Structural Mechanism of Mammalian Prion Infectivity

哺乳动物朊病毒感染性的结构机制

• 批准号: 10191067
• 负责人: Surachai Supattapone
• 金额: $ 59.69万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10191067
• 关键词: Alzheimer' s Disease; Amino Acids; Animals; Biochemical; Biological; Bovine Spongiform Encephalopathy; Chemicals; Coupling; Creutzfeldt-Jakob Syndrome; Data; Development; Diagnostic; Digestion; Dissociation; Electron Microscopy; Elements; Endopeptidase K; Goals; Human; Infectious Agent; Isotope Labeling; Isotopes; Laboratories; Lead; Light; Link; Livestock; Maintenance; Maps; Mass Spectrum Analysis; Measurement; Membrane Lipids; Methods; Molecular; Molecular Conformation; Molecular Structure; Neurodegenerative Disorders; Parkinson Disease; Pathogenesis; Pattern; Phosphatidylethanolamine; Positioning Attribute; PrP; Prion Diseases; Prions; Process; Property; Protein Subunits; Proteins; Recombinants; Registries; Research; Resolution; Role; Rotation; Sampling; Shapes; Side; Structural Models; Structure; System; Techniques; Time; Vaccines; Variant; Vertebral column; Work; beta pleated sheet; cofactor; conformer; experimental study; nervous system disorder; novel; pathogenic isoform; protein misfolding; protein protein interaction; protein structure; public health relevance; recombinant PrP; solid state; solid state nuclear magnetic resonance; tool

Project Summary Mammalian prions, which cause fatal neurodegenerative diseases in humans and livestock animals, are unorthodox infectious agents that replicate by the autocatalytic conformational change of the host prion protein (PrPC) into a pathogenic isoform (PrPSc). Taking a reductionist biochemical approach, we made the surprising discovery that endogenous cofactors such as the membrane lipid phosphatidylethanolamine (PE) are required to produce infectious PrPSc molecules. For example, autocatalytic PrPSc molecules formed with a combination of recombinant PrP and PE substrates display a specific infectivity >105-fold greater than that of similar autocatalytic PrPSc molecules formed from PrP alone. We have taken advantage of this specific information to produce, for the first time, sufficient quantities of fully infectious, isotopically labeled, and conformationally homogeneous recombinant prions to perform high-resolution solid state (ss) NMR analysis of PrPSc structure. Here, our collaborative team proposes to use ssNMR, electron microscopy, and mass spectrometry analysis to elucidate the important structural elements that lead to infectivity. ssNMR will be used to determine and compare the secondary structure maps of infectious and non-infectious PrPSc molecules. This work will help identify and characterize specific infectivity-associated domains as well as crucial NMR residue-peak assignments. Together with constraints from electron microscopy and mass spectrometry analysis to identify the position of cut points using digestion by proteinase K, these data will eventually enable structural determination of the entire PrPSc molecule. We will also use ssNMR to determine precisely the structural mechanism by which PE induces the infectious prion conformation. Finally, we will use 15N-13C transferred- echo double resonance (TEDOR) to determine the symmetry pattern of PrPSc subunits in isotopically mixed samples of infectious recombinant prions. These data will allow us to discriminate between competing b-solenoid and in-register b-sheet quaternary structure models of infectious PrPSc molecules. Overall, this proposal is a critical step towards determining the full high-resolution structural determination of infectious mammalian prions as well as the structural mechanism of prion infectivity.

项目概要 哺乳动物朊病毒，导致人类和牲畜致命的神经退行性疾病 动物，是通过自催化构象复制的非正统传染原 宿主朊病毒蛋白 (PrPC) 转变为致病亚型 (PrPSc)。采取还原论 通过生化方法，我们令人惊讶地发现内源性辅助因子，例如 膜脂磷脂酰乙醇胺 (PE) 是产生传染性 PrPSc 所必需的 分子。例如，自催化 PrPSc 分子由以下物质的组合形成： 重组 PrP 和 PE 底物表现出的特异性感染性比重组的 PrP 和 PE 底物高 105 倍 类似的自催化 PrPSc 分子仅由 PrP 形成。我们已经利用了 这些具体信息首次产生了足够数量的完全 感染性、同位素标记、构象均质的重组朊病毒 对 PrPSc 结构进行高分辨率固态 (ss) NMR 分析。在这里，我们的 合作团队建议使用单链核磁共振、电子显微镜和质谱 分析以阐明导致传染性的重要结构要素。 ssNMR 将是 用于确定和比较感染性和非感染性的二级结构图 PrPSc 分子。这项工作将有助于识别和描述与特定感染性相关的特征 结构域以及重要的 NMR 残基峰分配。加上来自的约束 使用电子显微镜和质谱分析来确定切割点的位置 通过蛋白酶 K 消化，这些数据最终将能够确定整个 PrPSc 分子。我们还将使用 ssNMR 来精确确定结构机制 PE 诱导感染性朊病毒构象。最后，我们将使用15N-13C转移- 回声双共振 (TEDOR) 以确定 PrPSc 亚基的对称模式 感染性重组朊病毒的同位素混合样品。这些数据将使我们能够 区分竞争的 b-螺线管模型和注册 b-片四元结构模型 感染性 PrPSc 分子。总体而言，该提案是朝着确定 传染性哺乳动物朊病毒的完整高分辨率结构测定 作为朊病毒感染性的结构机制。