Molecular Regulation of Apoprotein B Degradation

描述(由申请人提供)：血浆载脂蛋白B(ApoB)水平是冠状动脉疾病的最大危险因素之一；因此，了解apoB-脂蛋白产生的调节从根本上是有趣的，具有临床意义，并最终可能建议新的治疗方法来治疗血脂异常。载脂蛋白B100是由人类肝脏合成的载脂蛋白B，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)的主要蛋白质组分。肝细胞分泌apoB100主要受分泌前降解的控制。我们一直处于定义apoB100降解途径的前沿，在这项提议中，我们重点关注1)内质网相关降解(ERAD)，它由蛋白酶体介导；以及2)ER后，视网膜前蛋白分解(PERPP)，它是由临床上用来降低VLDL水平的饮食脂肪酸刺激的，我们认为它是由自噬介导的。在目标1中，我们建议描述ERAD针对新生载脂蛋白B的过程。我们以前已经证明，这个过程需要一组不同的伴侣和伴侣样蛋白。我们将继续使用体外和体内试验来确定控制载脂蛋白B在ER中生物生成的因素，以及一个新的载脂蛋白B酵母表达系统。在目标2中，我们建议确定自噬在基础代谢状态和扰动代谢状态下ER后apoB100周转中的作用。根据我们最近的数据，我们假设，在基础条件下以及当肝细胞与鱼油脂肪酸(如DHA)或其他降低VLDL水平的多不饱和脂肪酸(PUFA)孵育时，自噬调节apoB100的降解。为了解决这一假设，我们将研究apoB100在肝细胞和特定自噬因子活动已被操纵的小鼠中的周转。此外，基于胰岛素介导的载脂蛋白B降解的特点，我们还将检验自噬和胰岛素反应途径相交来调节载脂蛋白B100代谢的假设。在目标3中，我们建议确定负责降解载脂蛋白B100的氧化剂(S)以及活性氧物种(ROS)对极低密度脂蛋白组装过程的影响。当肝细胞与DHA和其他多不饱和脂肪酸孵育时，ROS和脂质过氧化增加，apoB100受损、聚集，并成为自噬的靶点。根据我们的初步数据，我们假设超氧化物(SO)在apoB100聚集/降解中起核心作用。我们将在肝细胞和氧化途径发生遗传变化的小鼠身上验证这一假设。其他初步数据表明，多不饱和脂肪酸减少了高尔基体中完全成熟的极低密度脂蛋白颗粒的数量。通过将脉冲追逐研究与亚细胞分离和脂质分析技术相结合，我们将确定这是由于VLDL组装失败还是由于VLDL组装后快速靶向自噬所致。综上所述，这些研究将导致对apoB100细胞内代谢的更详细的分子理解，并将有助于开发方法来控制正常和病理状态下致动脉粥样硬化脂蛋白的产生。公共卫生相关性：动脉粥样硬化是由含有胆固醇的颗粒(脂蛋白)积累引起的。这些颗粒的主要来源是肝脏，它将胆固醇与一种蛋白质apoB组装在一起，apoB是颗粒形成和离开肝脏所必需的。我们已经确定了肝脏可以破坏载脂蛋白B的方法，从而减少导致动脉粥样硬化的颗粒释放到血流中。该提案的主要目标是发现调节这种破坏的分子，以便最终能够开发出降低心脏病风险的治疗新靶点。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Atherosclerosis is caused by the accumulation of particles (lipoproteins) containing cholesterol. The major source of these particles is the liver, which assembles the cholesterol with a protein, apoB, which is absolutely required for formation of and exit from the liver of the particles. We have identified ways by which the liver can destroy apoB and thereby reduce the release of the atherosclerosis-causing particles into the blood stream. The proposal's main goal is to uncover the molecules that regulate this destruction, so that ultimately, new targets for therapy to lower heart disease risk can be developed.