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，最初的重点是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。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2011财年，我们重点关注三个领域： (1)对淀粉样原纤维模型中的β-拱廊基序和β-螺线管蛋白结构进行了系统分析。与天然蛋白质的不同折叠不同，它们的淀粉样蛋白在刚性、光滑的侧面和具有交叉β结构方面基本上是相似的。尽管获得高分辨率的实验确定的纤维结构存在困难，但通过集成来自多种技术的数据，正在获得越来越可信的模型。目前大多数疾病相关的淀粉样蛋白模型都使用了β-拱形结构，这是一种柱状结构，由贝塔-拱形结构堆积而成。β-Arch是一种链-转向链基序，其中两条β-链通过它们的侧链相互作用，而不是像传统的β-发夹那样通过多肽骨架相互作用。贝塔螺线管的晶体结构，一类具有淀粉样属性的蛋白质，提供了对拱门中发现的贝塔弧转弯的洞察。一般的热力学考虑表明，两个或多个β-拱形结构的复合体可能导致淀粉样蛋白纤维形成。在2011财年，我们发表了一篇关于β-拱形结构的综述(1)。 (2)感染酵母细胞中Ure3蛋白细丝的可视化。野生型Ure2p是一种可溶性二聚体蛋白，参与调节酿酒酵母的氮分解代谢。在其Prion形式中，Ure2p聚集并失去其活性。我们早期对过度表达Ure2p的感染细胞进行的薄片电子显微镜研究表明，聚集体具有丝状亚结构，通过免疫金标记法，这些细丝含有Ure2p。在体外，Ure2p组装成细丝，其淀粉样纤维骨架被球状结构域包围。在2011财年，我们扩展了这一研究路线，目的是更密切地比较体外组装和体内组装的细丝，并说明Ure2p的总细胞组成。为此，我们使用了电子断层扫描。在冷冻替代保存优化的样品中，可以看到直径为20 nm的非中空纤维。在体外组装中，以相同方式制备的长丝也是非中空的，并且直径基本相同。在聚集体中，细丝是随机取向的，偶尔会有交叉点。我们没有观察到聚集体与其他细胞质成分的任何联系；否则在细胞质中丰富的核糖体被排除在外。通过比较生物化学测定的每个细胞中Ure2p的数量与断层图像中细丝中的数量，我们得出结论，即使不是全部，也是大部分Ure2p存在于聚集体中。这些观察结果正在准备发表。 (3)蛋白高电荷中间结构域(M-结构域)的配置和作用。在感染PSI+Prion的酵母细胞中，Sup35p蛋白形成聚集体，其翻译终止活性下调但不会被消除。Sup35p有一个N-末端的Prion结构域；一个约125个残基的高电荷M-结构域；以及一个功能性的C-末端结构域。通过负染、冷冻-EM和扫描透射电子显微镜(STEM)，在体外组装的全长Sup35p细丝显示出细小的骨架纤维，周围环绕着弥漫的C-结构域云，获得了完整的直径为65 nm的纤维。在直径(8 Nm)和外观上，这些骨骼单独类似于N结构域的淀粉样纤维。单位长度的茎质量数据为N-原纤维、NM-原纤维和Sup35P细丝每0.47 nm产生一个亚基，进一步支持淀粉样原纤维主干模型。装饰性C-结构域径向跨度为30 nm，表明M-结构域具有高度延伸的构象。扩展的M结构域构象为感染细胞中残留的Sup35p活性提供了解释，即C结构域保持足够的自由，能够与核糖体进行一些相互作用。2011财年，该项目完成并发布(2)。 目前的研究主要集中在生产有序的Ure2p纤维，用于高分辨率的冷冻EM分析主干结构。

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, focussing 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. 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 FY11 we focussed on three areas:

 (1) Systematic analysis of beta-arcade motifs in amyloid fibril models and beta-solenoid protein structures. Unlike the diverse folds of native proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and in having cross-beta structures. Despite the difficulties attendant upon obtaining high resolution experimentally determined fibril structures, increasingly credible models are being derived by integrating data from multiple techniques. Most current models of disease-related amyloids invoke beta-arcades, columnar structures produced by in-register stacking of beta-arches. A beta-arch is a strand-turn-strand motif in which the two beta-strands interact via their side-chains, not via the polypeptide backbone as in a conventional beta;-hairpin. Crystal structures of beta-solenoids, a class of proteins with amyloid-like properties, offer insight into the beta-arc turns found in arches. General thermodynamic considerations suggest that complexes of two or more beta-arches may nucleate amyloid fibrillogenesis.
In FY11, we published a review of beta-arcades (1). (2) Visualization of Ure3 prion filaments in infected yeast cells. Wild-type Ure2p is a soluble dimeric protein that contributes to regulating nitrogen catabolism in S. cerevisiae. In its prion form, Ure2p aggregates and loses its activity. Our earlier work on thin section electron microscopy of infected cells over-expressing Ure2p showed that the aggregates have a filamentous substructure and, by immuno-gold labelling, that the filaments contain Ure2p. In vitro, Ure2p assembles into filaments with an amyloid fibril backbone surrounded by globular domains. In FY11, we extended this line of investigation with the aims of making a closer comparison between in vitro-assembled and in vivo=assemble filaments and accounting for the total cellular complement of Ure2p. To this end, we used electron tomography. In specimens whose preservation was optimized by freeze substitution, the filaments are seen to be non-hollow and 20 nm in diameter. In vitro-assembled filaments prepared in identical fashion are also non-hollow and of essentially the same diameter. In aggregates, the filaments are randomly oriented with occasional crossing points. We did not observe any connection of aggregates to other cytoplasmic components; ribosomes, otherwise abundant in the cytoplasm, are excluded. By comparing the amount of Ure2p per cell, determined biochemically, with the amount in filaments, quantitated from tomograms, we conclude that most if not all Ure2p is present in the aggregates. These observations are being prepared for publication. (3) Disposition and role of the highly charged middle domain (M-domain) of the Sup35p prion protein. In yeast cells infected with the PSI+ prion, the protein Sup35p forms aggregates and its activity in translation termination is down-regulated but not eliminated. Sup35p has an N-terminal prion domain; a highly charged M-domain of about 125 residues; and a functional C-terminal domain. By negative staining, cryo-EM, and scanning transmission EM (STEM), in vitro-assembled filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse cloud of C-domains, giving a full diameter of 65nm. In diameter (8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield 1 subunit per 0.47 nm for N-fibrils, NM-fibrils, and Sup35p filaments, further supporting the amyloid fibril backbone model. The 30nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations. The extended M-domain conformations offer an explanation for residual Sup35p activity in infected cells, whereby the C-domains remain free enough to be capable of some interaction with ribosomes. In FY11, this project was completed and published (2). Current research is focussed primarily on the production of well ordered filaments of Ure2p, intended for high resolution cryo-EM analyses of the backbone structure.