HYDROGEN EXCHANGE STUDIES ON A-BETA AMYLOID FIBRILS

描述（来自申请人的摘要）：发现淀粉样原纤维 与越来越多的人类疾病有关，包括一些 神经退行性疾病，其中最普遍的是阿尔茨海默病 （AD）。在每一种疾病中，蛋白质是蛋白质的主要成分， 淀粉样纤维是不同的。因此，淀粉样蛋白是一种典型的 聚合结构，而不是特定的蛋白质分子。果然 一些推测认为这种淀粉样蛋白类型的聚集结构可能是一种 蛋白质折叠基序，可以通过许多蛋白质序列访问下， 正确的情况下。由于它与人类疾病和 对蛋白质折叠的基本理解，特别重要的是， 更详细地了解淀粉样纤维的折叠结构。淀粉样纤维 不适合于标准的蛋白质测定技术 结构，结果是我们对这个结构知之甚少。 这些聚集体富含β折叠 二级结构我们选择研究与AD相关的淀粉样蛋白， 由肽A β组成。在这里提出的研究中，我们将使用 氢-氘交换（HX）技术来绘制 纤维中的A-β氢键，例如在多肽之间发现的 β折叠中骨架酰胺氢，保护酰胺氢不被交换 而大多数其它酰胺氢自由交换。具体目标是 应用程序是（1）开发收集A-β上HX数据的方法 掺入淀粉样蛋白原纤维，使用质谱法（MS）和 核磁共振（NMR）来量化交换水平，并使用这些 方法来映射保护和暴露的酰胺氢的A-β在 （2）对其他A-β聚集体进行类似的交换研究，如 与淀粉样蛋白装配和Abeta蛋白有关的原纤维 聚集体毒性;（3）使用与HX数据一致的计算方法 建立、测试和改进原丝和原纤维结构的模型; (4)对其他A-β聚集体进行交换实验，包括 由A-β片段以及神经炎斑块核心在体外制成的纤维 分离自人类AD脑材料。这些研究会让我们更深入地了解 淀粉样纤维的结构，这将使我们能够更好地 了解纤维如何生长以及它们如何破坏人体细胞和组织。一个 结构知识的提高也将提高我们识别和 设计治疗分子来抑制A-β的生长和毒性， 淀粉样蛋白和其它聚集体。

DESCRIPTION (From the Applicant's Abstract): Amyloid fibrils are found associated with a growing number of human diseases, including several neurodegenerative diseases, the most prevalent of which is Alzheimer's Disease (AD). In each of these diseases, the protein that is the main component of the amyloid fibril is different. Thus, amyloid is a characteristic type of aggregate structure, rather than a particular protein molecule. Indeed, there is some speculation that this amyloid type of aggregate structure may be a protein folding motif that can be accessed by many protein sequences under the right circumstances. Because of its relevance to both human disease and fundamental understanding of protein folding, it is particularly important to know in more detail the folded structure of the amyloid fibril. Amyloid fibrils do not lend themselves to standard techniques for determining protein structure, with the result that we know very little about this structure at the molecular level beyond the fact that these aggregates are rich in beta sheet secondary structure. We have chosen to work on the amyloid associated with AD, composed of the peptide A-beta. In the research proposed here we will use the technique of hydrogen-deuterium exchange (HX) to map the secondary structure of A-beta in the fibril. Hydrogen bonds, such as are found between polypeptide backbone amide hydrogens in beta sheet, protect amide hydrogens from exchange while most other amide hydrogens freely exchange. The specific aims of this application are to (1) Develop methods for collecting HX data on A-beta incorporated into amyloid fibrils, using both mass spectrometry (MS) and nuclear magnetic resonance (NMR) to quantify levels of exchange, and use these methods to map the protected and exposed amide hydrogens of A-beta in the fibril; (2) carry out similar exchange studies on other A-beta aggregates, like protofibrils, that are implicated in both amyloid assembly and in Abeta aggregate toxicity; (3) use computational methods in concert with the HX data to build, test, and refine models of protofilament and fibril structure; and (4) conduct exchange experiments on other aggregates of A-beta, including fibrils made in vitro from A-beta fragments as well as neuritic plaque cores isolated from human AD brain material. These studies will give us a closer look at the structure of the amyloid fibril, which will allow us to better understand how fibrils grow and how they disrupt human cells and tissue. An improved knowledge of structure will also improved our ability to identify and design therapeutic molecules for inhibiting the growth and toxicity of A-beta amyloid and other aggregates.