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强危险因素之一；因此，了解载脂蛋白产生的调控从根本上是有趣的，具有临床意义，并可能最终为血脂异常提供新的治疗方法。载脂蛋白100是人类肝脏产生的载脂蛋白ob的一种形式，是致动脉粥样硬化极低（VLDL）和低密度（LDL）脂蛋白的主要蛋白质成分。肝细胞分泌apoB100主要受分泌前降解控制。我们一直处于定义apoB100降解途径的前沿，并在本提案中重点关注1)内质网相关降解（ERAD），这是由蛋白酶体介导的；2)内质网后，分泌前蛋白水解（PERPP），这是由膳食脂肪酸刺激的，临床上用于降低VLDL水平，我们认为这是由自噬介导的。在目标1中，我们建议描述针对ERAD的新生载脂蛋白的过程。我们之前已经证明，这个过程需要一组独特的伴侣蛋白和伴侣蛋白样蛋白。我们将继续通过体外和体内实验，以及新的载脂蛋白酵母表达系统，确定控制内质网中载脂蛋白生物发生的因素。在目的2中，我们提出确定自噬在基础和紊乱代谢状态下er后apoB100转换中的作用。根据我们最近的数据，我们假设在基础条件下，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，我们将研究肝细胞和小鼠中apoB100的周转，其中特定自噬因子的活性已被操纵。此外，基于胰岛素介导的载脂蛋白ob降解的特点，我们还将验证自噬和胰岛素反应途径交叉调节载脂蛋白100代谢的假设。在目标3中，我们建议确定负责apoB100降解的氧化剂以及活性氧（ROS）对VLDL组装过程的影响。当肝细胞与DHA和其他PUFA孵育时，ROS和脂质过氧化作用增加，apoB100被破坏、聚集并靶向自噬。基于我们的初步数据，我们假设超氧化物（SO）在apoB100聚集/降解中起核心作用。我们将在氧化途径发生遗传改变的肝细胞和小鼠中测试这一假设。其他初步数据表明，PUFA减少了高尔基体中完全成熟的VLDL颗粒的数量。通过将脉冲追踪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组装失败还是由于组装后VLDL快速靶向自噬造成的。总之，这些研究将导致对apoB100细胞内代谢的更详细的分子理解，并将有助于在正常和病理状态下控制动脉粥样硬化脂蛋白产生的方法的发展。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）积聚引起的。这些颗粒的主要来源是肝脏，它将胆固醇与一种蛋白质载脂蛋白ob组装在一起，载脂蛋白ob对于颗粒的形成和从肝脏排出是绝对必需的。我们已经确定了肝脏破坏载脂蛋白b的方法，从而减少导致动脉粥样硬化的颗粒释放到血液中。该提案的主要目标是发现调节这种破坏的分子，从而最终开发出降低心脏病风险的新治疗靶点。

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.