Structural Biology of Amyloid and Amyloid-like Proteins

淀粉样蛋白和类淀粉样蛋白的结构生物学

• 批准号: 7964941
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 66.34万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964941
• 关键词: Agreement; Amyloid; Amyloid Fibrils; Area; Biochemical; Bovine Spongiform Encephalopathy; C-terminal; Caliber; Catabolism; Cells; Classification; Clinical; Coiled-Coil Domain; Collaborations; Complex; Cytoplasmic Protein; Data; Deposition; Disease; Disease model; Domestic Animals; Electrons; Filament; Freezing; Genetic; Genetic Polymorphism; Glutamine; Goals; Human; In Situ; In Vitro; Individual; Infection; Kuru; Literature; Measurement; Modeling; Molds; Molecular Conformation; Morphology; N-terminal; National Institute of Diabetes and Digestive and Kidney Diseases; Neurodegenerative Disorders; Nitrogen Fixation Genes; Non-Insulin-Dependent Diabetes Mellitus; Paper; Parkinson Disease; Peptide Hydrolases; Periodicity; Podospora anserina; Preparation; Prions; Property; Proteins; Publishing; Recombinants; Research; Resistance; Resolution; Rheumatoid Arthritis; Scrapie; Side; Source; Specimen; Structure; Surface; Techniques; Testing; Thermodynamics; Thick; Triplet Multiple Birth; Uncertainty; Vertebral column; Work; Yeasts; amyloid formation; base; beta pleated sheet; density; electron density; electron tomography; fibrillogenesis; gain of function; insight; islet amyloid polypeptide; monomer; polypeptide; programs; protein aggregate; protein folding; protein structure; response; solid state nuclear magnetic resonance; structural biology; yeast prion

We started studying the structures of yeast prions in 1998, in collaboration with R Wickner (NIDDK), 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 certain 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; exploring its range of applicability; and investigating fibril polymorphism. In FY09, we focussed on three areas: (1) Systematic analysis of beta-arcade motifs in amyloid fibril models and beta-solenoid protein structures. Amyloid fibrils accumulate in diseases, such as Alzheimers or type II diabetes. The amyloid-forming protein is disease-specific. Amyloids may also be formed in vitro from many other proteins. Unlike the diverse native folds of these proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and in having cross-β-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 β-arcades, columnar structures produced by in-register stacking of β-arches. A β-arch is a strand-turn-strand motif in which the two β-strands interact via their side-chains, not via the polypeptide backbone as in a conventional β-hairpin. Crystal structures of β-solenoids, a class of proteins with amyloid-like properties, offer insight into the β-arc turns found in arches. General thermodynamic considerations suggest that complexes of two or more β-arches may nucleate amyloid fibrillogenesis. (2) Formation of both infectious and non-infectious amyloid fibrils by the HET-s prion protein of the filamentous fungus, Podospora anserina. This prion differs from the yeast prions protein, Ure2p and Sup35p, in producing a gain-of-function prion, and in not having an abnormally high content of Asn/Gln residues. In 2007, we published a paper showing by electron diffraction that Het-s fibrils have a cross-beta structure (as anticipated), and by EM-based mass measurements that it has an axial packing density of 1 subunit per 0.94 nm - half the density of Ure2p and Sup35p fibrils, and in agreement with a published model, the stacked beta-solenoid. In another study, also completed in 2007, we compared the amyloids formed by the HET-s prion domain at different pH's. Fibrils formed at pH 7 from those formed at pH 2 on morphological grounds and in having higher specific infectivity. In FY09, we investigated the correlation between infectivity and fibril morphology further, by cryo-EM. Above pH 3, singlet fibrils are produced while, below pH 3, triplet fibrils are obtained. Singlets have an axial periodicity of 400 and a left-handed twist. Triplet fibrils have three supercoiled protofibrils whose diameter (50 ), and axial packing density (1 subunit per 9.4 ) resemble those of singlet fibrils but whose axial repeat and supercoiling differ. In triplet fibrils formed at higher pH, the interactions among protofibrils appear to loosen, eventually leading to their separation into individual protofibrils. By fitting a published atomic model derived from solid state NMR into tthe electron density of triplet fibrils, we investigated the inter-protofibril surface and suggest some key residues (E235, R238 and R274) that could be important in the interaction. (3) Fibrillation in situ and in vitro of the yeast prion protein Ure2p. For the most part, biochemical studies of amyloid formation are carried out in vitro with purified protein, often from exogenous recombinant sources. Genetic studies are carried out in situ. In this context, there has been long-standing uncertainty as to whether the same form of aggregate is being studied in both cases. With Ure2p, in vitro-formed fibrils have been electroporated into uninfected yeast cells and shown to elicit infection, albeit with low efficiency. However, it has not been possible to isolate fibrils from infected cells for structural analysis. In FY09, we have approached this problem by studying the Ure2p fibrils present in infected cells by electron tomography of freeze-substituted preparations. In this way, we have been able to estimate the total amount of fibrillar Urep present in these cells and found that it correlates with the amount per cells ascertained by biochemical quantitation. Thus all or almost all of the Ure2p protein in these cells is in fibrillar. The in situ fibrils appear to be slightly thicker than their in vitro-assembled counterparts. Our interpretation is that both kinds of fibrils have essentially the same structures and the in situ thickening probably represents a deposition of other cytoplasmic proteins onto the fibrils in response to specimen preparation for EM.

我们在1998年开始研究酵母朊病毒的结构，与R Wickner（NIDDK）合作，最初专注于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。拓扑相似的结构是某些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，探索其适用范围，并调查原纤维多态性。 在2009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。淀粉样蛋白原纤维在疾病中积累，如阿尔茨海默病或II型糖尿病。淀粉样蛋白是疾病特异性的。淀粉样蛋白也可以在体外由许多其他蛋白质形成。与这些蛋白质的多样性天然折叠不同，它们的淀粉样蛋白在刚性、光滑侧面和具有交叉结构方面基本相似。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是由-拱，-拱的对齐堆叠产生的柱状结构。A-弓是链-转角-链基序，其中两条链通过它们的侧链相互作用，而不是如在常规发夹中那样通过多肽主链相互作用。晶体结构的β-淀粉样蛋白，一类蛋白质与淀粉样蛋白的性质，提供深入了解的圆弧转向发现在拱门。一般的热力学考虑表明，两个或多个-拱的复合物可能使淀粉样纤维形成成核。 (2)丝状真菌鹅柄孢菌的HET-s朊病毒蛋白形成感染性和非感染性淀粉样纤维。这种朊病毒与酵母朊病毒蛋白Ure 2 p和Sup 35 p的不同之处在于产生功能获得性朊病毒，并且不具有异常高含量的Asn/Gln残基。2007年，我们发表了一篇论文，通过电子衍射显示Het-s原纤维具有交叉β结构（如预期的那样），并且通过基于EM的质量测量，它具有每0.94 nm 1个亚基的轴向堆积密度-Ure 2 p和Sup 35 p原纤维密度的一半，并且与已发表的模型一致，堆叠的β螺线管。在2007年完成的另一项研究中，我们比较了不同pH值下HET-s朊病毒结构域形成的淀粉样蛋白。在pH 7时形成的原纤维从在pH 2时形成的原纤维在形态学上看具有更高的特异性感染性。在2009财年，我们通过冷冻电镜进一步研究了感染性和原纤维形态之间的相关性。在pH 3以上，产生单线态原纤维，而在pH 3以下，获得三线态原纤维。单线态具有400的轴向周期性和左手扭转。三重态原纤维具有三个超螺旋原纤维，其直径（50）和轴向堆积密度（1个亚基/9.4）类似于单线态原纤维，但其轴向重复和超螺旋不同。在较高pH下形成的三重原纤维中，原纤维之间的相互作用似乎变松，最终导致它们分离成单个原纤维。通过拟合来自固态NMR的已发表的原子模型到三重态原纤维的电子密度，我们研究了间原纤维表面，并提出了一些关键残基（E235，R238和R274），可能是重要的相互作用。 (3)酵母朊病毒蛋白Ure 2 p的原位和体外原纤化。 在大多数情况下，淀粉样蛋白形成的生物化学研究是在体外用纯化的蛋白质进行的，通常来自外源重组来源。遗传学研究是在现场进行的。在这方面，长期以来一直不确定是否在这两种情况下研究相同形式的总量。使用Ure 2 p，体外形成的原纤维已被电穿孔到未感染的酵母细胞中，并显示出引起感染，尽管效率较低。然而，还不可能从受感染的细胞中分离原纤维用于结构分析。在2009财年，我们通过冷冻替代制剂的电子断层扫描研究感染细胞中存在的Ure 2 p原纤维来解决这个问题。通过这种方式，我们已经能够估计这些细胞中存在的纤维状Urep的总量，并发现它与通过生化定量确定的每个细胞的量相关。因此，这些细胞中所有或几乎所有的Ure 2 p蛋白都是纤维状的。原位原纤维似乎比它们在体外组装的对应物稍厚。我们的解释是，这两种纤维具有基本相同的结构和原位增厚可能代表了其他细胞质蛋白沉积到原纤维上，以响应标本制备EM。