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

描述(申请人提供)：载脂蛋白(Apo)A-IV是一种46 kDa的脂结合蛋白，最初是作为肠源性脂蛋白的一种成分分离出来的。从肠道淋巴管进入血浆后，人apoA-IV迅速从乳糜粒中解离出来，与高密度脂蛋白结合，或作为无脂apoA-IV的重要组成部分存在。这增加了apoA-IV的交替构象状态在脂蛋白代谢中发挥不同功能的可能性。事实上，除了在乳糜管组装中的潜在作用外，apoA-IV还被认为具有多种功能，包括参与胆固醇的反向运输、抑制炎症过程和调节食物摄入量。要了解单个蛋白质如何调节这些不同的影响，需要详细了解apoA-IV在其各种状态下的结构/功能关系。本应用的目标是a)推导出apoA-IV在脂相关和非结合状态下的所有原子分子模型，b)了解apoA-IV脂亲和力的分子决定因素，以及c)确定apoA-IV脂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得出，该技术结合了高分辨率质谱学和计算机化建模策略。由此产生的模型将用有限的蛋白质分解和光谱研究的实验数据进行严格的评估。我们还假设，人类载脂蛋白A-IV含有结构特征，削弱了其与脂质相互作用的能力。为了测试这一点，我们将使用突变策略来分离apoA-IV中介导脂质相互作用的特定区域。这些信息将被用来设计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。选定的突变体将被引入细胞培养和啮齿动物模型中，apoA-IV脂结合亲和力将与乳糜管组装和分解代谢相关。来自这些研究的信息不仅在载脂蛋白A-IV的功能和潜在的心血管疾病治疗用途方面将是重要的，而且将有助于理解整个可交换载脂蛋白家族的结构/功能。

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.