Genesis of lipid-poor HDL

贫脂 HDL 的起源

• 批准号: 6890945
• 负责人: PHOEBE E FIELDING
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890945
• 关键词: apolipoproteins; binding sites; bioenergetics; blood lipoprotein biosynthesis; blood lipoprotein metabolism; cholesterol; crosslink; high density lipoproteins; high performance liquid chromatography; human subject; mass spectrometry; membrane transport proteins; phosphatidylcholine sterol acyltransferase; phospholipids; protein binding; site directed mutagenesis; thin layer chromatography

DESCRIPTION: (Applicant's abstract) High density lipoprotein (HDL) is the major atheroprotective lipoprotein of human plasma. Successful formation of high density lipoprotein (HDL) from its major protein, apolipoprotein A-1 (apo A-1) depends on an initial, energy-dependent transfer of phospholipid (PL) from the cell surface which is catalyzed by the ATP-binding cassette-1 (ABC 1) transporter protein. PL transfer precedes FC transfer to apo A-1. In the absence of ABC-1 activity (as in human Tangier Disease) normal HDL are completely absent, despite the ability of PL to bind spontaneously to apo A-1 in vitro. Here the hypothesis is tested that ABC 1 modifies the conformation of apo A-1 binding to the cell surface to promote contact between PL and one or more specific PL-binding targets within its amino acid sequence. These contacts prime the nascent complex for the transfer of additional PL, and facilitate the efflux of cell surface FC. This is subsequently stabilized by direct FC-apo A-1 binding. In this Project, the binding sites of PL and FC to apo A- I in lipid-poor HDL formed at the surface of vascular cells will be characterized from the lipid-protein crosslinks generated by photoactivation from FC and PL analogs containing benzophenone groups at different points in their structure. Lipid-protein crosslinks will localized by protease digestion and HPLC/mass spectrometry. Lipid/lipid crosslinks will be identified by HPLC and TLC. The data obtained will be extended and confirmed using apo A-1 proteins from which individual PL and FC binding sites have been deleted by site- directed mutagenesis. Finally this project will identify the point at which nascent HDL become direct substrates for lecithin: cholesterol acyltransferase and cholesterol ester transfer protein, when cell-derived PL and FC are metabolized and appear in mature acceptor lipoproteins. These analyses, carried out at intervals during the initial lipidation of apo A- 1, will provide the most detailed picture available of the genesis of HDL at the surface of peripheral cells. Novel information on the spatial organization of FC and PL in lipoprotein complexes will be obtained, highly relevant to the molecular analysis of human HDL deficiency.

描述：（申请人的摘要） 高密度脂蛋白（HDL）是主要的手动保护脂蛋白 人血浆。从其成功形成高密度脂蛋白（HDL） 主要蛋白质，载脂蛋白A-1（APO A-1）取决于初始 磷脂（PL）的能量依赖性转移来自细胞表面 由ATP结合卡塞特-1（ABC 1）转运蛋白催化。 pl 将FC转移到APO A-1。在没有ABC-1活性的情况下（AS 在人类探针疾病中）尽管有 PL在体外自发与APO A-1结合的能力。这里的假设 测试ABC 1修改了Apo A-1与细胞结合的构象 表面促进PL和一个或多个特定PL结合之间的接触 在其氨基酸序列内的靶标。这些触点是新生的 复合物转移附加PL，并促进细胞外排 表面FC。随后通过直接的FC-APO A-1结合来稳定这一点。在 这个项目，PL和FC的绑定位点与脂质贫乏的HDL中的Apo A- i 在血管细胞表面形成的 FC和PL类似物产生的光激活产生的脂质 - 蛋白交联 在其结构的不同点中包含苯甲酮基团。 脂质 - 蛋白交联将通过蛋白酶消化和HPLC/质量定位 光谱法。脂质/脂质交联将通过HPLC和TLC鉴定。这 获得的数据将使用APO A-1蛋白进行扩展和确认 单个PL和FC结合位点已被站点定向删除 诱变。最后，该项目将确定新生HDL的点 成为卵磷脂的直接底物：胆固醇酰基转移酶和 当细胞衍生的PL和FC是胆固醇酯转移蛋白 代谢并出现在成熟的受体脂蛋白中。这些分析， 在Apo A-1的初始脂化期间，以隔离为零，将 提供最详细的HDL起源的图片 外围细胞的表面。有关空间组织的新信息 将获得脂蛋白复合物中的FC和PL，与 人HDL缺乏的分子分析。