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

支持这种PPG应用的全球假设是脂肪、肝脏和血管内之间的串扰 脂解作用调节致动脉粥样硬化的载脂蛋白B的生物发生，最显著的是极低密度脂蛋白。项目2 (P2)侧重于控制肝细胞中apoB/VLDL产生的机制。PI和之前的工作 已经出版了14份出版物的CO-I建立了一个控制流通的主要机制 载脂蛋白B/极低密度脂蛋白是载脂蛋白B在内质网(ER)中的受控降解。这在新陈代谢方面 精心编排的活动需要内质网相关降解(ERAD)途径，这是第一个命名的 由Co-I澄清。在低脂条件下，apoB的ERAD从分子选择开始 伴侣蛋白，随后是apoB泛素化和递送到细胞质蛋白酶体。相比之下， 当中性脂类(主要是三酰甘油)过多时，apoB被MTP共翻译为脂类，而 新生的载脂蛋白B/极低密度脂蛋白颗粒被内质网驻留脂滴(LDS)来源的脂类膨胀，而Re-Re。 模拟的载脂蛋白B/极低密度脂蛋白颗粒被包装成COPII囊泡，然后输送到高尔基体，然后进入循环。 相比之下，调节与载脂蛋白B组装的脂类的数量和组成的事件-以及 将充血的、富含脂质的载脂蛋白B/极低密度脂蛋白颗粒包装到COPII囊泡中的特征很差。对这些人 最后，Pi和他的同事产生的结果表明有两个因素，内质网驻留蛋白KLHL12和 FIT2在控制载脂蛋白B上的脂质聚集和载脂蛋白/极低密度脂蛋白的包埋中起重要作用 COPII囊泡的体外和体内实验。这些最近获得的数据支持几个新的假设：第一， KLHL12驻留在ER上的apoB出口位置，包括COPII组件，其次是效率低下的 将apoB整合到COPII囊泡中选择最近表征的ER吞噬途径的蛋白质， 这一点也得到了Co-I的澄清。一种新的肝脏特异性KLHL12基因敲除小鼠Will的特征 用于确定KLHL12在非酒精性脂肪性肝病和脂肪性肝炎中的作用。反过来，最近的其他 已获得的初步数据表明，FIT2缺乏会增加ER膜脂含量，并伴随着 产生耗尽脂质的apoB/VLDL。基于FIT2在胞质LDS形成中的作用，这些数据 强调另一个新的假设，FIT2将LD传递到内质网，然后它们被整合到内质网中 前载脂蛋白B/极低密度脂蛋白颗粒以MTP依赖或独立的方式表达。一种新的肝脏特异性FIT2基因敲除 老鼠将同时允许研究FIT2与脂肪性肝病和脂肪性肝炎之间的联系， 动脉粥样硬化(由于FIT2调节载脂蛋白B/极低密度脂蛋白的脂质含量和组成)。大体上说， 本申请的具体目的是：(1)定义KLHL12依赖的调节机制 将富含脂质的apoB/VLDL包裹到肝脏内质网衍生的高尔基体定向转运囊泡中，以及(2)确定 FIT2对载脂蛋白B/极低密度脂蛋白颗粒脂质负荷及其致动脉粥样硬化的影响 与P1、P3和核心设施的协作。

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.