Roles of apolipoprotein A-IV structure/lipid affinity

载脂蛋白 A-IV 结构/脂质亲和力的作用

• 批准号: 7140823
• 负责人: W Sean Davidson
• 金额: $ 30.7万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140823
• 关键词: X ray crystallography; apolipoproteins; blood lipoprotein metabolism; chemical binding; chemical models; chylomicrons; conformation; gastrointestinal epithelium; genetically modified animals; high density lipoproteins; intermolecular interaction; laboratory mouse; mass spectrometry; molecular dynamics; nuclear magnetic resonance spectroscopy; protein engineering; protein structure function; site directed mutagenesis; structural biology

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.

描述（由申请人提供）：载脂蛋白（apo）A-IV是一种46kDa的脂质结合蛋白，最初作为肠源性脂蛋白的成分被分离。从肠道淋巴管进入血浆后，人 apoA-IV 迅速与乳糜微粒分离，并与高密度脂蛋白结合或作为无脂 apoA-IV 的重要部分存在。这提出了 apoA-IV 的替代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发挥潜在作用外，apoA-IV 还被认为具有多种功能，包括参与反向胆固醇转运、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。本应用的目标是 a) 推导 apoA-IV 在脂质相关和非相关状态下的所有原子分子模型，b) 了解 apoA-IV 脂质亲和力的分子决定因素，以及 c) 确定 apoA 的作用-IV 脂质结合对体内脂蛋白代谢的影响。结构模型将使用结合高分辨率质谱和计算机建模策略的最先进技术得出。由此产生的模型将使用有限的蛋白水解和光谱研究的实验数据进行严格评估。我们还假设人类 apoA-IV 含有减弱其与脂质相互作用能力的结构特征。为了测试这一点，我们将使用诱变策略来分离 apoA-IV 中介导脂质相互作用的特定区域。该信息将用于设计 apoA-IV 突变体，其结构与野生型相似，但脂质关联不同。选择的突变体将被引入细胞培养和啮齿动物模型中，apoA-IV 脂质结合亲和力将与乳糜微粒组装和分解代谢相关。这些研究的信息不仅对于 apoA-IV 对抗心血管疾病的功能和潜在治疗用途很重要，而且对于了解整个可交换载脂蛋白家族的结构/功能也很有用。