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

PPG 应用的总体假设是脂肪、肝脏和血管内的细胞之间的串扰 脂肪分解调节含 apoB 的致动脉粥样硬化脂蛋白的生物发生，尤其是 VLDL。项目2 (P2) 重点关注肝细胞中控制 apoB/VLDL 生成的机制。 PI 之前的工作和 Co-I 已发表 14 份出版物，建立了控制流通的主要机制 apoB/VLDL 是 apoB 在内质网 (ER) 中受调节的降解。这在代谢上 精心策划的事件需要 ER 相关降解 (ERAD) 途径，该途径最初被命名 由 Co-I 阐明。在脂质匮乏的条件下，apoB 的 ERAD 从分子选择开始 分子伴侣，随后进行 apoB 泛素化并递送至细胞质蛋白酶体。相比之下， 当中性脂质（主要是三酰甘油）过量时，apoB 会被 MTP 共翻译脂质化，并且 新生的 apoB/VLDL 颗粒通过来自 ER 驻留脂滴 (LD) 的脂质进行扩展，并且重新 模型化的 apoB/VLDL 颗粒被包装到 COPII 囊泡中，输送到高尔基体，然后进入循环。 相比之下，调节与 apoB 组装的脂质数量和组成的事件以及 将充盈的富含脂质的 apoB/VLDL 颗粒包装到 COPII 囊泡中的过程尚不清楚。对这些 最后，PI 和他的同事产生的结果表明，两个因素，ER 驻留蛋白 KLHL12 和 FIT2 在控制脂质组装到 apoB 以及将 apoB/VLDL 封装到 COPII 囊泡在体外和体内。这些最近获得的数据支持几个新假设：首先， KLHL12 位于 ER 上的 apoB 出口位点，其中包括 COPII 成分，其次是低效的 将 apoB 整合到 COPII 囊泡中，为最近表征的 ER 吞噬途径选择了蛋白质， Co-I 也对此进行了阐述。新型肝脏特异性 KLHL12 敲除小鼠的表征将 用于定义 KLHL12 在非酒精性脂肪肝和脂肪性肝炎中的作用。反过来，其他最近 获得的初步数据表明，FIT2 缺乏会增加内质网膜脂含量，同时也会增加内质网膜脂含量。 产生脂质耗尽的 apoB/VLDL。基于 FIT2 在形成胞质 LD 中的功能，这些数据 强调了另一个新颖的假设，即 FIT2 将 LD 传递到 ER 中，然后将它们整合到 前apoB/VLDL颗粒以MTP依赖或独立的方式。一种新的肝脏特异性 FIT2 敲除方法 小鼠将同时允许研究 FIT2 与脂肪肝疾病和脂肪性肝炎之间的联系， 还有动脉粥样硬化（因为 FIT2 调节 apoB/VLDL 脂质含量和组成）。从广义上讲， 本申请的具体目标是：(1) 定义 KLHL12 依赖性调节的潜在机制 富含脂质的 apoB/VLDL 封装到肝 ER 衍生的、高尔基定向的转运囊泡中，以及 (2) 定义 FIT2 对 apoB/VLDL 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 与 P1、P3 和核心设施的合作。

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.