Structural Studies of Prion Fibrils and Other Protein Fibrils

朊病毒原纤维和其他蛋白质原纤维的结构研究

• 批准号: 8148954
• 负责人: ROBERT TYCKO
• 金额: $ 32.05万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/8148954
• 关键词: Prions; Proteins

Progress in FY2010 was in the following areas: 1. DETERMINATION OF BETA-SHEET STRUCTURES IN YEAST PRIONS. We showed that three yeast prion proteins adopt in-register parallel beta-sheet structures in their prion-active, amyloid fibril states. These include the Ure2p protein that produces the URE3 phenotype, the Sup35 protein that produces the PSI+ phenotype, and the Rnq1 protein that produces the PIN+ phenotype. We showed that the in-register parallel beta-sheet structure is maintained in mutants of Ure2p and Sup35 that have scrambled amino acid sequences in their prion domains, and that this structure is not dependent on hydration. These results should quell suggestions in the literature that yeast prions have beta-helix-like structures, rather than in-register parallel beta-sheet structures. 2. INVESTIGATIONS OF FUNCTIONAL AMYLOIDS. We carried out initial studies of two proteins that are believed to have biological functions in their amyloid state, namely CsgA (which forms so-called curli fibrils on the surface of bacteria, which have an adhesive function) and Pmel17 (which catalyzes melanin production in mammalian melanocytes). Neither CsgA nor Pmel17 fibrils appear to have the in-register parallel beta-sheet structures found in yeast prion fibrils and in beta-amyloid fibrils. Instead, they may have beta-helical structures. An extensive study of Pmel17 fibrils shows that these fibrils are highly polymorphic, and that different polymorphs differ in the identity of pseudo-repeat sequences that comprise the fibril core. 3. INSULIN FIBRIL STRUCTURES. Solid state NMR measurements on fibrils formed by human insulin indicate conversion of native alpha-helices to beta-strands (collaboration with M. Weiss). The solid state NMR data place constraints on possible molecular structures, showing that models for insulin fibrils in the literature (developed in absence of detailed experimental data) are not correct. Insulin fibril formation is a serious public health problem, particularly in areas that lack refrigeration, as stored insulin tends to convert to fibrils at elevated temperatures. 4. MAMMALIAN PRION PROTEIN FIBRILS. Solid state NMR measurements on fibrils formed by recombinant mammalian PrP (full-length, Syrian hamster sequence) show that these fibrils contain parallel beta-sheets, and that the fibril core is formed primarily by a 30-residue segment near the C-terminus (collaboration with I.V. Baskakov). Although these fibrils are not infectious as prions, they may resemble infectious PrP aggregates in many respects.

2010财政年度的进展如下： 1.酵母朊病毒β-片层结构的测定 我们发现，三种酵母朊病毒蛋白在朊病毒活性淀粉样原纤维状态下采用了平行的β折叠结构。 这些包括产生URE 3表型的Ure 2 p蛋白、产生PSI+表型的Sup 35蛋白和产生PIN+表型的Rnq 1蛋白。 我们发现，在登记的平行β-折叠结构是保持在突变体的Ure 2 p和Sup 35，在他们的朊病毒结构域的乱序氨基酸序列，这种结构是不依赖于水化。 这些结果应该平息了文献中的建议，即酵母朊病毒具有β-螺旋样结构，而不是平行的β-折叠结构。 2. 功能性淀粉样蛋白的表达 我们对两种蛋白质进行了初步研究，这两种蛋白质被认为在淀粉样蛋白状态下具有生物学功能，即CsgA（在细菌表面形成所谓的卷曲纤维，具有粘附功能）和Pmel 17（催化哺乳动物黑色素细胞中的黑色素生成）。 CsgA和Pmel 17原纤维似乎都不具有在酵母朊病毒原纤维和β-淀粉样蛋白原纤维中发现的配准平行β-折叠结构。 相反，它们可能具有β-螺旋结构。 对Pmel 17原纤维的广泛研究表明，这些原纤维是高度多态性的，并且不同的多晶型物在组成原纤维核心的假重复序列的同一性方面不同。 3. 胰岛素纤维结构。 对人胰岛素形成的原纤维的固态NMR测量表明天然α-螺旋转化为β-链（与M.韦斯）。 固态NMR数据对可能的分子结构进行了限制，表明文献中的胰岛素原纤维模型（在缺乏详细实验数据的情况下开发）不正确。 胰岛素原纤维形成是一个严重的公共卫生问题，特别是在缺乏冷藏的地区，因为储存的胰岛素在高温下往往会转化为原纤维。 4.哺乳动物朊病毒蛋白纤维。 对重组哺乳动物PrP（全长，叙利亚仓鼠序列）形成的原纤维的固态NMR测量表明，这些原纤维含有平行的β-折叠，并且原纤维核心主要由C-末端附近的30个残基片段形成（与I. V. Baskakov合作）。 虽然这些纤维不像朊病毒那样具有感染性，但它们在许多方面可能类似于感染性PrP聚集体。