Relation of free cholesterol and caveolae

描述：（申请人摘要） 小窝是富含游离胆固醇和鞘脂的微区 大量存在于血管细胞表面。小窝参与信号传递 从质膜转导作为支架结合的中间体 MAP激酶和PK-A通路。他们也被牵连到FC 体内平衡，并可能作为细胞表面FC水平的传感器。洞穴， Caveolae的主要结构蛋白是FC结合蛋白。在这 预测FC与小窝蛋白结合的分子基础， 其对FC传输和信令的影响将被分析。假设， FC流出到生理受体（载脂蛋白） A-1/磷脂复合物）与FC的解离偶联， 细胞器内残余FC和SPH构象的变化。在 初步研究，FC和鞘磷脂（SPH）的光活化类似物将被 用于定义生物膜小囊内的脂质-蛋白质结合位点， 细胞蛋白质序列内交联的位置将是 在蛋白酶消化后通过HPLC/MS测定。 FC流出与信号转导的关系。钒酸盐增加 小窝蛋白磷酸化，并在相同条件下下调 表达细胞表面小窝和小窝蛋白，抑制FC流出（>80%） 从血管细胞。本项目将确定FC是否与 小窝蛋白启动信号传导到细胞核， FC中膜脂组成与转录调控之间的联系 感应FC外排的第二种抑制剂7-酮基胆固醇，浓度为 与存在于人类动脉粥样硬化斑块中的那些类似， 小窝和小窝蛋白表达和FC流出。假设将被检验 氧化固醇可以取代FC进入细胞从小窝蛋白固醇结合 位点，导致不适当的FC储存和小窝蛋白下调 表情本项目的研究探讨了如何 正常的血管细胞感知并调节它们的FC含量。结果 获得的结果对于理解正常的FC稳态具有重要意义， 以及胆固醇负荷在病理生理条件下的后果。 条件

DESCRIPTION: (Applicant's Abstract) Caveolae are free cholesterol (FC) and sphingolipid -rich microdomains abundant at the surface of vascular cells. Caveolae take part in signal transduction from the plasma membrane as scaffolds binding intermediates of the MAP kinase and PK-A pathways. They have also been implicated in FC homeostasis, and potentially, as sensors of cell surface FC levels. Caveolin, a major structural protein of caveolae, is a FC- binding protein. In this project the molecular basis of FC binding to caveolin, and the mechanism of its effects on FC transport and signaling, will be analyzed. The hypothesis to be tested is that FC efflux to a physiological acceptor (apolipoprotein A-1/phospholipid complex) is coupled to dissociation of FC the caveola, and changes in the conformation of residual FC and SPH within the organelle. In initial studies, photoactivable analogs of FC and sphingomyelin (SPH) will be used to define lipid-protein binding sites within the caveolae of living cells. The location of crosslinks within the protein sequence will be determined after protease digestion by HPLC/MS. Subsequent studies deal with the relationship between FC efflux and signal transduction. Vanadate increases caveolin phosphorylation, and under the same conditions downregulates expression of cell surface caveolae and caveolin and inhibits FC efflux (>80%) from vascular cells. This Project will determine if FC dissociation from caveolin initiates signal transduction to the nucleus to provide the missing link between membrane lipid composition and transcriptional regulation in FC sensing. A second inhibitor of FC efflux, 7-ketocholesterol, at concentrations similar to those present in human atherosclerotic plaques, also downregulates caveolae and caveolin expression and FC efflux. The hypothesis will be tested that oxysterols can displace FC into the cell from the caveolin sterol binding site, leading to inappropriate FC storage and downregulation of caveolin expression. The studies in this Project explore fundamental issues in how normal vascular cells sense and regulate their FC content. The results obtained will have significance for understanding normal FC homeostasis, as well as for the consequences of cholesterol loading under pathophysiologic conditions.