New Avenues in GPIHBP1 Research

描述(由申请人提供)：这项建议的目的是确定GPIHBP1对于含apo-B48的脂蛋白的脂解过程是否特别重要，了解Gpihbp1缺陷(Gpihbp1-/-)小鼠的动脉粥样硬化，并建立和分析人类自然发生的GPIHBP1突变的敲入小鼠模型。我们最近发现，Gpihbp1-/-小鼠患有乳糜粒微粒症，即使在饮食中也是如此，其血浆甘油三酯水平高达8000 mg/dl，血浆胆固醇水平高达800 mg/dl。由于GPIHBP1位于“脂解组织”(如心脏、肌肉、脂肪)的毛细血管表面，并且GPIHBP1与脂蛋白脂肪酶(LPL)和乳糜粒结合，我们怀疑GPIHBP1可作为富含甘油三酯的脂蛋白的代谢处理平台。值得注意的是，GPIHBP1包含一个带强负电荷的氨基末端结构域，这对于结合LPL和脂蛋白是重要的。Gpihbp1-/-小鼠的血浆中含有apo-B48的脂蛋白水平升高，但含有apo-B100的脂蛋白水平正常。我推测GPIHBP1可能在含apo-B48的脂蛋白的加工过程中起着特别重要的作用。为了探索这一假设，我将检查Gpihbp1-/-小鼠的表型，这些表型在ApoB中是纯合子的“apo-B100-only”或“apo-B48-only”突变。大的脂蛋白，如乳糜粒，通常被认为是非致动脉粥样硬化的，但我已经证明，喂食Gpihbp1-/-的小鼠会发生自发的动脉粥样硬化损害。为了确定Gpihbp1-/-小鼠的动脉粥样硬化是否是由富含甘油三酯的脂蛋白的胆固醇含量驱动的，我将研究ezetimibe和NPC1L1缺乏对Gpihbp1-/-小鼠动脉粥样硬化易感性的影响。最近，在乳糜粒微粒血症患者中发现了GPIHBP1基因G56R和Q115P纯合子突变。这两种突变都发生在高度保守的残基中。我发现Q115P突变取消了GPIHBP1与LPL或乳糜粒结合的能力，但G56R突变没有检测到影响--正如体外试验所判断的那样。这些发现让我不确定GPIHBP1的体外检测是否真的是GPIHBP1体内功能的可靠指标。为了探索这个问题，我将建立和表征G56R和Q115P突变的敲入小鼠模型。这项建议的三个具体目的是：(1)确定GPIHBP1对于清除含有apo-B48的脂蛋白是否特别重要；(2)确定喂养Gpihbp1-/-小鼠的动脉粥样硬化病变是否由“富含甘油三酯”的脂蛋白的胆固醇含量驱动；以及(3)建立GPIHBP1中G56R和Q115P突变的敲入小鼠模型。与公众健康相关：了解脂蛋白代谢很重要，因为血浆脂蛋白将甘油三酯燃料输送到重要组织，而且脂蛋白是动脉粥样硬化形成的致病因素。这个项目的一个关键目标是了解GPIHBP1在脂蛋白脂肪酶介导的富含甘油三酯的脂蛋白加工中的作用。拟议的研究可能会提供有关脂质营养如何输送到重要组织的新信息；我的实验还将探索Gpihbp1缺乏背景下动脉粥样硬化的机制，并确定特定的人类GPIHBP1错义突变在体内的相关性。

DESCRIPTION (provided by applicant): The objectives of this proposal are to determine if GPIHBP1 is particularly important for the lipolytic processing of apo-B48-containing lipoproteins, to understand the atherosclerosis in Gpihbp1-deficient (Gpihbp1-/-) mice, and to create and analyze knock-in mouse models for naturally occurring GPIHBP1 mutations in humans. We recently found that Gpihbp1-/- mice have chylomicronemia, even on chow diet, with plasma triglyceride levels as high as 8000 mg/dl and plasma cholesterol levels as high as 800 mg/dl. Because GPIHBP1 is located on the luminal surface of capillaries of "lipolytic tissues" (e.g., heart, muscle, fat) and because GPIHBP1 binds both lipoprotein lipase (LPL) and chylomicrons, we suspect that GPIHBP1 serves as a platform for the metabolic processing of triglyceride-rich lipoproteins. Of note, GPIHBP1 contains a strongly negatively charged amino-terminal domain that is important for binding both LPL and lipoproteins. Gpihbp1-/- mice have elevated levels of apo-B48-containing lipoproteins in their plasma, but normal levels of apo-B100-containing lipoproteins. I hypothesize that GPIHBP1 may have a particularly important role in the processing of apo-B48-containing liporoteins. To explore this hypothesis, I will examine the phenotypes of Gpihbp1-/- mice that are homozygous for the "apo-B100-only" or "apo-B48-only" mutations in Apob. Large lipoproteins such as chylomicrons are generally assumed to be nonatherogenic, but I have demonstrated that chow-fed Gpihbp1-/- mice develop spontaneous atheroscerotic lesions. To determine if the atherosclerosis in Gpihbp1-/- mice is driven by the cholesterol content of the "triglyceride-rich" lipoproteins, I will examine the impact of ezetimibe and Npc1l1 deficiency on the susceptibility of Gpihbp1-/- mice to atherosclerosis. Recently, homozygous G56R and Q115P mutations in GPIHBP1 were identified in patients with chylomicronemia. Both mutations occurred in highly conserved residues. I found that the Q115P mutation abolishes the ability of GPIHBP1 to bind to LPL or chylomicrons, but the G56R mutation had no detectable effect-as judged by in vitro assays. These findings left me with uncertainty regarding whether the in vitro assays of GPIHBP1 were truly reliable indicators of GPIHBP1 function in vivo. To explore this issue, I will generate and characterize knock-in mouse models for the G56R and the Q115P mutations. The three specific aims of this proposal are: (1) to determine if GPIHBP1 is particularly important for the clearance of apo-B48-containing lipoproteins; (2) to determine if atherosclerotic lesions in chow-fed Gpihbp1- /- mice are driven by the cholesterol content of "triglyceride-rich" lipoproteins; and (3) to create knock-in mouse models for the G56R and Q115P mutations in GPIHBP1. RELEVANCE TO PUBLIC HEALTH: Understanding lipoprotein metabolism is important because plasma lipoproteins deliver triglyceride fuel to vital tissues and because lipoproteins are causal factors in atherogenesis. A key goal of this project is to understand the role of GPIHBP1 in the lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins. The proposed studies are likely to provide new information on how lipid nutrients are delivered to vital tissues; my experiments will also explore mechanisms for atherosclerosis in the setting of Gpihbp1 deficiency and define the in vivo relevance of specific human GPIHBP1 missense mutations.