New Avenues in GPIHBP1 Research

描述（由申请人提供）：本提案的目的是确定GPIHBP1是否对含载脂蛋白b48的脂溶过程特别重要，了解GPIHBP1缺陷（GPIHBP1 -/-）小鼠的动脉粥样硬化，并创建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-/-小鼠有乳糜微粒血症，即使是吃鼠粮，血浆甘油三酯水平高达8000mg /dl，血浆胆固醇水平高达800mg /dl。由于GPIHBP1位于“溶脂组织”（如心脏、肌肉、脂肪）毛细血管的管腔表面，并且GPIHBP1结合脂蛋白脂肪酶（LPL）和乳糜微粒，我们怀疑GPIHBP1作为富含甘油三酯的脂蛋白代谢加工的平台。值得注意的是，GPIHBP1含有一个带强负电荷的氨基末端结构域，这对LPL和脂蛋白的结合都很重要。Gpihbp1-/-小鼠血浆中含载脂蛋白b48水平升高，但含载脂蛋白b100水平正常。我推测GPIHBP1可能在含有载脂蛋白b48的脂蛋白的加工中起着特别重要的作用。为了探索这一假设，我将研究Gpihbp1-/-小鼠的表型，这些小鼠是载脂蛋白中“仅载脂蛋白b100”或“仅载脂蛋白b48”突变的纯合子。像乳糜微粒这样的大脂蛋白通常被认为不会导致动脉粥样硬化，但我已经证明，喂饱的Gpihbp1-/-小鼠会发生自发性动脉粥样硬化病变。为了确定Gpihbp1-/-小鼠的动脉粥样硬化是否由“富含甘油三酯”的脂蛋白的胆固醇含量驱动，我将研究依zetimibe和Npc1l1缺乏对Gpihbp1-/-小鼠动脉粥样硬化易感性的影响。最近，GPIHBP1的G56R和Q115P纯合子突变在乳糜小铁血症患者中被发现。这两种突变都发生在高度保守的残基上。我发现Q115P突变消除了GPIHBP1与LPL或乳糜微粒结合的能力，但G56R突变没有可检测到的影响-通过体外试验判断。这些发现让我不确定GPIHBP1的体外检测是否真的是GPIHBP1在体内功能的可靠指标。为了探讨这个问题，我将为G56R和Q115P突变生成并表征敲入小鼠模型。该提案的三个具体目的是：(1)确定GPIHBP1对含载脂蛋白b48的清除是否特别重要；(2)确定鼠粮Gpihbp1- /-小鼠的动脉粥样硬化病变是否由“富甘油三酯”脂蛋白的胆固醇含量驱动；(3)建立GPIHBP1基因中G56R和Q115P突变的敲入小鼠模型。与公共卫生相关：了解脂蛋白代谢是很重要的，因为血浆脂蛋白将甘油三酯燃料输送到重要组织，因为脂蛋白是动脉粥样硬化的诱因。该项目的一个关键目标是了解GPIHBP1在脂蛋白脂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关于脂质营养素如何输送到重要组织的新信息；我的实验还将探索Gpihbp1缺乏情况下动脉粥样硬化的机制，并定义特定的人类Gpihbp1错义突变的体内相关性。

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.