Molecular Regulation of Apoprotein B Degradation

描述(申请人提供)：这是一个续期申请，以继续研究载脂蛋白B(ApoB)降解的分子调控。载脂蛋白B是致动脉粥样硬化脂蛋白的主要蛋白质组分。因此，了解载脂蛋白B-脂蛋白的组装和分泌的调节不仅具有基本意义，而且具有医学意义。肝来源细胞的载脂蛋白B净分泌的一个重要控制因素是其分泌前降解的水平，这是载脂蛋白B生物合成和组装成脂蛋白颗粒的许多不寻常特征之一。我们最近已经建立了强大的无细胞系统来补充在培养细胞和动物模型中的研究，以确定和研究靶向载脂蛋白B通过蛋白酶体和非蛋白酶体机制降解的分子因素，以及那些介导载脂蛋白B作为脂蛋白颗粒的一部分从内质网退出的分子因素。应用分为3个拟议的目的：目的1是确定调节蛋白酶体介导的内质网相关降解(ERAD)载脂蛋白B的因子的谱。我们已证实胞内Hsp70和Hsp90促进apoB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确定氧化应激在n-3脂肪酸诱导的内质网后非蛋白酶体降解载脂蛋白B中的作用。N-3脂肪酸是已知的降血脂药物，我们最近的数据表明，在肝细胞中，它们刺激ER后的、非蛋白酶体的降解途径(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B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两种人类疾病的小鼠模型中进行测试，其中一种是家族性混合性高脂血症(并增加了肝脏抗氧化水平)，另一种是胰岛素抵抗(Akt2 KO小鼠)。目的3是确定调控载脂蛋白内质网退出的因素。基于蛋白质转运的经典研究，apoB脂蛋白的大小和其他性质使其有可能存在新的特征，从而驱动其包装和退出内质网。使用无细胞模型系统(据我们所知，这是第一次为此目的而建立)和肝细胞，我们将识别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.