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

摘要 存在三种主要的载脂蛋白E同种型（称为apoE 2、apoE 3和apoE 4），其产生于 单个基因位点，彼此仅存在单个氨基酸的差异。现在已经确定， 发展为迟发性阿尔茨海默病的主要危险因素（约占90%） 痴呆症病例中）存在apoE 4同种型。这种蛋白质是如此阴险， 具有两个apoE 4拷贝的个体将在70岁时患上阿尔茨海默病。我们的假设是 apoE亚型与淀粉样蛋白-b（淀粉样蛋白中的主要蛋白质）相互作用的差异 斑块，负责apoE亚型之间的功能差异。有相当大 有证据表明，抗体从细胞中清除是阿尔茨海默病发展的决定性因素 并且是apoE同种型依赖性的。我们的目标是了解apoE-Ab相互作用。有三 具体目的：了解抗体聚集的动力学和机制，了解apoE- 抗体和apoE-Ab-脂质相互作用，并找到干扰这些相互作用的小分子。的 对apoE-Ab相互作用了解甚少。因此，apoE上的Ab结合位点，即与apoE结合的Ab形式， apoE、脂质在Ab结合中的作用以及Ab与apoE结合的机制在很大程度上是未知的。 我们已经证明，apoE只与抗体聚集过程中发生的中间体相互作用 理解Ab聚集本身的机制是必要的。我们问这些中间体 以及他们如何使用我们开发的新荧光分析来定义与apoE的相互作用 首先确定在Ab过程中形成低聚物的速率和平衡常数 聚集，其次检查低聚物形成以外的聚集过程。这些 方法将用于了解脂质对apoE-Ab相互作用的作用。我们将用我们 最近观察到apoE 3和apoE 4之间的结构差异， 单个氨基酸变化的位点，作为检查相互作用差异的基础 apoE亚型和Ab之间的关系。apoE和Ab两者的特定区域/残基参与它们的表达。 相互作用将通过定点突变和/或氢/氘交换来确定 程序.这些方法，沿着我们提出的高通量筛选实验， 用于区分apoE 4相对于apoE 3的行为， AB.这一结果可能会导致新的想法有关的治疗药物的发展，以延迟 老年痴呆症的发病与华盛顿大学的其他人合作， 将测试这样做的分子化合物影响小鼠脑中Ab清除的能力。的 至关重要的是apoE 3的全长单体形式（34 kDa）的结构的可用性， 唯一已知的apoE同种型全长结构。

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.