REGULATION OF THE SECRETION OF APOB-LIPOPROTEINS

描述：肝脏载脂蛋白B100(ApoB)分泌增加 参与某些高脂血症和动脉粥样硬化的发展。 最近的研究表明，肝脏载脂蛋白B在细胞内转运。 分泌途径是一个独特的动态过程，包括apoB的分选 分子的分泌或降解途径。这条路的候选者 降解载脂蛋白B的是泛素-蛋白酶体系统。这个项目的总体目标是 研究是为了了解肝脏载脂蛋白B-脂蛋白的分泌机制， 包括新生载脂蛋白B在细胞内转运的准确途径 通过分泌途径的不同隔间，以及 泛素-蛋白酶体系统对载脂蛋白B的降解。细胞内 内源性和表达载脂蛋白B的转运将在HepG2和McArdle中进行研究 RH-7777肝癌细胞株。还将对初级猪进行研究 肝细胞，以证实在细胞系中的观察结果，并研究 共翻译或翻译后载脂蛋白B降解的生理作用。我会瞄准的 研究新生细胞的位置、取向和细胞内转运 ApoB在内质网(ER)和高尔基体分泌室 1)嘌呤霉素同步化细胞途径和载脂蛋白B的免疫细胞化学 和其他蛋白质；2)绿色荧光蛋白标记apoB的运输 以及3)纳米金免疫电子显微镜，以更精确地 在细胞器内或细胞器附近定位载脂蛋白B。目标2将决定：1)机制 载脂蛋白B转运到蛋白酶体；2)蛋白酶体群体(胞浆或 内质网相关)参与载脂蛋白B的降解；3)转运子在 载脂蛋白B的逆行转运；4)载脂蛋白B的泛素化位点。目标3 将利用我们最新开发的渗透性细胞降解试验 为载脂蛋白B，并鉴定兔网织红细胞裂解液中的蛋白(S) 刺激apoB蛋白酶体的快速降解。意义：整个 将详细阐明载脂蛋白B的分泌途径以及参与的蛋白质。 载脂蛋白B的降解将被表征。全面了解 载脂蛋白B分泌的调节将有助于揭示高脂血症与 载脂蛋白B的过度生产，可能是由于体内 蛋白酶体途径降解低脂载脂蛋白B。

DESCRIPTION: Increased hepatic secretion of apolipoprotein B100 (apoB) is involved in the development of certain hyperlipidemias and atherosclerosis. Recent findings have demonstrated that hepatic apoB intracellular transport in the secretory pathway is a unique dynamic process that includes sorting of apoB molecules to secretion or degradation pathways. A candidate for the pathway that degrades apoB is the ubiquitin-proteasome system. The overall goal of this research is to understand the mechanism of hepatic apoB-lipoprotein secretion, including the precise pathway of intracellular transport of nascent apoB through the various compartments of the secretory pathway, and the pathway of degradation of apoB by the ubiquitin-proteasome system. The intracellular transport of endogenous and expressed apoB will be studied in HepG2 and McArdle Rh-7777 hepatoma cells. Studies will also be performed with primary pig hepatocytes to confirm observations made in cell lines and to study the physiological role of co- or post-translational apoB degradation. Aim I will investigate the location, orientation, and intracellular transport of nascent apoB in the endoplasmic reticulum (ER) and Golgi compartments of the secretory pathway using 1) puromycin-synchronized cells and immunocytochemistry of apoB and other proteins; 2) transport of apoB tagged with green fluorescent protein in live cells; and 3) nanogold immuno electron microscopy to more precisely localize apoB in or near organelles. Aim 2 will determine: 1) the mechanism of apoB transport to the proteasome; 2) the proteasome population (cytosol or ER-associated) involved in apoB degradation; 3) the role of the translocon in retrograde transport of apoB; and 4) the sites of ubiquitination in apoB. Aim 3 will take advantage of our newly developed permeabilized cell degradation assay for apoB and identify the protein(s) in rabbit reticulocyte lysate that stimulate the rapid proteasomal degradation of apoB. Significance: The entire secretory pathway of apoB will be elucidated in detail and proteins involved in the degradation of apoB will be characterized. A comprehensive understanding of the regulation of apoB secretion will shed light on hyperlipidemias involving overproduction of apoB-lipoproteins, possibly the result of inefficiency in the degradation of lipid-poor apoB by the proteasome pathway.