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INTERMEDIATE FILAMENTS IN CHOLESTEROL METABOLISM

胆固醇代谢中的中间丝

基本信息

批准号：
2228836
负责人：
ROBERT M EVANS
金额：
$ 18.06万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 1998-08-31
项目状态：
已结题

项目摘要

The ability of cells to utilize cholesterol derived from lipoprotein is important in normal steroidogenesis, membrane biosynthesis, and regulation of sterol synthesis. Pathological accumulation of esterified cholesterol is a central feature of atherosclerosis. While the endocytosis of lipoprotein derived cholesterol has been well characterized, the subsequent events that mediate the post-lysosomal intracellular transport of cholesterol derived from lipoprotein are not understood. This proposal presents evidence that the capacity of human adrenal tumor cells to esterify lipoprotein derived cholesterol is affected by vimentin-type intermediate filaments (IFs), a major component of the cytoskeleton. Preliminary studies indicate that the intracellular transport of lipoprotein-derived cholesterol from the lysosome to the site of esterification in these cells is a vimentin IF dependent process, but the mechanism is unknown. In the proposed experiments, human SW-13 adrenal tumor cell lines that either contain vimentin-type IFs, or completely lack any detectable IF network will be studied to define and characterize the step in the intracellular transport of cholesterol that is affected by vimentin IF expression. The specificity of vimentin effects on cholesterol transport in these studies will be established by examination of stable cell lines derived from IF negative cells that express a mouse vimentin cDNA, and cell lines with IF networks specifically disrupted by expression of a dominant negative mutant vimentin. Studies following the fate of radiolabeled LDL-cholesteryl linoleate will determine whether IFs affect cholesterol movement from the lysosome to the plasma membrane, from the plasma membrane to the site of esterification, or perhaps from the lysosome to the site of esterification by a mechanism that can bypass the plasma membrane. The intracellular movement of a fluorescent cholesteryl ester will also be studied in cells that contain or lack IFs to follow the intracellular movement of cholesterol from the lysosome using fluorescence microscopy. Adrenal tumor cell lines that express keratin-type IFs will be derived from cells that do not express endogenous cytoplasmic IFs by stable transfection with keratin cDNA expression plasmids. The ability of these cells to esterify lipoprotein derived cholesterol will be compared with cell lines that contain or lack vimentin IFs to determine whether keratin IFs, characteristic of epithelial cells, have a similar function. The expression and cytoplasmic localization of sterol carrier protein-2 (SCP- 2), and apolipoprotein E (apoE) will also be examined in SW-13 cells that contain or lack IFs. Studies will be conducted using immunoprecipitation and immunofluorescence microscopy with anti-SCP-2 and anti-apoE antibodies to determine if the effect of IFs on the transport of lysosomal cholesterol could involve an association with either SCP-2 or apoE. The long-term goal of this project is to define and understand the molecular details of this process, its regulation, and potential involvement in steroidogenesis and pathological cholesterol accumulation.

细胞利用来自脂蛋白的胆固醇的能力是在正常类固醇生成、膜生物合成和甾醇合成的调节。酯化的病理性蓄积胆固醇是动脉粥样硬化的主要特征。而脂蛋白衍生的胆固醇的内吞作用已经很好地特征，随后的事件，介导后溶酶体来自脂蛋白胆固醇的细胞内转运不明白这一建议表明，人类的能力肾上腺肿瘤细胞对脂蛋白衍生的胆固醇的降解作用，受主要成分波形蛋白型中间丝（IF）的影响的细胞骨架。初步研究表明，脂蛋白衍生的胆固醇从溶酶体转运到这些细胞中的酯化位点是波形蛋白IF依赖性过程，但其机制尚不清楚。在拟议的实验中，人类SW-13 肾上腺肿瘤细胞系含有波形蛋白型IF，或完全缺乏任何可检测的IF网络将被研究，表征胆固醇细胞内转运的步骤，受波形蛋白IF表达的影响。波形蛋白的特异性这些研究中对胆固醇转运的影响将通过以下方式确定：检查源自IF阴性细胞的稳定细胞系，表达小鼠波形蛋白cDNA，以及具有IF网络的细胞系通过显性失活突变体的表达而被特异性破坏波形蛋白放射性标记LDL-胆固醇的去向研究亚油酸酯将决定IF是否影响胆固醇运动，溶酶体到质膜，从质膜到酯化，或者可能从溶酶体到酯化反应通过一种可以绕过质膜的机制进行。的荧光胆固醇酯的细胞内移动也将在含有或缺乏IFs的细胞中研究，使用荧光显微镜观察胆固醇从溶酶体中的移动。将衍生表达角蛋白型IFs的肾上腺肿瘤细胞系来自不表达内源性胞质IFs的细胞，用角蛋白cDNA表达质粒转染。这些能力将比较用于纯化脂蛋白衍生胆固醇的细胞与含有或缺乏波形蛋白IF的细胞系，以确定角蛋白作为上皮细胞特征的IFs具有类似的功能。的固醇载体蛋白-2（SCP-2）的表达和细胞质定位 2）和载脂蛋白E（apoE）也将在SW-13细胞中进行检查，包含或缺少IF。将使用免疫沉淀法进行研究用抗SCP-2和抗apoE进行免疫荧光显微镜检查抗体，以确定IFs对转运的影响，溶酶体胆固醇可能与SCP-2或 apoE 本项目的长期目标是定义和理解这一过程的分子细节，其调节和潜力参与类固醇生成和病理性胆固醇蓄积。