Structural Biology of Amyloid and Amyloid-like Proteins

淀粉样蛋白是以交叉β结构为特征的异常折叠蛋白质的丝状聚合物。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朊病毒。我们于1998年开始研究酵母朊病毒结构，最初集中在Ure 2 p，一种氮催化剂的负调节剂。我们发现，它的N-末端结构域是负责朊病毒发生，而C-末端结构域，执行其调节功能仍然折叠在细丝，但失活的空间机制。在我们的淀粉样蛋白骨架概念中，朊病毒结构域形成丝状骨架，并被C-末端结构域包围。2005年，我们发表了淀粉样蛋白骨架的平行重叠β结构模型。它设想通过堆叠具有平面β-蛇形折叠的单体产生的平行β-片层阵列。拓扑相似的结构是其他淀粉样纤维的良好候选者，包括胰淀素，并且越来越多的科学文献中出现了对这种模型的支持。正在进行的工作旨在测试和完善这一模型，调查原纤维多态性，并与淀粉样蛋白的天然构象。在2011财年，我们专注于三个领域：

 (1)淀粉样蛋白原纤维模型中β-拱廊基序和β-螺线管蛋白结构的系统分析。与天然蛋白质的多样折叠不同，它们的淀粉样蛋白在刚性、光滑侧面和具有交叉β结构方面基本相似。尽管获得高分辨率实验确定的原纤维结构的困难，越来越可信的模型正在通过整合来自多种技术的数据。目前大多数与疾病相关的淀粉样蛋白模型都涉及β-拱，β-拱的对齐堆叠产生的柱状结构。β-拱形是链-转角-链基序，其中两条β-链通过其侧链相互作用，而不是如在常规β-发夹中那样通过多肽主链相互作用。β-淀粉样蛋白是一类具有淀粉样性质的蛋白质，它的晶体结构提供了对在拱门中发现的β-弧转弯的深入了解。一般的热力学考虑表明，两个或多个β-拱的复合物可能使淀粉样纤维形成成核。8232;在2011财年，我们发表了一篇关于beta-arcades的综述（1）。 (2)感染的酵母细胞中Ure 3朊病毒丝的可视化。野生型Ure 2 p是一种可溶性二聚体蛋白，有助于调节S.啤酒。在朊病毒形式下，Ure 2 p聚集并失去活性。我们早期的工作薄切片电子显微镜感染的细胞过度表达Ure 2 p表明，聚集体有一个丝状的子结构，并通过免疫金标记，该丝含有Ure 2 p。在体外，Ure 2 p组装成具有被球状结构域包围的淀粉样原纤维骨架的细丝。在2011财年，我们扩展了这一研究路线，目的是在体外组装和体内组装细丝之间进行更密切的比较，并解释Ure 2 p的总细胞补体。为此，我们使用了电子断层扫描。在通过冷冻置换优化保存的标本中，可以看到细丝是非中空的，直径为20 nm。以相同方式制备的体外组装的长丝也是非中空的并且具有基本上相同的直径。在聚集体中，细丝随机取向，偶尔有交叉点。我们没有观察到聚集体与其他细胞质成分的任何联系;核糖体，否则丰富的细胞质中，被排除在外。通过比较每个细胞的Ure 2 p的量，确定生物化学，与细丝中的量，从断层扫描定量，我们得出结论，大多数，如果不是所有的Ure 2 p是存在于聚集体。这些意见正在编写中，以便发表。 (3)Sup 35 p朊病毒蛋白的高电荷中间结构域（M-结构域）的分布和作用。在感染PSI+朊病毒的酵母细胞中，蛋白质Sup 35 p形成聚集体，其翻译终止活性下调但未消除。Sup 35 p具有N-末端朊病毒结构域;约125个残基的高电荷M-结构域;和功能性C-末端结构域。通过负染色、冷冻电镜和扫描透射电镜（STEM），全长Sup 35 p的体外组装的细丝显示出被C结构域的弥散云包围的细骨架原纤维，给出65 nm的全直径。在直径（8 nm）和外观上，骨架类似于单独的N结构域的淀粉样纤维。对于N-原纤维、NM-原纤维和Sup 35 p细丝，STEM每单位长度质量数据产生每0.47 nm 1个亚基，进一步支持淀粉样蛋白原纤维骨架模型。装饰C-结构域的30 nm径向跨度表明M-结构域呈现高度延伸的构象。扩展的M-结构域构象提供了一个解释剩余的Sup 35 p活性在感染的细胞，其中C-结构域保持足够的自由，能够与核糖体的一些相互作用。在2011财年，该项目已完成并发布（2）。 目前的研究主要集中在生产良好有序的细丝的Ure 2 p，旨在高分辨率cryo-EM分析的骨干结构。

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.