Novel regulatory mechanisms controlling hepatic apoB-Lp lipid loading and secretion

该PPG应用的总体假设是脂肪、肝脏和血管内 脂解调节致动脉粥样硬化的含apoB的脂蛋白，最显著的是VLDL的生物发生。计划2 (P2)主要关注肝细胞中apoB/VLDL生成的控制机制。PI的既往工作， 第一委员会已出版了14份出版物，确定了一个主要的机制， apoB/VLDL是apoB在内质网（ER）中的调节性降解。这种新陈代谢 协调的事件需要ER相关降解（ERAD）途径，该途径被命名为 由Co-I解释。在低脂条件下，apoB的ERAD开始于其被分子选择， 分子伴侣，随后是apoB泛素化并递送至细胞质蛋白酶体。与此相反， 当中性脂质（主要是三酰甘油）过量时，载脂蛋白B被MTP共同脂化， 新生apoB/VLDL颗粒被来自ER驻留脂滴（LD）的脂质扩增， 模型化的apoB/VLDL颗粒被包装到COPII囊泡中，用于递送到高尔基体，然后进入循环。 相反，调节与apoB组装的脂质的数量和组成的事件以及 将充胀的、富含脂质的apoB/VLDL颗粒包装到COPII囊泡中的特性很差。对这些 PI和他的同事产生的结果表明，两个因素，ER驻留蛋白KLHL 12和 FIT 2在控制脂质组装到apoB上和apoB/VLDL包封到apoB/VLDL中中起重要作用。 COPII囊泡在体外和体内。这些最近获得的数据支持了几个新的假设：首先， KLHL 12位于ER上的apoB出口位点，包括COPII组分，其次，低效的 apoB整合到COPII囊泡中为最近表征的ER-吞噬途径选择蛋白质， 这也是由Co-I阐明的。一种新的肝脏特异性KLHL 12基因敲除小鼠的特征将 用于确定KLHL 12在非酒精性脂肪性肝病和脂肪性肝炎中的作用。另一方面，最近 获得的初步数据表明，FIT 2缺乏增加ER膜脂质含量沿着， 脂质耗尽的apoB/VLDL的产生。基于FIT 2在形成细胞溶质LD中的功能，这些数据 强调了另一个新的假设，即FIT 2将LD递送到ER中，然后将其整合到ER中。 前apoB/VLDL颗粒以MTP依赖性或非依赖性方式。一种新的肝脏特异性FIT 2基因敲除 小鼠将同时允许研究FIT 2与脂肪肝疾病和脂肪性肝炎之间的联系， 以及动脉粥样硬化（因为FIT 2调节apoB/VLDL脂质含量和组成）。广义上讲是 本申请的具体目的是：（1）确定KLHL 12依赖性调节的潜在机制， 富含脂质的apoB/VLDL包封到肝ER衍生的高尔基体定向转运囊泡中，和（2）为了定义 FIT 2对载脂蛋白B/极低密度脂蛋白颗粒的脂质负荷及其致动脉粥样硬化性的影响，这两者都需要关键的 与P1、P3和核心设施合作。

The global hypothesis underlying this PPG application is that crosstalk between adipose, liver, and intravascular lipolysis regulates the biogenesis of atherogenic apoB-containing lipoproteins, most notably VLDLs. Project 2 (P2) focuses on mechanisms that control the generation of apoB/VLDL in liver cells. Prior work from the PI and Co-I, which has resulted in 14 publications, established that a major mechanism controlling circulating apoB/VLDL is the regulated degradation of apoB in the endoplasmic reticulum (ER). This metabolically orchestrated event requires the ER-associated degradation (ERAD) pathway, which was named and first elucidated by the Co-I. Under lipid-poor conditions, the ERAD of apoB begins with its selection by molecular chaperones, which is followed by apoB ubiquitinylation and delivery to the cytoplasmic proteasome. In contrast, when neutral lipids (primarily triacylglycerols) are in excess, apoB is co-translationally lipidated by MTP, and the nascent apoB/VLDL particles are expanded by lipids sourced from ER resident lipid droplets (LDs), and the re- modelled apoB/VLDL particle is packaged into COPII vesicles for delivery to the Golgi and then into circulation. In contrast, the events that regulate the number and composition of lipids assembled with apoB—as well as the packaging of engorged, lipid-rich apoB/VLDL particles into COPII vesicles—are poorly characterized. To these ends, results generated by the PI and his colleagues indicate that two factors, ER-resident proteins KLHL12 and FIT2, play a significant role in controlling lipid assembly onto apoB and the encapsulation of apoB/VLDL into COPII vesicles in vitro and in vivo. These recently acquired data support several new hypotheses: First, that KLHL12 resides at apoB exit sites on the ER that include COPII components, and second that the inefficient integration of apoB into COPII vesicles selects the protein for the recently characterized ER-phagy pathway, which was also elucidated by the Co-I. A characterization of a new liver-specific KLHL12 knockout mouse will be used to define the role of KLHL12 in non-alcoholic fatty liver disease and steatohepatitis. In turn, other recently acquired preliminary data indicate that FIT2 deficiency increases ER membrane lipid content along with the generation of lipid-depleted apoB/VLDLs. Based on the function of FIT2 in forming cytosolic LDs, these data underscore another novel hypothesis, that FIT2 delivers LDs into the ER, where they are then integrated into pre-apoB/VLDL particles in either an MTP-dependent or independent manner. A new liver-specific FIT2 knockout mouse will concomitantly allow for a study of the links between FIT2 and fatty liver disease and steatohepatitis, and also atherosclerosis (because of FIT2 regulation of apoB/VLDL lipid content and composition). Broadly, the Specific Aims of this application are: (1) To define the mechanisms underlying KLHL12-dependent regulation of lipid-rich apoB/VLDL encapsulation into hepatic ER-derived, Golgi-directed transport vesicles, and (2) To define the impact of FIT2 on lipid loading of apoB/VLDL particles and their atherogenicity, both of which require critical collaborations with P1, P3, and Core facilities.