Relation of free cholesterol and caveolae

描述：(申请人摘要) 小窝是游离胆固醇(FC)和富含鞘脂的微域 丰富的分布在维管细胞表面。小凹病毒参与信号传递 从质膜转导作为结合中间体的支架 MAP激酶和PK-A通路。他们还被牵连到FC 动态平衡，并潜在地作为细胞表面Fc水平的传感器。卡维林， 小窝的主要结构蛋白是Fc结合蛋白。在这 推测Fc与小窝蛋白结合的分子基础，以及Fc与小窝蛋白结合的机制 将分析其对FC传输和信令的影响。假设是为了 被检测的是Fc外流到一个生理受体(载脂蛋白 A-1/磷脂复合体)偶联到小凹Fc的解离，并且 细胞器内残留的Fc和SPH的构象变化。在……里面 初步研究，FC和鞘磷脂(SPH)的光激活类似物将是 用来确定活体小窝内的脂蛋白结合部位 细胞。蛋白质序列中的交联链的位置将是 用高效液相色谱/质谱仪测定酶消化后的含量。 Fc外流与信号转导的关系。钒酸根含量增加 小窝蛋白磷酸化，并在相同条件下下调 细胞表面小窝和小窝蛋白的表达并抑制Fc外流(&gt；80%) 来自血管细胞。该项目将确定FC是否会从 Caveolin启动信号转导到细胞核以提供缺失的 Fc膜脂组成与转录调控的关系 感官。Fc外流的第二种抑制剂，7-酮胆固醇 类似于人类动脉粥样硬化斑块中存在的那些，也下调了 小窝和小窝蛋白表达与Fc外流。这一假设将得到检验。 氧合甾醇可以将Fc从小窝内酯结合转移到细胞内 站点，导致不适当的FC存储和降低对CAVERABLE的监管 表情。该项目中的研究探索了如何 正常的血管细胞感知并调节其Fc含量。结果是 该结果将对理解正常的Fc动态平衡具有重要意义 以及在病理生理条件下胆固醇负荷的后果 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.