Structural Mechanism of Mammalian Prion Infectivity

哺乳动物朊病毒感染性的结构机制

• 批准号: 10610392
• 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/10610392
• 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; In Vitro; Infectious Agent; Isotope Labeling; Isotopes; Laboratories; Link; Livestock; Maintenance; Maps; Mass Spectrum Analysis; Measurement; Membrane Lipids; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Neurodegenerative Disorders; Nuclear Magnetic Resonance; Parkinson Disease; Pathogenesis; Pattern; Phosphatidylethanolamine; Positioning Attribute; PrP; Prion Diseases; Prions; Process; Property; Protein Subunits; Proteins; Recombinants; Registries; Research; Resolution; Role; Sampling; Shapes; Side; Structural Models; Structure; System; Techniques; Time; Vaccines; Variant; Vertebral column; Visualization; Wild Type Mouse; 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 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prions，引起人类和牲畜的致命神经退行性疾病 动物是非正统的传染剂，通过自催化构象复制 将宿主prion蛋白（PRPC）变成致病性同工型（PRPSC）。扮演还原主义者 生化方法，我们提出了令人惊讶的发现，内源性辅助因子（例如 需要膜脂质磷脂酰乙醇胺（PE）才能产生感染性PRPSC 分子。例如，自催化PRPSC分子形成，结合了 重组PRP和PE底物的特定感染率> 105倍以上 单独由PRP形成的类似的自催化PRPSC分子。我们已经利用了 这种特定信息首次产生足够数量的全部信息 感染性，同位素标记和构象同质的重组王子 对PRPSC结构进行高分辨率固态（SS）NMR分析。在这里，我们的 协作团队建议使用SSNMR，电子显微镜和质谱法 分析以阐明导致感染性的重要结构元素。 SSNMR会 用于确定和比较传染和非感染的二级结构图 PRPSC分子。这项工作将有助于识别并表征与特定感染相关的特定 域以及至关重要的NMR残留峰分配。加上限制 电子显微镜和质谱分析，以确定使用切口的位置 蛋白酶K的消化，这些数据最终将实现整个整个结构确定 PRPSC分子。我们还将使用SSNMR确切确定结构机制 PE引起了传染性的pr构构象。最后，我们将使用15n-13c转移 - 回声双共振（TEDOR）确定PRPSC亚基的对称模式 传染性重组王室的同位素混合样品。这些数据将使我们能够 区分竞争的B-甲状腺素和注册b-sheet Quaternary结构模型 传染性PRPSC分子。总体而言，该提议是确定的关键一步 传染性哺乳动物prions的完整高分辨率结构性确定 作为王室感染的结构机制。