Cell Cholesterol Efflux and HDL Formation

申请人（Applicant？的摘要） 该计划项目结合了脂质光化学技术， 生物化学和分子和细胞生物学来表征结构， 游离胆固醇（FC）和 磷脂（PL）与两个脂质结合蛋白。Caveolin是一个主要的 细胞表面小窝结构蛋白。载脂蛋白A-1（apo A-1）是 高密度脂蛋白（HDL）的主要成分， 人血浆抗动脉粥样硬化脂蛋白。可光活化的FC和PL类似物 在其结构中的不同点用二苯甲酮基团改性， 被合成并整合到活细胞中，并与小窝蛋白交联 和载脂蛋白A-1。将确认脂质结合位点的同一性 使用定点诱变。在对小窝的进一步研究中， 钒酸盐，蛋白质磷酸酪氨酸磷酸酶的抑制剂， 减少FC外排。我们将探讨以下假设： 小窝蛋白的磷酸化从其结合位点置换FC， 对细胞表面信号转导的影响， 小窝蛋白转录。氧化固醇如何抑制FC外排也将是 确定，特别是这些脂质是否取代了小窝蛋白中的FC。 在apo A-1-PL复合物形成的研究中，ABC 1 转运蛋白将磷脂酰胆碱转运至无脂质载脂蛋白A-1， 要详细分析。FC掺入的来源和机制 这些复合物将被确定，特别是，FC是否结合 直接或仅通过PL与apo A-1结合。最后，他们将调查FC是否 在细胞表面形成的贫脂apo A- 1 /PL/FC复合物中， 直接酯化的卵磷脂：胆固醇酰基转移酶，和酯 转移到其他HDL颗粒上。尽管它在定义 细胞膜的性质，很少有研究， 蛋白质-FC结合。因此，在本报告中获得的信息 程序将是新颖的，高度相关的理解结构 和小窝的功能，FC和PL流出的分子基础， HDL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.