INTERMEDIATE FILAMENTS IN CHOLESTEROL METABOLISM

细胞利用来自脂蛋白的胆固醇的能力是 在正常的类固醇合成、膜生物合成和 类固醇合成的调节。酯化病理性堆积 胆固醇是动脉粥样硬化的主要特征。而当 脂蛋白胆固醇的内吞作用一直很好 具有特征性的、调节溶酶体后的后续事件 来自脂蛋白的胆固醇在细胞内的运输不是 明白了。这一提议提供了证据，表明人类的能力 肾上腺肿瘤细胞酯化脂蛋白衍生胆固醇 受主要成分--波形蛋白类中间丝(IF)的影响 细胞骨架的。初步研究表明，细胞内 脂蛋白衍生的胆固醇从溶酶体到 这些细胞中的酯化部位是一个波动相关的过程， 但其机制尚不清楚。在拟议的实验中，人类的sw-13 肾上腺肿瘤细胞系，要么含有Vimentin类型的IF，要么 完全缺乏任何可检测到的网络是否将被研究来定义和 描述胆固醇在细胞内运输的步骤 受Vimentin IF表达的影响。波形蛋白的特异性 在这些研究中对胆固醇运输的影响将通过 来自IF阴性细胞的稳定细胞系的检测 表达小鼠Vimentin基因，构建含IF网络的细胞系 被显性负性突变体的表达特异性干扰 波形蛋白。关于放射性标记低密度脂蛋白-胆固醇命运的研究 亚油酸盐将决定IFs是否影响胆固醇从 溶酶体到质膜，从质膜到部位 酯化，或者可能是从溶酶体到 通过一种可以绕过质膜的机制进行酯化。这个 荧光胆固醇酯在细胞内的运动也将是 在含有或缺乏IF的细胞中研究以跟随细胞内 用荧光显微镜观察胆固醇从溶酶体中的运动。 表达角蛋白IfS的肾上腺肿瘤细胞株将被衍生 来自不表达内源性胞质IFs的细胞 转染角蛋白基因表达载体。这些人的能力 细胞将脂蛋白衍生的胆固醇酯化将与 含有或缺乏Vimentin的细胞系确定角蛋白 具有上皮细胞特征的IF具有类似的功能。这个 固醇载体蛋白-2(SCP-2)的表达和胞浆定位 2)，载脂蛋白E(ApoE)也将在SW-13细胞中检测 包含或不包含IF。研究将使用免疫沉淀进行。 抗SCP-2和抗apoE免疫荧光显微镜检查 抗体以确定IFs是否对转运有影响 溶酶体胆固醇可能与SCP-2或 APOE.该项目的长期目标是定义和理解 这一过程的分子细节，它的调节和潜力 参与类固醇合成和病理性胆固醇蓄积。

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.