The roles of apolipoprotein A-IV structure and lipid affinity in its function

描述（由申请方提供）：载脂蛋白（apo）A-IV是一种46 kDa的脂质结合蛋白，最初分离时作为母体衍生脂蛋白的组分。当从肠道消化道进入血浆时，人apoA-IV迅速从乳糜微粒上解离，并与高密度脂蛋白结合或作为无脂质apoA-IV的重要部分存在。这就提出了apoA-IV的交替构象状态可能在脂蛋白代谢中发挥不同功能的可能性。事实上，除了在乳糜微粒组装中的潜在作用之外，还假定apoA-IV执行多种功能，包括参与胆固醇逆向转运、抑制炎症过程和调节食物摄入。要了解单个蛋白质如何介导这些不同的作用，需要详细了解apoA-IV在其各种状态下的结构/功能关系。本申请的目的是a）推导出apoA-IV在脂质缔合和非缔合状态下的所有原子分子模型，B）理解apoA-IV脂质亲和力的分子决定因素，和c）确定apoA-IV脂质结合在体内脂蛋白代谢中的作用。结构模型将使用最先进的技术，结合联合收割机高分辨率质谱和计算机建模策略。由此产生的模型将严格评估有限的蛋白水解和光谱研究的实验数据。我们还假设，人类载脂蛋白A-IV包含的结构特征，削弱其与脂质相互作用的能力。为了验证这一点，我们将使用诱变策略来分离介导脂质相互作用的apoA-IV内的特定区域。这些信息将用于工程设计apoA-IV突变体，这些突变体在结构上与野生型相似，但在脂质结合方面有所不同。将选择的突变体引入细胞培养物和啮齿动物模型中，并将apoA-IV脂质结合亲和力与乳糜微粒组装和catalysts相关联。这些研究的信息不仅在apoA-IV对心血管疾病的功能和潜在治疗用途方面很重要，而且对于理解整个可交换载脂蛋白家族的结构/功能也很有用。

DESCRIPTION (provided by applicant): Apolipoprotein (apo) A-IV is a 46 kDa lipid-binding protein initially isolated as a component of intestinally derived lipoproteins. Upon entry into plasma from the intestinal lymphatics, human apoA-IV rapidly dissociates from chylomicrons and either associates with high density lipoproteins or exists as a significant fraction of lipid-free apoA-IV. This raises the possibility that alternate conformational states of apoA-IV may perform distinct functions in lipoprotein metabolism. Indeed, in addition to a potential role in chylomicron assembly, apoA-IV has also been postulated to perform diverse functions including participation in reverse cholesterol transport, inhibition of inflammatory processes, and regulation of food intake. To understand how a single protein can mediate these varied effects requires detailed knowledge of the structure/function relationships of apoA-IV in its various states. The goals of this application are a) to derive all atom molecular models for apoA-IV in both lipid-associated and unassociated states, b) to understand the molecular determinants of apoA-IV lipid affinity, and c) to determine the role of apoA-IV lipid binding on lipoprotein metabolism in vivo. The structural models will be derived using state-of-the-art techniques that combine high resolution mass spectrometry and computerized modeling strategies. The resulting models will be rigorously evaluated with experimental data from limited proteolysis and spectroscopic studies. We also hypothesize that human apoA-IV contains structural features that attenuate its ability to interact with lipids. To test this, we will use mutagenesis strategies to isolate specific regions within apoA-IV that mediate lipid interactions. This information will be used to engineer apoA-IV mutants that are structurally similar to wild-type but vary in their lipid association. Select mutants will be introduced into cell culture and rodent models and the apoA-IV lipid binding affinity will be correlated with chylomicron assembly and catabolism. The information from these studies will be important not only in terms of the function and potential therapeutic use of apoA-IV against cardiovascular disease, but will also be useful for understanding the structure/function of the entire family of exchangeable apolipoproteins.