Structural Biology of Amyloid and Amyloid-like Proteins

淀粉样蛋白是由异常折叠的蛋白质组成的丝状聚合物，其特征是具有交叉β结构。淀粉样蛋白的积累与大约20种人类疾病有关，包括阿尔茨海默氏症、2型糖尿病和类风湿性关节炎。淀粉样蛋白分为两大类：感染性和非感染性。传染性淀粉样蛋白被称为普恩。我们从1998年开始研究酵母菌的Pron结构，最初专注于Ure2p，一种氮分解代谢的负调控因子。我们发现它的N-末端结构域负责原基的发生，而执行其调节功能的C-末端结构域仍然折叠在丝状结构中，但被立体机制灭活。在我们的淀粉样蛋白主干概念中，Pron结构域形成细丝主干，并被C-末端结构域包围。2005年，我们发表了淀粉样蛋白主干的平行超折叠贝塔结构模型。在这种模式下，它设想了通过将单体与平面的β-蛇形折叠堆叠而产生的平行β-折叠的阵列。拓扑相似的结构是其他淀粉样纤维的很好候选者，包括淀粉蛋白，科学文献中对这种模型的支持越来越多。正在进行的工作旨在测试和完善这一模型；调查纤维多态；并将淀粉样蛋白与天然构象联系起来。在2013财年，我们专注于两个项目。 (1)富含聚谷氨酰胺的淀粉样蛋白。许多神经退行性疾病，如亨廷顿病，表现为脑内聚集的多肽沉积，并伴有异常延长的串联谷氨酰胺残基(PolyQ)。这些蛋白质在体内和体外都是高度不溶的，形成含有淀粉样原纤维的聚集体。这些纤维的高分辨率结构对于研究这些疾病的分子基础和进展机制具有重要价值。然而，多聚Q纤维的分析仍然很困难。一个主要的挑战是它们的极端不溶性，这些多肽迅速聚集成更高阶的复合体，在这些复合体中，单个纤维几乎无法区分。为了生产出适合于结构分析的纤维，我们设计了合适长度(和RT；30个氨基酸)的多聚Q多肽，其中包括一些L赖氨酸残基以提高溶解性，以及两个D-赖氨酸通过插入反向旋转来阻断推测的β-链。合成了两种多肽：PolyQKd33(33个残基)和PolyQKd32(32个残基)，其中央部分少了一个谷氨酸胺。两种多肽在中性pH条件下均可溶，在pH为11-12的条件下可聚集成分散良好的纤维，适合于EM分析。布鲁克海文STEM实验室的未染色标本和未染色的冷冻干燥标本的暗场STEM用负染色EM、冷冻EM和电子衍射对这些原纤维进行成像。经电子衍射仪分析，两种纤维均呈锐利的环形，间距为(0。47 nm)-1(图1A，B)，表明交叉β结构，淀粉样蛋白的标志。然而，通过负染色EM和冷冻-EM，PolyQKd32纤维略厚于PolyQKd33，但始终比PolyQKd33厚。此外，暗视野STEM成像显示了纤维直径的相同趋势。每长度质量分析显示，亚基堆积密度从1.0个分子/轴向重复(0.47 nm)增加到1.5个分子(0.47 nm)。在整个纤维结构的这些变化的基础上，在多肽的中央部分插入了一个额外的谷氨酰胺残基。这些正在准备发表的观察结果为这些和其他含多聚Q的纤维的三维结构提供了模型。 2)含α-突触核蛋白的淀粉样蛋白。帕金森病是一种影响运动功能的慢性进行性神经退行性疾病。帕金森病的特点是多巴胺能神经元死亡和路易小体的存在。α-突触核蛋白(AS)纤维是路易小体的主要成分，以往的研究表明其纤颤是疾病病理的一部分。正常情况下，140个氨基酸的长蛋白具有膜重塑功能，我们也在研究，如AR027015-18项目所述。当与脂类和溶液中的无规卷曲结合时，AS是α螺旋的。在纤维形成过程中，蛋白质聚合成交叉β结构。尽管它们具有很高的临床相关性，但关于含砷的淀粉样纤维的结构信息一直难以捉摸，这类信息是本项目的目标。重组AS在大肠杆菌中表达，纯化后组装成纤维，用我们实验室的低温电子显微镜和Brookaven STEM设备的暗场STEM观察到。低温电子显微镜图像显示，AS纤维直径为5 nm，大致笔直，轴向重复长度为75 nm。由STEM数据进行的每长度质量的测量给出了单峰分布，平均密度相当于每0.47 nm轴向上升两个亚单位。这表明原纤维实际上是一对原纤维--每个原纤维都有平行的超褶皱结构--包裹在一个共同的轴上。横截面重建表明，每个原纤维都有两个组分。这些观察结果为建模研究提供了基础，目前正在准备出版。

Amyloids are filamentous polymers of aberrantly folded proteins distinguished by cross-beta structure. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, type-2 diabetes, and rheumatoid arthritis. Amyloids are distinguished into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focusing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. With this model, it envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin, and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model; investigating fibril polymorphism; and relating amyloids to native conformations. In FY 13 we focused on two projects. (1) Polyglutamine-rich amyloids. Many neurodegenerative disorders such as Huntingtons disease exhibit intracerebral deposition of aggregated polypeptides with abnormally extended tracts of tandem glutamine residues (polyQ). These proteins are highly insoluble and form amyloid fibril-containing aggregates in vivo and in vitro. High-resolution structures for these fibrils would be of great value for investigating the molecular basis and the mechanism of progression of these diseases. However, analysis of polyQ fibrils has remained difficult. A major challenge has been their extreme insolubility whereby these polypeptides rapidly aggregate into higher-order complexes in which individual fibrils are barely distinguishable. Aiming to produce fibrils suitable for structural analysis, we designed polyQ-containing peptides of suitable length ( and RT; 30 amino acids) in which some L-lysine residues were included to enhance solubility and two D-lysines to interrupt the putative beta-strand by inserting reverse turns. Two polypeptides were synthesized: polyQKd33 (33 residues) and polyQKd32 (32 residues), which has one fewer glut amines in the central part. Both peptides were found to be soluble at neutral pH and to assemble into well dispersed fibrils at pH 11-12 suitable for EM analysis. These fibrils were imaged by negative staining EM, cryo-EM, and electron diffraction from unstained specimens and by dark-field STEM of unstained freeze-dried specimens at the Brookhaven STEM laboratory. When analyzed by electron diffraction, both fibrils showed a sharp ring at a spacing of (0. 47nm)-1 (Fig. 1A, B), indicative of cross-beta structure, the hallmark of amyloid. However, the polyQKd32 fibrils were slightly but consistently thicker than polyQKd33 by both negative staining EM and cryo-EM. Furthermore, dark-field STEM imaging revealed the same trend in fibril diameters. Mass-per-length analyses revealed an accompanying increase in subunit packing density from 1.0 to 1.5 molecules per axial repeat (0.47 nm) of the cross-beta structure. Underlying these changes in overall fibril architecture is the insertion of a single additional glutamine residue in the central part of the polypeptide. These observations - which are being prepared for publication - suggest models for the three-dimensional structures of these and other polyQ-containing fibrils. 2) Alfa-synuclein-containing amyloids. Parkinsons disease (PD) is a chronic and progressive neurodegenerative disease affecting motor function. PD is characterized by dopaminergic neuronal cell death and by the presence of Lewy bodies. Alfa-synuclein (aS) fibrils are the main component of Lewy bodies, and previous research suggests that its fibrillation is part of the disease pathology. Normally, the 140 aa long protein has a membrane remodeling function which we are also researching, as reported in project AR027015-18. aS is alfa-helical when associated with lipid and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. Despite their high clinical relevance, structural information on aS-containing amyloid fibrils has been elusive and such information is the goal of this project. Recombinant aS was expressed in E. coli, purified and assembled into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at the Brookhaven STEM facility. The resulting cryo-EM images revealed that aS fibrils are 5 nm in diameter and more or less straight, and they have an axial repeat of 75 nm. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise. This suggests that the fibril is in fact a pair of protofibrils - each with a parallel superpleated structure - wrapping around a common axis. Reconstruction of the cross-section indicates that each protofibril has two components. These observations, which afford a basis for modeling studies, are currently being prepared for publication.