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 具有感染性的、同位素标记的重组PrP进行固态(Ss)核磁共振 PrPSc结构分析。在这里，我们的协作团队建议使用ss核磁共振来 感染性和非感染性PrPSc二级结构图谱的测定和比较 分子。这项工作将有助于识别和表征特定的传染性相关区域。 以及关键的核磁共振残基峰分配，最终将使结构 整个PrPSc分子的测定。我们还将使用ss核磁共振精确地确定 PE维持传染性Pron构象的结构机制。最后，我们会 用15N-13C传递回波双共振(TEDOR)测定心脏的对称性 感染性重组蛋白的同位素混合样本中的PrPSc亚基。这些数据将 允许我们区分竞争的β电磁阀和寄存器中的β-Sheet四元 感染性PrPSc分子的结构模型。总体而言，这项提议是关键的一步。 朝着确定感染性的全高分辨结构确定的方向 哺乳动物的Pron以及Pron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, recombinant prions to perform solid state (ss) NMR analysis of PrPSc structure. Here, our collaborative team proposes to use ssNMR 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 that will eventually enable structural determination of the entire PrPSc molecule. We will also use ssNMR to determine precisely the structural mechanism by which PE maintains 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 β-solenoid and in-register β-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.