Structural Mechanism of Mammalian Prion Infectivity

哺乳动物朊病毒感染性的结构机制

• 批准号: 10386899
• 负责人: Surachai Supattapone
• 金额: $ 53.92万
• 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/10386899
• 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; Modeling; 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; diagnostic tool; 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

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.

项目摘要 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，是非正统的传染性病原体，通过自催化构象复制 宿主朊病毒蛋白（PrPC）转变为致病性同种型（PrPSc）。把一个简化论者 通过生物化学方法，我们令人惊讶地发现，内源性辅因子，如 膜脂质磷脂酰乙醇胺（PE）是产生感染性PrPSc所必需 分子。例如，用以下物质的组合形成的自催化PrPSc分子 重组PrP和PE底物显示出比重组PrP和PE底物高>105倍的特异性感染性。 类似的自催化PrPSc分子由单独的PrP形成。我们充分发挥 这一具体的信息，以生产，第一次，足够数量的充分 感染性、同位素标记和构象均一的重组朊病毒 进行PrPSc结构的高分辨率固态（ss）NMR分析。这里我们的 一个合作小组建议使用ssNMR、电子显微镜和质谱法 分析以阐明导致传染性的重要结构要素。ssNMR将是 用于确定和比较传染性和非传染性的二级结构图 PrPSc分子。这项工作将有助于识别和表征特定的感染性相关 域以及关键的NMR残留峰分配。加上来自 电子显微镜和质谱分析，以确定切割点的位置， 通过蛋白酶K消化，这些数据最终将使整个结构的确定成为可能。 PrPSc分子。我们还将使用ssNMR来精确地确定结构机制， 该PE诱导感染性朊病毒构象。最后，我们将使用15 N-13 C转移- 回波双共振（TEDOR），以确定PrPSc亚基的对称性模式， 感染性重组朊病毒的同位素混合样本。这些数据将使我们能够 区分竞争的b-螺线管和配准的b-片四级结构模型 感染性PrPSc分子总的来说，这一提议是确定 传染性哺乳动物朊病毒的高分辨率结构测定 作为朊病毒感染性的结构机制。