Structural Biology of Amyloid Disease

描述（由申请人提供）：将通过结构和计算生物学的方法阐明阿尔茨海默病、帕金森病、朊病毒病、亨廷顿病和其他淀粉样蛋白样疾病中的蛋白质聚集。通过微晶学的背景研究已经表明，淀粉样蛋白样纤维的基本结构单元是一组β片层，其中相邻片层的氨基酸侧链相互啮合，称为空间拉链。片材的面之间的立体拉链界面是完全干燥的。形成片层的蛋白质片段长度短至4-8个残基，平行或反平行堆叠以生长原纤维，但片段可以更长，并且一些蛋白质含有几个这样的片段。为了了解疾病相关蛋白质的淀粉样纤维的结构，同样的方法将应用于微晶生长的短节的A？和阿尔茨海默病的Tau蛋白质，朊病毒疾病的PrP蛋白质，帕金森病的突触核蛋白，以及参与ALS和2型糖尿病的蛋白质。为了了解蛋白质的其余部分在原纤维形成过程中发生了什么，还将使用晶体筛选和微晶学的新方法对较大的片段和整个原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的哪些片段是纤维形成的片段，并且可以生长成适合结构测定的微晶。该过程基于3D剖面方法，用于使用能量函数找到适合给定折叠（在这种情况下为空间拉链）的序列。该程序将被扩展并应用于淀粉样蛋白。一旦发现了形成原纤维的片段，并且其结构已通过晶体学确定为属于空间拉链型结构，则可以通过片段是否可以将完整蛋白质接种到原纤维中来评估完整蛋白质的片段和原纤维之间的连接。通过突变蛋白质中对应于片段残基的残基，并寻找减少的纤维化，可以获得完整蛋白质的片段和原纤维连接的进一步证据。这些结构是由微晶学的新方法得到的，是淀粉样蛋白状态的第一个高分辨率（高达0.85 A分辨率），完全精细的原子结构。他们表明，淀粉样纤维的空间拉链棘至少有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.