Interrogation of novel pathways regulating VLDL production and plasma lipids

描述（由申请人提供）：含有载脂蛋白B （apoB-LP）的脂蛋白是冠状动脉疾病（CAD）的重要因果危险因素。apoB-LP升高的最常见原因是肝脏VLDL的过量产生，但调节肝脏VLDL组装和分泌的分子过程仍不完全清楚。人类遗传学已经确定了以前未被怀疑的基因，这些基因与血浆载脂蛋白脂蛋白水平以及CAD密切相关。与apoB-LP和CAD相关的两个最引人注目的新基因是SORT1和TRIB1，前者编码一种名为sortilin的蛋白质，后者编码一种名为tribles -1的蛋白质。我们最近发表的关于SORT1位点的研究表明，小鼠肝脏中sortilin的过表达会减少VLDL分泌和LDL-C水平，而sortilin的敲低会增加VLDL分泌和LDL-C水平。同时，我们研究了TRIB1位点，发现tribles -1的过表达减少了VLDL的分泌，而敲除TRIB1的小鼠则增加了VLDL的分泌。因此，这两个基因座编码调节肝脏VLDL生成的蛋白质。然而，它们影响这一复杂过程的分子机制尚不清楚。本提案的目标是进行一系列详细的实验，旨在阐明这些蛋白质调节肝脏VLDL组装和分泌的分子和细胞机制。关于sortilin，我们将验证sortilin作为一种分选蛋白的假设，将新生的VLDL从高尔基体结合并分选到溶酶体，将其从分泌途径转移。关于tribles -1，我们将测试tribles -1调节肝细胞MAP激酶活性的假设，以及tribles -1靶向C/EBP1和/或2在肝细胞中进行翻译后降解的假设。我们将在肝细胞培养系统中进行实验，以及在sortilin和tribles -1功能获得和丧失的小鼠中进行实验。此外，我们将测试SORT1和TRIB1中自然发生的突变的功能影响。成功完成上述目标有望产生关于调节VLDL产生和人类血浆脂质的新生物学途径的新见解，并可能为降低VLDL产生和心血管风险提供新的治疗靶点。

DESCRIPTION (provided by applicant): Lipoproteins containing apolipoprotein B (apoB-LP) are important causal risk factors for coronary artery disease (CAD). The most common cause of elevated apoB-LP is overproduction by the liver of VLDL, but the molecular processes regulating hepatic VLDL assembly and secretion remain incompletely understood. Human genetics has identified previously unsuspected genes that are strongly associated with plasma levels of apoB-LP as well as with CAD. Two of the most compelling new genes associated with apoB-LP and CAD are SORT1, encoding a protein called sortilin, and TRIB1, encoding a protein known as tribbles-1. We recently published studies of the SORT1 locus, demonstrating in mice that overexpression of sortilin in the liver reduces VLDL secretion and LDL-C levels whereas knockdown of sortilin increases VLDL secretion and LDL-C levels. In parallel, we investigated the TRIB1 locus, and found that overexpression of tribbles-1 reduced VLDL secretion and Trib1 knockout mice had increased VLDL secretion. Thus, both of these loci encode proteins that regulate the hepatic production of VLDL. However, the molecular mechanisms by which they influence this complex process remain unknown. The goal of this proposal is to perform a series of detailed experiments intended to elucidate the molecular and cellular mechanisms by which these proteins modulate hepatic VLDL assembly and secretion. With regard to sortilin, we will test the hypothesis that sortilin acts as a sorting protein to bind and sort nascent VLDL from Golgi to lysosome, diverting it from the secretory pathway. With regard to tribbles-1, we will test the hypotheses that tribbles-1 regulates MAP kinase activity in hepatocytes, and that tribbles-1 targets C/EBP1 and/or 2 for post-translational degradation in hepatocytes. We will perform experiments in hepatocyte cell culture systems, as well as in mice with gain and loss of function of sortilin and tribbles-1. In addition, we will test naturally-occurring mutations in SORT1 and TRIB1 for functional effects. Successful completion of the proposed aims promises to yield novel insights regarding new biological pathways regulating VLDL production and thus plasma lipids in humans, and could provide novel therapeutic targets for reducing VLDL production and cardiovascular risk.