Structural Biology of Amyloid Disease

描述(申请人提供)：阿尔茨海默氏症、帕金森氏症、普里恩病、亨廷顿氏症和其他淀粉样类疾病中的蛋白质聚集将通过结构和计算生物学的方法来阐明。背景微晶学研究表明，淀粉样原纤维的基本结构单元是一组β片状结构，其中相邻片状的氨基酸侧链相互交织在一起，形成空间拉链。床单表面之间的立体拉链界面是完全干燥的。形成片状的蛋白质片段的长度与4-8个残基一样短，平行或反平行堆积以形成原纤维，但片段可以更长，一些蛋白质包含几个这样的片段。为了从疾病相关蛋白中了解淀粉样纤维的结构，同样的微晶学方法将被应用于从A？以及阿尔茨海默病的Tau蛋白、普鲁恩病的PrP蛋白、帕金森病的β-突触核蛋白以及ALS和2型糖尿病的相关蛋白。为了了解在纤维形成过程中对其余蛋白质的影响，还将使用晶体筛选和微晶学的新方法，对更大片段和整个纤维形成蛋白进行结构研究。初步工作表明，计算能量学可以识别蛋白质中哪些片段是形成纤维的片段，并可以生长成适合结构确定的微晶体。该程序基于3D轮廓方法，使用能量函数来寻找符合给定折叠(在这种情况下是立体拉链)的序列。该程序将扩展并应用于淀粉样蛋白。一旦发现了形成纤维的片段，并通过结晶学确定其结构属于立体拉链类型的结构，就可以通过该片段是否能将完整的蛋白质种成纤维来评估该片段与完整蛋白质的纤维之间的联系。通过突变蛋白质中与片段残基相对应的残基，并寻找减少的纤维化，可以获得完整蛋白质片段和纤维连接的进一步证据。这些结构是由新的微晶学方法得出的，是第一个高分辨率(高达0.85A分辨率)的、完全精炼的淀粉样态原子结构。他们表明，淀粉样纤维的空间拉链脊椎至少有4种基本模式，可能多达7种。这些结构为设计这些毁灭性的神经退行性疾病的诊断和治疗提供了坚实的基础。

DESCRIPTION (provided by applicant): The aggregation of proteins in Alzheimer's, Parkinson's, prion diseases, Huntington's, and other amyloid-like diseases will be illuminated by methods of structural and computational biology. Background research by microcrystallography has shown that the fundamental structural unit of amyloid-like fibrils is a set of beta sheets, in which the amino acid sidechains of neighboring sheets intermesh, in what is termed a steric zipper. The steric zipper interface between the faces of the sheets is completely dry. The protein segments that form the sheets are as short as 4-8 residues in length, stacking either in parallel or antiparallel to grow a fibril, but the segments can be longer and some proteins contain several such segments. To learn the structures of amyloid fibrils from disease-associated proteins, the same methods of microcrystallography will be applied to microcrystals grown from short segments of the A? and Tau proteins of Alzheimer's disease, from the PrP protein of the prion diseases, from ?-synuclein of Parkinson's disease, and from proteins involved in ALS and diabetes type 2. To learn what happens during fibril formation to the rest of the protein, structural studies will also be conducted on larger segments and entire fibril-forming proteins, using novel methods of crystal screening and microcrystallography. Preliminary work shows that computational energetics can identify which segments from proteins are those that are fibril-forming and can be grown into microcrystals, suitable for structural determination. This procedure is based on the 3D Profile method for finding sequences that fit a given fold (in this case the steric zipper), using energy functions. The procedure will be extended and applied to amyloids. Once a segment has been discovered which forms fibrils, and its structure has been determined by crystallography as belonging to the steric-zipper type of architecture, the connection between the segment and fibrils of the full protein can be assessed by whether the segment can seed the full protein into fibrils. Further proof of the connection of the segment and fibrils of the full protein can be obtained by mutating residues in the protein that correspond to residues of the segment, and looking for diminished fibrillization. These structures, derived by novel methods of microcrystallography, are the first high-resolution (up to 0.85 A resolution), fully refined atomic structures for the amyloid state. They show that there are at least 4 basic patterns for the steric zipper spines of amyloid fibrils, and perhaps up to 7 such patterns. These structures offer a solid foundation on which to devise diagnostics and therapeutics for these devastating neurodegenerative diseases.