REGULATION OF THE SECRETION OF APOB-LIPOPROTEINS

描述：载脂蛋白B100（apoB）的肝脏分泌增加， 参与某些高血压和动脉粥样硬化的发展。 最近的研究结果表明，肝载脂蛋白B细胞内转运， 分泌途径是一个独特的动态过程， 分子分泌或降解途径。一个候选人的途径 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apoB-脂蛋白分泌的机制， 包括新生apoB细胞内转运的精确途径 通过分泌途径的各个隔室，以及 通过泛素-蛋白酶体系统降解apoB。细胞内 将在HepG 2和McArdle中研究内源性和表达的apoB的转运 Rh-7777肝癌细胞。还将对原代猪进行研究 肝细胞，以确认在细胞系中观察到的结果，并研究 共翻译或翻译后apoB降解的生理作用。我会瞄准的 研究新生血管的位置、方向和细胞内转运， apoB在内质网（ER）和高尔基体室的分泌 途径使用1）嘌呤霉素同步化细胞和apoB的免疫细胞化学 2）绿色荧光蛋白标记的apoB转运 在活细胞中;和3）纳米金免疫电子显微镜，以更精确地 apoB定位于细胞器内或细胞器附近。目标2将确定：1） apoB转运到蛋白酶体; 2）蛋白酶体群体（细胞质或 ER相关）参与apoB降解; 3）易位子在 apoB的逆行转运; 4）apoB中的泛素化位点。目标3 将利用我们新开发的透化细胞降解试验 并鉴定兔网织红细胞裂解物中 刺激apoB的快速蛋白酶体降解。意义：整个 apoB的分泌途径将被详细阐明， 将表征apoB的降解。全面了解 apoB分泌调节将有助于阐明高脂血症， 载脂蛋白B-脂蛋白的过量产生，可能是由于在 通过蛋白酶体途径降解脂质贫乏的apoB。

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.