Molecular Regulation of Apoprotein B Degradation

血浆载脂蛋白B(ApoB)水平是冠心病的最强危险因素之一；因此， 了解载脂蛋白B-脂蛋白产生的调节从根本上讲是有趣的，具有临床意义，而且 可能最终会提出治疗血脂异常的新方法。ApoB100是apoB的形式，由 是致动脉粥样硬化的极低密度脂蛋白(VLDL)和低密度脂蛋白(LDL)的主要蛋白质成分 脂蛋白。肝细胞分泌apoB100主要受分泌前降解的控制。我们 一直站在定义apoB100降解途径的前沿，在本提案中重点关注1) 内质网相关降解(ERAD)，它由蛋白酶体介导；以及，2)后 Er，视网膜前蛋白分解(PERPP)，由临床上用于降低极低密度脂蛋白的饮食脂肪酸刺激 水平，我们认为这是由自噬介导的。在目标1中，我们建议描述这一过程 ERAD的目标是新生的载脂蛋白B。我们以前已经表明，这一过程需要一组不同的 伴侣和伴侣样蛋白。我们将继续确定控制载脂蛋白B生物发生的因素 ER的体外和体内检测，以及一种新的apoB酵母表达系统。在目标2中，我们 提出在基础代谢和受扰代谢下确定自噬在ER后apoB100转换中的作用 各州。根据我们最近的数据，我们假设自噬调节apoB100在 基础条件和肝细胞与鱼油脂肪酸(如DHA)或其他 降低极低密度脂蛋白水平的多不饱和脂肪酸(PUFA)。为了解决这个假设，我们将研究apoB100 肝细胞和特定自噬因子活性已被激活的小鼠的周转 被操纵了。此外，基于胰岛素介导的载脂蛋白B降解的特点，我们还将测试 假设自噬和胰岛素反应通路相交来调节apoB100的新陈代谢。在……里面 目的3.确定导致载脂蛋白B100降解的氧化剂(S)以及反应性的影响 极低密度脂蛋白组装过程中的氧物种(ROS)。当肝细胞与DHA和其他 多不饱和脂肪酸、ROS和脂质过氧化增加，apoB100受损、聚集和靶向 自噬。根据我们的初步数据，我们假设超氧化物(SO)在 ApoB100聚集/降解。我们将在肝细胞和携带基因的小鼠身上验证这一假设。 氧化途径的改变。其他初步数据表明，多不饱和脂肪酸完全减少了 高尔基体内成熟的极低密度脂蛋白颗粒。通过将脉冲追逐研究与亚细胞分离和脂质分离相结合 分析技术，我们将确定这是由于极低密度脂蛋白组装中止还是由于 将极低密度脂蛋白定位于组装后的自噬。总而言之，这些研究将导致更详细的 对载脂蛋白B100细胞内代谢的分子理解将有助于 方法控制正常和病理状态下致动脉粥样硬化性脂蛋白的产生。

The plasma level of apoprotein B (apoB) is among the strongest risk factors for coronary artery disease; thus, understanding the regulation of apoB-lipoprotein production is fundamentally interesting, clinically relevant, and may ultimately suggest new therapeutic approaches to dyslipidemias. ApoB100 is the form of apoB made by human liver and is the predominant protein component of atherogenic very low (VLDL) and low density (LDL) lipoproteins. Secretion of apoB100 from hepatic cells is controlled primarily by pre-secretory degradation. We have been at the forefront of defining apoB100 degradative pathways, and in this proposal focus on 1) endoplasmic reticulum-associated degradation (ERAD), which is mediated by the proteasome; and, 2) post- ER, presecretory proteolysis (PERPP), which is stimulated by dietary fatty acids used clinically to reduce VLDL levels, and which we believe is mediated by autophagy. In aim 1, we propose to characterize the process that targets nascent apoB for ERAD. We have previously shown that this process requires a distinct cohort of chaperones and chaperone-like proteins. We will continue to identify the factors that control apoB biogenesis in the ER using in vitro and in vivo assays, as well as a new apoB yeast expression system. In aim 2, we propose to determine the role of autophagy in post-ER apoB100 turnover under basal and perturbed metabolic states. Based on our recent data, we hypothesize that autophagy regulates the degradation of apoB100 under basal conditions and when hepatic cells are incubated with fish oil fatty acids (such as DHA) or other polyunsaturated fatty acids (PUFA) that lower VLDL levels. To address this hypothesis, we will study apoB100 turnover in hepatic cells and in mice in which the activities of specific autophagic factors have been manipulated. Further, based on the characteristics of insulin-mediated apoB degradation, we will also test the hypothesis that autophagy and the insulin-responsive pathways intersect to modulate apoB100 metabolism. In aim 3, we propose to determine the oxidant(s) responsible for apoB100 degradation and the effects of reactive oxygen species (ROS) on the VLDL assembly process. When hepatic cells are incubated with DHA and other PUFA, ROS and lipid peroxidation increase and apoB100 becomes damaged, aggregated, and targeted for autophagy. Based on our preliminary data, we hypothesize that superoxide (SO) plays a central role in apoB100 aggregation/degradation. We will test this hypothesis in hepatic cells and mice with genetic alterations in oxidant pathways. Other preliminary data suggest that PUFA decrease the amount of fully matured VLDL particles in the Golgi. By combining pulse-chase studies with sub-cellular fractionation and lipid analytical techniques, we will determine whether this results from aborted VLDL assembly or from the rapid targeting of VLDL to autophagy after assembly. In summary, these studies will lead to a more detailed molecular understanding of the intracellular metabolism of apoB100 and will contribute to the development of methods to control the production of atherogenic lipoproteins in normal and pathological states.