝细胞中探索控制该功能的机制。此外，本发明还 虽然先前的研究表明，Trib 1的肝脏过表达降低了小鼠的血浆脂质， 治疗范例尚未在心脏代谢疾病的临床前模型中进行测试。最后，方向 TRIB 1在人类中的作用仍然未知，阻碍了这种基因的翻译和治疗潜力。 基因及相关通路。我们在这里提出的初步数据表明，肝脏TRIB 1确实促进 转录因子C/EBPα的COP 1依赖性泛素化，这一过程需要一种新的相互作用 用另一种假激酶STK 40此外，我们还建立了多种体内动物模型， 在代谢性疾病动物模型中增加肝脏Trib 1活性的治疗潜力。最后我们 在一个高度新颖的近亲群体中利用全外显子组测序来确定预测的丢失- TRIB 1中的pLoF变体可以帮助确定其在人类中的功能。这项实验计划旨在 通过实现以下具体目标来弥补上述知识差距：1） 确定STK 40如何调节肝脏TRIB 1介导的靶蛋白泛素化; 2）确定 Trib 1过表达在CAD和NAFLD动物模型中的治疗益处，以及 C/EBPα对这些益处的影响;以及3）通过以下方法确定TRIB 1的作用方向和治疗潜力： 携带TRIB 1 pLoF变体的受试者的鉴定和代谢表型分析。完成这些 目的不仅是阐明围绕E3连接酶、假激酶的新生物学， TRIB 1，而且还进一步加深了我们对无数遗传关联的机械基础的理解， 与TRIB 1基因的关系我们的最终目标是深入了解肝脏TRIB 1的功能， 希望这可以为针对血脂异常和由此产生的CAD、脂肪变性和 NAFLD和人类代谢综合征。

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.