New Avenues in GPIHBP1 Research

描述（由申请人提供）：本提案的目的是确定GPIHBP 1是否对含载脂蛋白B48的脂蛋白的脂解加工特别重要，以了解GPIHBP 1缺陷（GPIHBP 1-/-）小鼠中的动脉粥样硬化，并创建和分析人类天然GPIHBP 1突变的基因敲入小鼠模型。我们最近发现，Gpihbp 1-/-小鼠有乳糜微粒血症，即使在普通饲料中，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。因为GPIHBP 1位于“脂解组织”（例如，心脏、肌肉、脂肪），并且由于GPIHBP 1结合脂蛋白脂肪酶（LPL）和乳糜微粒，我们怀疑GPIHBP 1充当富含磷脂酰肌醇脂蛋白的代谢加工平台。值得注意的是，GPIHBP 1含有一个带强负电荷的氨基末端结构域，该结构域对于结合LPL和脂蛋白都很重要。Gpihbp 1-/-小鼠血浆中含apo-B48的脂蛋白水平升高，但含apo-B100的脂蛋白水平正常。我推测GPIHBP 1可能在含载脂蛋白B48的脂蛋白的加工中具有特别重要的作用。为了探索这一假设，我将检查Gpihbp 1-/-小鼠的表型，这些小鼠是Apob中“apo-B100-only”或“apo-B48-only”突变的纯合子。大的脂蛋白如乳糜微粒通常被认为是非致动脉粥样硬化的，但我已经证明，饲料喂养的Gpihbp 1-/-小鼠自发性动脉粥样硬化病变。为了确定Gpihbp 1-/-小鼠的动脉粥样硬化是否由“富含胆固醇”脂蛋白的胆固醇含量驱动，我将研究依折麦布和Npc 1 l1缺乏对Gpihbp 1-/-小鼠动脉粥样硬化易感性的影响。最近，在乳糜微粒血症患者中发现了GPIHBP 1的G56 R和Q115 P纯合突变。这两种突变都发生在高度保守的残基上。我发现Q115 P突变消除了GPIHBP 1与LPL或乳糜微粒结合的能力，但G56 R突变没有检测到这种影响--这是通过体外试验判断的。这些发现让我对GPIHBP 1的体外测定是否是GPIHBP 1体内功能的真正可靠指标感到不确定。为了探索这个问题，我将生成并表征G56 R和Q115 P突变的敲入小鼠模型。该提案的三个具体目标是：（1）确定GPIHBP 1是否对含apo-B48的脂蛋白的清除特别重要;（2）确定喂食饲料的Gpihbp 1- /-小鼠中的动脉粥样硬化病变是否由“富含胆固醇”脂蛋白的胆固醇含量驱动;和（3）创建GPIHBP 1中G56 R和Q115 P突变的敲入小鼠模型。与公共卫生的关系：了解脂蛋白代谢是很重要的，因为血浆脂蛋白提供甘油三酯燃料的重要组织，因为脂蛋白是动脉粥样硬化的致病因素。该项目的一个关键目标是了解GPIHBP 1在脂蛋白脂肪酶介导的富含磷脂酰肌醇的脂蛋白加工中的作用。拟议的研究可能会提供新的信息，脂质营养物质是如何传递到重要组织;我的实验还将探讨动脉粥样硬化的机制，在设置GPIHBP 1缺陷和定义特定的人类GPIHBP 1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.