Molecular Regulation of Apoprotein B Degradation

描述（由申请方提供）：载脂蛋白B（apo B）的血浆水平是冠状动脉疾病最强的风险因素之一;因此，了解apo B-脂蛋白产生的调节是非常有趣的，具有临床相关性，并可能最终为血脂异常提供新的治疗方法。ApoB 100是由人类肝脏产生的apoB的形式，并且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组分。apoB 100从肝细胞的分泌主要受分泌前降解的控制。我们一直处于定义apoB 100降解途径的最前沿，在本提案中，重点关注1）内质网相关降解（ERAD），由蛋白酶体介导;和2）ER后分泌前蛋白水解（PERPP），由临床上用于降低VLDL水平的膳食脂肪酸刺激，我们认为是由自噬介导的。在目标1中，我们建议描述ERAD靶向新生apoB的过程。我们以前已经表明，这一过程需要一个独特的伴侣蛋白和伴侣蛋白样蛋白的队列。我们将继续确定的因素，控制载脂蛋白B的生物合成在ER使用体外和体内试验，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和扰动代谢状态下ER后apoB 100周转中的作用。根据我们最近的数据，我们假设自噬调节apoB 100的降解在基础条件下，当肝细胞与鱼油脂肪酸（如DHA）或其他多不饱和脂肪酸（PUFA），降低VLDL水平孵育。为了解决这一假设，我们将研究载脂蛋白B100的营业额在肝细胞和小鼠中的特定自噬因子的活动已被操纵。此外，基于胰岛素介导的apoB降解的特征，我们还将检验自噬和胰岛素应答途径交叉调节apoB 100代谢的假设。在目标3中，我们建议确定负责apoB 100降解的氧化剂和活性氧（ROS）对VLDL组装过程的影响。当肝细胞与DHA和其他PUFA一起孵育时，ROS和脂质过氧化增加，apoB 100受损，聚集并成为自噬的目标。基于我们的初步数据，我们假设超氧化物（SO）在apoB 100聚集/降解中起着核心作用。我们将在肝细胞和氧化途径发生遗传改变的小鼠中检验这一假设。其他初步数据表明，PUFA减少高尔基体中完全成熟的VLDL颗粒的量。通过将脉冲追踪研究与亚细胞分级分离和脂质分析技术相结合，我们将确定这是否是由于VLDL组装失败或组装后VLDL快速靶向自噬的结果。总之，这些研究将导致一个更详细的了解apoB 100的细胞内代谢的分子，并将有助于控制在正常和病理状态下的致动脉粥样硬化脂蛋白的生产方法的发展。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，肝脏将胆固醇与蛋白质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，从而减少动脉粥样硬化引起的颗粒释放到血流中的方法。该提案的主要目标是揭示调节这种破坏的分子，以便最终开发出降低心脏病风险的新靶点。

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.