Deciphering Mechanisms for Triglyceride and Cholesterol Transport

项目1：甘油三酯和胆固醇运输的破译机制 摘要/摘要 项目1研究人员致力于探索脂蛋白代谢的基本机制。 健康和疾病。他们发现一种名为GPIHBP1的内皮细胞蛋白负责运输 脂蛋白脂酶(LPL)对毛细血管管腔的作用；LPL-GPIHBP1复合体对血管的边际作用至关重要 富含甘油三酯的脂蛋白(TRL)沿毛细血管；GPIHBP1保护LPL免受自发性和 ANGPTL4催化的去折叠/失活。他们的努力已经产生了60种出版物，其中许多反映了 致力于了解人类疾病。例如，他们发现GPIHBP1突变会导致 乳杆菌微粒症，并从GPIHBP1自身抗体中发现了一种新的人类疾病-乳杆菌微粒症。 最近，项目1的调查人员和同事确定了LPL-GPIHBP1复合体的结构。在.期间 在接下来的5年里，项目1的调查人员将追求两个独立的目标。第一个是追求持续 血管内脂肪分解的研究，建立在GPIHBP1-LPL复合体结构的见解基础上。那 结构，加上新的试剂，新的方法和专家的合作者，使血管内 脂肪分解比以往任何时候都令人兴奋。关键目标包括定义GPIHBP1-LPL所需的氨基酸残基 相互作用，探索潜在的特定“乳糜体微粒子血症突变”的机制，了解- 脂蛋白脂酶的功能多态，确定GPIHBP1‘S酸性结构域在稳定脂蛋白脂酶活性中的作用 解离/灭活GPIHBP1‘S酸性结构域在从硫酸乙酰肝素中募集LPL中的作用 蛋白多糖在内皮下间隙的结合位点，并研究ANGPTL4如何启动去折叠 和LPL的失活。我们还将确定与FAB相关的GPIHBP1和LPL的结构 LPL特异性单抗5D2的片段。我们的第二个目标是调查 巨噬细胞中的胆固醇和巨噬细胞处理胆固醇的机制。在预赛中 研究发现，项目1研究人员发现，巨噬细胞通过质膜萌发释放大量30- 70 nm的颗粒。通过NanoSIMS成像，这些颗粒高度富含胆固醇，包括 新陈代谢活性的“可及胆固醇”，可被细菌细胞溶血素(如Alo-D4)检测到。这一发现 巨噬细胞中富含胆固醇的颗粒“萌芽”引发了许多问题。粒子的作用是什么？ 萌芽？这些粒子的组成是什么？颗粒萌发受调控吗？与项目协作 2和3，项目1将调查不同环境下巨噬细胞颗粒的数量和组成 (例如，类固醇饥饿、胆固醇负荷、LXR激动剂治疗以及LXRs、ABCA1或 Abcg1)。初步的NanoSIMS成像研究表明，高密度脂蛋白在卸载方面是有效的 巨噬细胞衍生颗粒中的胆固醇，这意味着巨噬细胞颗粒的萌发可能与 动脉粥样硬化斑块中胆固醇的反向运输和载脂细胞的出现。

Project 1: Deciphering Mechanisms for Triglyceride and Cholesterol Transport SUMMARY/ABSTRACT Project 1 investigators have devoted their careers to exploring basic mechanisms of lipoprotein metabolism in health and disease. They discovered that an endothelial cell protein, GPIHBP1, is responsible for transporting lipoprotein lipase (LPL) to the capillary lumen; that the LPL–GPIHBP1 complex is crucial for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries; and that GPIHBP1 protects LPL from spontaneous and ANGPTL4-catalyzed unfolding/inactivation. Their efforts have resulted in >60 publications, many reflecting a commitment to understanding human disease. For example, they identified GPIHBP1 mutations causing chylomicronemia and uncovered a new human disease—chylomicronemia from GPIHBP1 autoantibodies. Recently, Project 1 investigators and coworkers determined the structure of the LPL–GPIHBP1 complex. During the next 5 years, Project 1 investigators will pursue two independent objectives. The first is to pursue ongoing studies of intravascular lipolysis, building on insights from the structure of the GPIHBP1–LPL complex. That structure, along with new reagents, new methodologies, and expert collaborators, have made intravascular lipolysis more exciting than ever. Key goals include defining amino acid residues required for GPIHBP1–LPL interactions, exploring mechanisms underlying specific “chylomicronemia mutations,” understanding a gain-of- function polymorphism in LPL, defining the role of GPIHBP1’s acidic domain in stabilizing LPL from unfolding/inactivation, examining the function of GPIHBP1’s acidic domain in recruiting LPL from heparan sulfate proteoglycan binding sites in the subendothelial spaces, and investigating how ANGPTL4 initiates the unfolding and inactivation of LPL. We will also determine the structure of GPIHBP1 and LPL in association with an Fab fragment of the LPL–specific monoclonal antibody 5D2. Our second objective is to investigate the distribution of cholesterol in macrophages and the mechanisms by which macrophages dispose of cholesterol. In preliminary studies, Project 1 investigators found that macrophages release, by plasma membrane budding, numerous 30– 70-nm particles. By NanoSIMS imaging, these particles are highly enriched in cholesterol, including the metabolically active “accessible cholesterol” detectable by bacterial cytolysins (e.g., ALO-D4). The finding that cholesterol-rich particles “bud” from macrophages raises many questions. What is the function of particle budding? What is the composition of these particles? Is particle budding regulated? In collaboration with projects 2 and 3, project 1 will investigate the numbers and composition of macrophage particles in different settings (e.g., sterol starvation, cholesterol loading, LXR agonist treatment, and deficiencies of LXRs, ABCA1, or ABCG1). Preliminary NanoSIMS imaging studies showed that high-density lipoproteins are effective in unloading cholesterol from macrophage-derived particles, implying that macrophage particle budding could be relevant to reverse cholesterol transport and the emergence of cholesterol-laden cells in atherosclerotic plaques.