Cell Cholesterol Efflux and HDL Formation

建议项目(申请人？S摘要) 该项目结合了来自脂质光化学的技术， 生物化学和分子和细胞生物学来表征结构和 游离胆固醇(FC)与游离胆固醇形成的络合物的性质 磷脂(PL)与两种脂结合蛋白。卡维林是一家 细胞表面小凹的结构蛋白。载脂蛋白A-1(apo A-1)是 高密度脂蛋白(HDL)的主要成分，主要是 人血浆中的抗动脉粥样硬化脂蛋白。可光激活的FC和PL类似物 在其结构中的不同位置用二苯甲酮基团修饰将 被合成并结合到活细胞中并与小窝蛋白交联物 并鉴定出载脂蛋白A-1。将确认脂类结合部位的身份 使用定点突变。在对凹陷的进一步研究中，其机制 通过它，蛋白酪氨酸磷酸酶的抑制剂钒酸， 将确定减少FC外流。我们将探索这样的假设： 小窝蛋白的磷酸化取代了Fc的结合部位，并产生了影响 细胞表面信号转导途径对细胞周期的影响 洞穴转录。氧甾醇如何抑制Fc外流也将是 确定，特别是这些脂质是否取代了小窝蛋白中的Fc。 在载脂蛋白A-1-磷脂复合体形成的研究中，ABC1的形成机制 转运蛋白将磷脂酰胆碱转移到无脂载脂蛋白A-1将 进行了详细的分析。功能界别并入的由来及机制 这些络合物将被确定，特别是Fc是否结合 直接到载脂蛋白A-1或仅经PL。最后，他们将调查FC是否 在脂质缺乏的情况下，在细胞表面形成的载脂蛋白A-1/PL/Fc复合体可以 卵磷脂直接酯化：胆固醇酰基转移酶及其酯 转移到其他高密度脂蛋白颗粒上。尽管它在定义 对于细胞膜的性质，目前还鲜有研究 蛋白质-Fc结合。因此，在此过程中要获得的信息 计划将既新颖又与理解结构高度相关 和小窝的功能，Fc和PL外排的分子基础，以及 高密度脂蛋白的结构。

PROPOSED PROGRAM (Applicant?s abstract) This Program Project combines techniques from lipid photochemistry, biochemistry and molecular and cell biology to characterize the structure and properties of the complexes formed between free cholesterol (FC) and phospholipid (PL) with two lipid binding proteins. Caveolin is a major structural protein of cell surface caveolae. Apolipoprotein A-1 (apo A-1) is the major component of high density lipoprotein (HDL), the major atheroprotective lipoprotein of human plasma. Photoactivable FC and PL analogs modified with benzophenone groups at different points in their structure will be synthesized and incorporated into living cells and crosslinks to caveolin and apo A-1 identified. The identity of lipid binding sites will be confirmed using site-directed mutagenesis. In further studies on caveolae, the mechanism by which vanadate, an inhibitor of protein phosphotyrosine phosphatases, reduces FC efflux will be identified. The hypothesis will be explored that phosphorylation of caveolin displaces FC from its binding site, with effects on signal transduction from the cell surface that lead to suppression of caveolin transcription. How oxysterols inhibit FC efflux will also be determined, and in particular, whether these lipids displace FC from caveolin. In studies of apo A-1-PL complex formation, the mechanism by which the ABC1 transporter protein transfers phosphatidyl choline to lipid-free apo A-1 will be analyzed in detail. The origin and mechanism of incorporation of FC into these complexes will be determined, and in particular, whether FC binds directly to apo A-1 or only via PL. Finally, they will investigate whether FC within lipid-poor apo A- 1 /PL/FC complexes formed at the cell surface can be directly esterified by lecithin: cholesterol acyltransferase, and the esters transferred to other HDL particles. In spite of its significance in defining the properties of the cell membrane, there has been little investigation of protein-FC binding. As a result, the information to be obtained in this program will be both novel and highly relevant to understanding the structure and functions of caveolae, the molecular basis of both FC and PL efflux, and the structure of HDL.