Refining Physiologic Mechanisms for Intravascular Triglyceride Metabolism

在这个多重首席研究员R01授予的第二个周期中，我们研究了脂蛋白脂酶(LPL)和GPIHBP1(将LPL运送到毛细血管管腔的内皮细胞蛋白)，以及这两种蛋白在富含甘油三酯的脂蛋白(TRL)的脂解过程中的作用。我们卓有成效，发表了20篇手稿，全部发表在顶级期刊上。值得注意的是，我们从GPIHBP1自身抗体中发现了一种新的人类疾病-乳糜粒微粒症。我们定义了这种疾病的病理生理学，并与我们的合作者表明，利妥昔单抗可以相当有效地治疗这种疾病，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肪分解的基本生理机制。我们证明了LPL作为单体是活性的，消除了几十年来认为LPL是同源二聚体的教条。我们发现GPIHBP1保留了LPL的水解酶结构域的结构和活性。我们解决了LPL-GPIHBP1复合体的原子结构，这为GPIHBP1如何稳定LPL以及特定的LPL和GPIHBP1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。第一个是验证GPIHBP1‘S酸性结构域对LPL-GPIHBP1复合体从毛细血管内皮细胞腔内膜到腔内膜(PM)的运动起关键作用的假设。我们最近创造了表达缺乏酸性结构域的突变GPIHBP1的基因编辑小鼠。突变体GPIHBP1‘S的LU结构域未发生改变，因此能够与LPL结合。出乎意料的是，酸性结构域的缺失阻碍了GPIHBP1-LPL复合体从内皮细胞的腔面PM向毛细血管腔的运输。我们怀疑LPL-GPIHBP1复合体的运动被GPIHBP1结合的LPL和邻近的硫酸乙酰肝素蛋白多糖(HSPGs)之间持续的静电相互作用所阻碍。结合表面等离子体共振研究和体内GPIHBP1转运研究，我们将检验这一假设，即GPIHBP1‘S酸性结构域的一个关键功能是破坏GPIHBP1结合的LPL和附近的HSPG之间沿着腔质膜的相互作用，从而释放LPL跨内皮细胞移动到毛细血管腔。我们的第二个具体目标是利用先进的显微镜来探索最近的一个发现，即由GPIHBP1转运到毛细血管中的一些LPL随后从GPIHBP1中分离出来，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，我们将沿着毛细血管内皮细胞表征与GPIHBP1结合的和与糖萼结合的LPL池。我们还将研究糖萼结合的LPL与TRL沿毛细血管的边际作用和TRL加工的相关性。最后，我们将开发所需的工具来测试糖萼LPL与人类疾病的相关性。我们处于理想的位置，拥有所有的试剂、实验方法和专家合作者，以解决我们的特定目标。我们预计，我们的研究将改变教科书上的血管内甘油三酯代谢模型。

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.