Refining Physiologic Mechanisms for Intravascular Triglyceride Metabolism

在这个多个主要研究者R 01资助的第二个周期中，我们研究了脂蛋白脂肪酶（LPL）和GPIHBP 1（将LPL穿梭到毛细血管腔的内皮细胞蛋白）以及这两种蛋白质在富含脂蛋白的脂蛋白（TRL）的脂解加工中的作用。我们是富有成效的，发表了>20篇手稿，都在顶级期刊上。值得注意的是，我们从GPIHBP 1自身抗体中发现了一种新的人类疾病-乳糜微粒血症。我们定义了这种疾病的病理生理学，并与我们的合作者一起表明，这种疾病可以用利妥昔单抗有效治疗，导致自身抗体消失和血浆甘油三酯水平正常化。我们还探讨了血管内脂解的基本生理机制。我们发现LPL作为单体是有活性的，这消除了几十年来认为LPL是同二聚体的教条。我们发现GPIHBP 1保留了LPL水解酶结构域的结构和活性。我们解决了LPL-GPIHBP 1复合物的原子结构，这为GPIHBP 1如何稳定LPL以及特定的LPL和GPIHBP 1突变如何导致人类疾病提供了见解。对于第三个赠款周期，我们有两个具体目标。第一个是测试的假设，GPIHBP 1的酸性结构域是至关重要的LPL-GPIHBP 1复合物的运动从近腔到管腔质膜（PM）的毛细血管内皮细胞。我们最近创建了基因编辑小鼠，表达缺乏酸性结构域的突变GPIHBP 1。GPIHBP 1的LU结构域没有改变;因此，突变体GPIHBP 1能够结合LPL。出乎意料的是，酸性结构域的缺乏阻碍了GPIHBP 1-LPL复合物从内皮细胞的近腔PM到毛细血管腔的运输。我们怀疑LPL-GPIHBP 1复合物远离近腔质膜的运动受到GPIHBP 1结合的LPL和相邻的硫酸乙酰肝素蛋白聚糖（HSPGs）之间持续的静电相互作用的阻碍。使用表面等离子体共振研究和体内GPIHBP 1转运研究的组合，我们将测试的假设，GPIHBP 1的酸性结构域的一个重要功能是破坏GPIHBP 1结合的LPL和附近的HSPGs之间的相互作用，沿着近腔质膜，从而释放LPL移动通过内皮细胞的毛细血管腔。我们的第二个具体目标是探索最近的发现，使用先进的显微镜，一些LPL的GPIHBP 1运输到毛细血管随后从GPIHBP 1分离，并进入富含HSPG的糖萼。利用超分辨率共聚焦显微镜，电子显微镜和NanoSIMS成像，我们将表征GPIHBP 1结合和糖萼结合池LPL沿着毛细血管内皮细胞。我们还将研究糖萼结合LPL与TRL沿沿着毛细血管边缘化和TRL加工的相关性。最后，我们将开发测试糖萼LPL与人类疾病相关性所需的工具。我们拥有理想的位置，所有的试剂，实验方法和专家合作者，以实现我们的特定目标。我们预期我们的研究将改变血管内甘油三酯代谢的教科书模型。

During the second cycle of this Multiple Principal Investigator R01 grant, we investigated lipoprotein lipase (LPL) and GPIHBP1 (the endothelial cell protein that shuttles LPL to the capillary lumen) and the roles of both proteins in the lipolytic processing of triglyceride-rich lipoproteins (TRLs). We were productive, publishing >20 manuscripts, all in top-tier journals. Of note, we discovered a new human disease—chylomicronemia from GPIHBP1 autoantibodies. We defined the pathophysiology of that disease and with our collaborators showed that the disease can be treated quite effectively with rituximab, resulting in disappearance of autoantibodies and normalization of plasma triglyceride levels. We also explored basic physiologic mechanisms for intravascular lipolysis. We showed that LPL is active as a monomer, dispelling decades-old dogma that LPL is a homodimer. We showed that GPIHBP1 preserves the structure and activity of LPL's hydrolase domain. We solved the atomic structure of the LPL–GPIHBP1 complex, which provided insights into how GPIHBP1 stabilizes LPL and how specific LPL and GPIHBP1 mutations cause human disease. For the third grant cycle, we have two specific aims. The first is to test the hypothesis that GPIHBP1's acidic domain is crucial for the movement of LPL–GPIHBP1 complexes from the abluminal to the luminal plasma membrane (PM) of capillary endothelial cells. We recently created gene-edited mice expressing a mutant GPIHBP1 lacking the acidic domain. GPIHBP1's LU domain was unaltered; hence, the mutant GPIHBP1 was able to bind LPL. Unexpectedly, the absence of the acidic domain impeded trafficking of the GPIHBP1–LPL complex from the abluminal PM of endothelial cells to the capillary lumen. We suspect that the movement of LPL–GPIHBP1 complexes away from the abluminal plasma membrane was impeded by persistent electrostatic interactions between the GPIHBP1-bound LPL and adjacent heparan sulfate proteoglycans (HSPGs). Using a combination of surface plasmon resonance studies and in vivo GPIHBP1 transport studies, we will test the hypothesis that a crucial function of GPIHBP1's acidic domain is to disrupt interactions between GPIHBP1-bound LPL and nearby HSPGs along the abluminal plasma membrane, thereby freeing LPL to move across endothelial cells to the capillary lumen. Our second specific aim is to explore a recent discovery, using advanced microscopy, that some of the LPL that is transported into capillaries by GPIHBP1 subsequently detaches from GPIHBP1 and enters the HSPG-rich glycocalyx. Taking advantage of super-resolution confocal microscopy, electron microscopy, and NanoSIMS imaging, we will characterize GPIHBP1-bound and glycocalyx-bound pools of LPL along capillary endothelial cells. We will also examine the relevance of the glycocalyx-bound LPL to TRL margination along capillaries and TRL processing. Finally, we will develop the tools required to test the relevance of glycocalyx LPL to human disease. We are ideally positioned, with all of the reagents, experimental approaches, and expert collaborators, to address our specific aims. We anticipate that our studies will transform textbook models of intravascular triglyceride metabolism.e metabolism.