Homeostatic role of IRE1a-XBP1-PDI1 in hepatic lipid metabolism

 描述(由申请人提供)：大约20%的美国人患有非酒精性脂肪性肝病(NAFLD)，这种疾病可以发展为肝骨病、纤维化、肝硬变 和肝细胞癌。肝脏分泌的极低密度脂蛋白在调节肝内和血管内脂类平衡方面起着重要作用。肝脏极低密度脂蛋白的过度产生是肥胖和糖尿病中高脂血症的特征，并显著增加了心血管疾病的风险。尽管极低密度脂蛋白分泌异常对人类健康有重大影响，但驱动极低密度脂蛋白聚集的基本细胞机制尚不清楚。由于内质网(ER)中的蛋白质错误折叠，肝脏VLDL的组装和分泌显着减少，而适应性未折叠蛋白反应(UPR)则显着增加。这项拟议的研究将阐明UPR在维持极低密度脂蛋白组装的ER管腔中甘油三酯(TG)水平方面的机制作用。我们将大力检验这一假说，即肝细胞ER蛋白折叠环境的变化激活肌醇需要蛋白1(IRE1)，以剪接XBP1mRNA来响应营养刺激而产生XBP1s，这是非酒精性脂肪肝、异常脂肪分泌和高脂血症的病因中的关键。我们的假设是基于最近关于极低密度脂蛋白组装的三个基本发现。首先，我们已经证明，营养过剩改变了肝细胞中的脂质平衡并激活了UPR。目标1将揭示内质网中代谢压力与蛋白质错误折叠之间的联系机制。目标2和3集中在XBP1s如何在维持内质网脂类平衡方面发挥重要作用。我们已经证明，XBP1为组装富含甘油三酯的极低密度脂蛋白提供了两个独立的功能。首先，XBP1s激活蛋白质二硫键异构酶(Pdi1)的转录，以维持微粒体TG Transfer Protein(MTP)的活性，促进脂质与载脂蛋白B(ApoB)的组装。目的2将研究营养应激如何以依赖PDI1的方式降低MTP活性。目的3将确定第二个机制，其中XBP是必不可少的促进富含甘油三酯的极低密度脂蛋白组装以一种MTP不依赖的方式使用mRNA-Seq和质谱仪相结合的方法。最后，重要的是，我们的数据表明，PDI1促进了ApoB的氧化折叠，从而首次在体内发现了PDI1的蛋白质折叠底物。Aim 4将使用肝细胞特异性Pdi1缺失的小鼠来研究PDI1如何促进ApoB100的氧化折叠并改变与蛋白质折叠环境(即分子伴侣)的相互作用。这项研究的结果将阐明IRE1VBP1/XBP1通过将甘油三酯分配到内质网管腔以进行极低密度脂蛋白组装来维持肝细胞脂质平衡的机制。这些发现将提供对极低密度脂蛋白成熟调控机制的基本了解，这将有助于开发治疗肝骨病和高脂血症的新疗法。

 DESCRIPTION (provided by applicant): Approximately 20% of the US population suffers from non-alcoholic fatty liver disease (NAFLD) that can progress to hepatosteatosis, fibrosis, cirrhosis and hepatocellular carcinoma. Hepatic secretion of VLDL plays an essential role in regulating intrahepatic and intravascular lipid homeostasis. The overproduction of hepatic VLDL characterizes the pathogenesis of hyperlipidemia in obesity and diabetes and contributes significantly to an increased risk of cardiovascular disease. Despite the significant impact of abnormalities in VLDL secretion on human health, fundamental cellular mechanisms that drive VLDL assembly are yet to be understood. Hepatic VLDL assembly and secretion is significantly reduced by protein misfolding in the endoplasmic reticulum (ER) and increased by the adaptive unfolded protein response (UPR). The proposed research will elucidate the mechanistic role of the UPR in maintaining triglyceride (TG) levels in the ER lumen for VLDL assembly. We will vigorously test the hypothesis that changes in the ER protein-folding environment of the hepatocyte activate inositol-requiring kinase1 (IRE1) to splice Xbp1 mRNA to generate XBP1s in response to a nutritional stimulus are key in the etiology of NAFLD and abnormal lipid secretion and hyperlipidemia. Our hypothesis is based on three recent fundamental findings regarding assembly of VLDL. First, we have demonstrated that nutritional excess alters lipid homeostasis and activates the UPR in hepatocytes. Aim 1 will uncover the mechanism that links metabolic stress with protein misfolding in the ER. Aims 2 and 3 focus on how XBP1s is essential role to maintain ER lipid homeostasis. We have demonstrated that XBP1s provides two independent functions for assembly of TG-rich VLDL. First, XBP1s activates transcription of protein disulfide isomerase (Pdi1) to maintain microsomal TG transfer protein (MTP) in an active form to promote lipid assembly with apolipoprotein B (ApoB). Aim 2 will study how nutritional stress reduces MTP activity in a PDI1-dependent manner. Aim 3 will identify the second mechanism by which XBP1s is essential to promote TG-rich VLDL assembly in an MTP-independent manner using a combination of mRNA-Seq and mass spectrometry approaches. Finally, and significantly, our data show that PDI1 promotes oxidative folding of ApoB, thus for the first time uncovering a protein folding substrate for PDI1 in vivo. Aim 4 will use hepatocyte-specific Pdi1-null mice to study how PDI1 promotes ApoB100 oxidative folding and alters interactions with the protein-folding environment (i.e., molecular chaperones). Results from the proposed studies will elucidate the mechanisms by which IRE1/XBP1 maintains hepatocyte lipid homeostasis by partitioning TG into the ER lumen for VLDL assembly. The findings will provide fundamental understanding of mechanisms that govern VLDL maturation, which will facilitate the development of novel therapeutic approaches to treat hepatosteatosis and hyperlipidemia.