Structure/function analysis of A-beta fibril assembly

描述(由申请人提供)：各种实验方法都表明，在阿尔茨海默病特征的神经功能进行性丧失的过程中，Abeta肽在皮质中的有序聚集是一个关键步骤。目前的想法倾向于对快速形成的Abeta多肽聚集体(称为原纤维)具有毒性作用，这些聚集体的残基为41到43。然而，由这些多肽组成的成熟淀粉样蛋白纤维，以及在第39或40位结束的较短版本也可能影响疾病，作为有毒或保护性实体。尽管我们对Aβ淀粉样蛋白结构的了解目前还处于中等水平，但在过去的四年里，在分析Aβ蛋白聚集体的结构和结构能量学方面取得了长足的进步，并开发了用于聚集体结构分析的新工具。这一建议建立在先前工作的基础上，侧重于(A)Aβ(1-42)淀粉样蛋白的结构和稳定性；(B)Aβ(1-42)原纤维的结构和稳定性；以及(C)最近报道的不同构象类型的Aβ淀粉样纤维的结构和性质。丙氨酸和脯氨酸突变体将聚集，并将测定和分析由这些突变体形成的淀粉样原纤维和原纤维稳定性的变化。将对1-42个突变体进行单半胱氨酸和双半胱氨酸突变的交联分析，以确定纤维的内部结构。这些方法和其他方法也将用于分析纤维的替代构象的结构和稳定性(Aβ纤维的构象改变可能会影响它们的生物活性，就像酵母蛋白的淀粉样蛋白一样)。这项建议涉及到与几个分子建模小组的合作，将使用实验数据来测试当前的纤维和原纤维结构和组装模型，并将允许设计额外的实验来测试开发中的结构模型。这些研究将有助于理解Aβ淀粉样原纤维的结构、组装和淀粉样原纤维结构稳定的基本基础，这对于建立Aβ和其他与神经退行性疾病相关的多肽的结构模型将是重要的。此外，这些研究应该继续推动我们对Aβ原纤维及其在疾病中的作用的认识的前沿。这些数据将增强我们对AD分子基础的了解，并在此过程中有助于开发药物设计的分子靶点。

DESCRIPTION (provided by applicant): A variety of experimental approaches implicate the ordered aggregation of the Abeta peptide in the cortex as a key step in the progressive loss of neuronal function characteristic of Alzheimer's disease. Current thinking favors a toxic role for rapidly formed aggregates, called protofibrils, of Abeta peptides ending at residue 41 to 43. However, it is also possible that mature amyloid fibrils composed of these peptides, as well as shorter versions ending at positions 39 or 40, may also influence disease, as either toxic or protective entities. Although our knowledge of Abeta amyloid structure is at present only at modest resolution, great strides have been made in the last four years in the analysis of the structure and structural energetics of Abeta aggregates, and new tools for structural analysis of aggregates have been developed. This proposal builds on previous work by focusing on (a) the structure and stabilization of Abeta (1-42) amyloid; (b) the structure and stabilization of Abeta (1-42) protofibrils; and (c) the structure and properties of different conformational types of Abeta amyloid fibrils recently reported. Alanine and proline mutants will be aggregated, and the change in stability of amyloid fibrils and protofibrils formed from these mutants will be determined and analyzed. Single and double cysteine mutant crosslinking analysis will be conducted on 1-42 mutants, to determine the internal structure of the fibrils. These and other methods will also be applied to the analysis of the structure and stability of alternative conformations of the fibrils (altered conformational forms of Abeta fibrils may have influenced their biological activity, as is the case for amyloid forms of yeast prions). Involving collaborations with several molecular modeling groups, this proposal will use experimental data to test current models for fibril and protofibril structure and assembly, and will allow for design of additional experiments to test structural models as they are developed. These studies should contribute to the understanding of Abeta amyloid fibril structure, assembly, and the fundamental basis of amyloid fibril structural stabilization, which will be important in developing structural models for amyloid fibrils of Abeta and other peptides responsible for neurodegenerative diseases. In addition, these studies should continue to push the frontiers of our knowledge of Abeta protofibrils and their role in disease. This data will enhance our knowledge of the molecular basis of AD and in the process contribute to the development of molecular targets for drug design.