Structural Mechanism of Mammalian Prion Infectivity

项目摘要 哺乳动物的Prion，它会导致人和牲畜的致命神经退行性疾病 动物是一种非正统的感染体，通过自身催化构象进行复制 将宿主蛋白(PrPC)转变为致病异构体(PrPSc)。接受一个简化论者 生化方法，我们有一个令人惊讶的发现，内源性辅助因子，如 膜脂磷脂酰乙醇胺(PE)是产生感染性PrPSc所必需的 分子。例如，自催化PrPSc分子由以下物质组合形成 重组PrP和PE底物显示出比 由PrP单独生成类似的自催化PrPSc分子。我们已经利用了 这一特定信息第一次产生了足够数量的充分 传染性的、同位素标记的和构象均一的重组普鲁恩 进行PrPSc结构的高分辨固体(Ss)核磁共振分析。在这里，我们的 合作团队建议使用单核磁共振、电子显微镜和质谱学 分析以阐明导致传染性的重要结构要素。SS核磁共振将是 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表征与特定传染性相关的 结构域以及关键的核磁共振残基峰归属。以及来自以下方面的约束 用电子显微镜和质谱仪分析确定切割点的位置 被K酶消化，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ss核磁共振通过以下方式精确确定结构机理 哪种PE诱导具有感染性的朊病毒构象。最后，我们将使用15N-13C转移- 回声双共振(TEDOR)确定PrPSc亚基的对称性 具有感染性的重组普恩病毒的同位素混合样本。这些数据将使我们能够 竞争的b-螺线管和寄存器内b-薄片四元结构模型的区分 具有传染性的PrPSc分子。总体而言，这项提议是朝着确定 感染性哺乳动物朊病毒的全高分辨结构测定 作为普恩病毒感染性的结构机制。

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.