Molecular Regulation of Apoprotein B Degradation

描述（由申请人提供）：这是一份更新申请，旨在继续研究载脂蛋白B （apoB）降解的分子调控。载脂蛋白ob是致动脉粥样硬化脂蛋白的主要蛋白质成分。因此，了解载脂蛋白的组装和分泌的调节不仅具有根本的意义，而且具有医学意义。肝脏细胞净分泌载脂蛋白ob的一个重要控制因素是其分泌前降解水平，这是载脂蛋白ob生物合成和组装成脂蛋白颗粒的许多不寻常特征之一。我们最近建立了强大的无细胞系统，以补充培养细胞和动物模型的研究，以识别和研究靶载脂蛋白ob通过蛋白酶体和非蛋白酶体机制降解的分子因素，以及介导载脂蛋白ob作为脂蛋白颗粒从内质网退出的分子因素。该应用分为3个拟议目的：目的1是确定调节蛋白酶体介导的载脂蛋白er相关降解（ERAD）的因子谱。我们之前已经证实细胞质Hsp70和Hsp90促进载脂蛋白ob的ERAD，并提出已知的Hsp70和Hsp90共伴侣和辅助因子也起重要作用。目的2是确定氧化应激在n-3脂肪酸诱导的er后载脂蛋白ob非蛋白酶体降解中的作用。N-3脂肪酸是已知的降血脂剂，我们最近有数据表明，它们在肝细胞中刺激内质网后非蛋白酶体降解途径（与胰岛素诱导的途径非常相似），该途径被抗氧化剂阻断。n-3刺激的载脂蛋白ob降解的分子特征，特别是关于氧化应激，及其与胰岛素刺激过程的关系，将在细胞培养系统和最近描述的2种人类疾病小鼠模型中进行测试，一种是家族性合并高脂血症（并增加肝脏抗氧化水平），另一种是胰岛素抵抗（Akt2 KO小鼠）。目的3是确定调节载脂蛋白er出口的因素。基于对蛋白质运输的经典研究，载脂蛋白的大尺寸和其他特性可能使其具有驱动其包装和退出内质网的新特征。利用无细胞模型系统（据我们所知，这是第一次为此目的而建立）和肝细胞，我们将确定er退出过程的分子特征。

DESCRIPTION (provided by applicant): This is a renewal application to continue studies on the molecular regulation of apoprotein B (apoB) degradation. ApoB is the predominant protein component of the atherogenic lipoproteins. Thus, knowledge of the regulation of the assembly and secretion of apoB-lipoproteins is not only of fundamental interest, but is also medically relevant. An important control of the net secretion of apoB from cells of hepatic origin is the level of its pre-secretory degradation, one of the many unusual features of the biosynthesis and assembly of apoB into lipoprotein particles. We have recently established powerful cell-free systems to complement studies in cultured cells and animal models to identify and investigate the molecular factors that target apoB to degradation by proteasomal and non-proteasomal mechanisms as well as those that mediate apoB exit from the ER as part of a lipoprotein particle. The application is divided into 3 proposed aims: Aim 1 is to determine the spectrum of factors regulating the proteasome-mediated ER-associated degradation (ERAD) of apoB. We have previously established that cytosolic Hsp70 and Hsp90 promote ERAD of apoB and propose that known Hsp70 and Hsp90 co-chaperones and accessory factors also play important roles. Aim 2 is to determine the role of oxidant stress in the post-ER, non-proteasomal degradation of apoB induced by n-3 fatty acids. N-3 fatty acids are known hypolipidemic agents and we have recent data that in hepatic cells they stimulate a post-ER, non-proteasomal, degradative pathway (with remarkable similarity to the one induced by insulin) that is blocked by anti-oxidants. The molecular characteristics of n-3-stimulated apoB degradation, particularly with regard to oxidant stress, and its relationship to the insulin-stimulated process, will be tested in cell culture systems and in 2 recently described mouse models of human diseases, one of familial combined hyperlipidemia (and which has increased hepatic antioxidant levels) and one of insulin resistance (the Akt2 KO mouse). Aim 3 is to determine the factors regulating the ER-exit of apoB-containing lipoproteins. Based on classical studies of protein trafficking, the large size and other properties of apoB lipoproteins make it likely that there are novel features that drive their packaging and exit from the ER. Using cell-free model systems (established, to our knowledge, for the first time for this purpose), and hepatic cells, we will identify the molecular characteristics of the ER-exit process.