ALZHEIMER'S DISEASE: DEFINING THE APOE-AMYLOID-BETA INTERACTION

摘要 载脂蛋白E有三种主要亚型(称为载脂蛋白2、载脂蛋白E3和载脂蛋白E4)，它们产生于 单基因座，彼此之间只有单一氨基酸的差异。现在公认的是 迟发性阿尔茨海默病的主要危险因素(约占90% 痴呆症病例)是apoE4亚型的存在。这种蛋白质是如此隐蔽，以至于 携带两个载脂蛋白E4副本的人将在70岁前患上阿尔茨海默病。我们的假设是 载脂蛋白E亚型与淀粉样蛋白中的主要蛋白质淀粉样蛋白-b相互作用的差异 斑块，是导致载脂蛋白E亚型之间功能差异的原因。有相当多的 细胞抗体清除是阿尔茨海默病发生的决定性因素的证据 并且是载脂蛋白E亚型依赖的。我们的目标是了解apoE-Ab的相互作用。一共有三个 具体目的：了解抗体聚集的动力学和机制，了解载脂蛋白E- AB和apoE-Ab-脂质相互作用，并找到干扰这些相互作用的小分子。这个 载脂蛋白E-抗体的相互作用知之甚少。因此，载脂蛋白E上的抗体结合部位，结合抗体的形式 载脂蛋白E、脂类在抗体结合中的作用以及抗体与载脂蛋白E结合的机制尚不清楚。 我们已经证明载脂蛋白E只与抗体聚集过程中的中间产物相互作用 了解抗体聚集本身的机制是非常必要的。我们问这些中间体是什么 以及他们如何使用我们开发的新的荧光分析方法定义与载脂蛋白E的相互作用 它首先确定了在抗体过程中形成低聚物的速率和平衡常数 首先是聚合，然后考察齐聚物以外的聚合过程。这些 然后将使用各种方法来了解脂质在apoE-Ab相互作用中的作用。我们将使用我们的 最近观察到apoE3和apoE4的结构差异，差异远离 单个氨基酸变化的位置，作为检查相互作用差异的基础 ApoE亚型与Ab.载脂蛋白E和抗体参与其功能的特定区域/残基 相互作用将通过定点突变和/或氢/氢交换来确定。 程序。这些方法，连同我们提出的高通量筛选实验，将是 用于区分apoE4与apoE3之间的相互作用 阿布。这一结果可能会导致关于延缓治疗剂开发的新想法 阿尔茨海默氏症的发病。与华盛顿大学的其他人合作 这样做的分子化合物将被测试它们影响小鼠大脑中抗体清除的能力。的 关键的重要性是获得全长单体形式的apoE3(34 KDa)的结构， 唯一已知的载脂蛋白E亚型的全长结构。

Abstract There are three major isoforms of apolipoprotein E (called apoE2, apoE3 and apoE4) that arise from a single gene locus and differ from each other only by single amino acids. It is now well-established that the major risk factor for the development of late-onset Alzheimer's disease (accounting for about 90% of cases of dementia) is the presence of the apoE4 isoform. This protein is so insidious that individuals with two copies of apoE4 will develop Alzheimer's disease by age 70. Our hypothesis is that the difference in the interaction of apoE isoforms with amyloid-b, the major protein in amyloid plaques, is responsible for the functional differences between apoE isoforms. There is considerable evidence that Ab clearance from cells is a defining factor in the development of Alzheimer's disease and is apoE isoform dependent. Our goal is to understand the apoE-Ab interaction. There are three specific aims: To understand the kinetics and mechanism of Ab aggregation, to understand the apoE- Ab and apoE-Ab-lipid interaction and to find small molecules that perturb these interactions. The apoE-Ab interaction is poorly understood. Thus, the Ab binding site on apoE, the forms of Ab that bind apoE, the role of lipid in Ab binding and the mechanism of Ab binding to apoE are largely unknown. We have shown that apoE interacts only with intermediates that occur during Ab aggregation making it is essential to understand the mechanism of Ab aggregation itself. We ask what those intermediates are and how they define the interaction with apoE using new fluorescence assays we have developed that first determine the rate and equilibrium constants for the formation of oligomers during the Ab aggregation and secondly examine the aggregation process beyond oligomer formation. These methods will be then used to understand the role of lipid on the apoE-Ab interaction. We will use our recent observation of structural differences between apoE3 and apoE4, differences distant from the site of the single amino acid change, as the basis for examining the differences in the interaction between apoE isoforms and Ab. Specific regions/residues of both apoE and Ab involved in their interaction will be determined by site-directed mutations and/or hydrogen/deuterium exchange procedures. These methods, along with high throughput screening experiments we propose, will be used to differentiate the behavior of apoE4 relative to that of apoE3 with respect to the interaction with Ab. The results may lead to the new ideas concerning the development of therapeutic agents to delay the onset of Alzheimer's disease. In collaboration with others at Washington University any small molecule compounds that do so will be tested for their ability to affect Ab clearance in mouse brain. Of critical importance is the availability of the structure of a full-length monomeric form of apoE3 (34 kDa), the only known full-length structure of any apoE isoform.