Biochemistry of Infectious Prions

描述（由申请人提供）：感染性朊病毒的生物化学“仅蛋白”假说认为，朊病毒是感染性蛋白，缺乏信息核酸。最近对丝状真菌和酵母菌的研究证实了这一假说对特定真菌蛋白的影响。哺乳动物朊病毒也已在体外生成。然而，对于哺乳动物朊病毒，科赫的假设仍然没有实现，因为在野生型朊病毒的体外传播研究中，使用的是粗均质物，而不是纯化的蛋白质；截断的合成PrP分子被重新折叠成淀粉样原纤维，当直接接种在非转基因动物身上时，迄今尚未引起疾病。我们的实验室最近开发了一种体外系统，该系统仅使用纯化的PrPC和合成的核酸分子来扩增PrPres， PrPres是与传染性哺乳动物朊病毒相关的蛋白酶抗性蛋白构象。值得注意的是，初步数据表明，该纯化系统可以在体外自催化形成PrPres分子。我们的具体目标将集中在严格测试“仅蛋白”假设，并在体外研究翻译后修饰对prpc到prpres转化的影响。目的1。在体外纯化的系统中产生和繁殖传染性哺乳动物朊病毒。目标2。目的观察纯化的PrP分子能否在体外忠实地传播哺乳动物朊病毒的菌株特性。目标3。目的探讨PrP翻译后修饰对朊病毒增殖效率和特异性的影响。总之，我们建议严格检验非正统的观点，即疾病的传染因子，如克雅氏病（CJD）可以由单一蛋白质组成。

DESCRIPTION (provided by applicant): Biochemistry of Infectious Prions The "protein-only" hypothesis proposes that prions are infectious proteins, which lack informational nucleic acids. Recent studies in filamentous fungi and yeast have proven this hypothesis for specific fungal proteins. Mammalian prions have also been generated in vitro. However, Koch's postulates remain unfulfilled for mammalian prions because in vitro prion propagation studies of wild type prions have used crude homogenates rather than purified proteins; and truncated, synthetic PrP molecules refolded into amyloid fibrils have thus far not caused disease when directly inoculated in non-transgenic animals. Our laboratory has recently developed an in vitro system that amplifies PrPres, the protease-resistant protein conformer associated with infectious mammalian prions, using only purified PrPC and synthetic nucleic acid molecules. Significantly, preliminary data indicate that this purified system can autocatalytically form PrPres molecules in vitro. Our specific aims will focus upon rigorously testing the "protein-only" hypothesis, and upon investigating the effects of post-translational modifications on PrPC-to-PrPres transformation in vitro. Aim 1. To generate and propagate infectious mammalian prions in a purified system in vitro. Aim 2. To determine whether strain properties of mammalian prions can be faithfully propagated by purified PrP molecules in vitro. Aim 3. To investigate the effects of PrP post-translational modifications on the efficiency and specificity of prion propagation. In summary, we propose to test rigorously the unorthodox idea that the infectious agent of diseases such as Creutzfeldt-Jakob disease (CJD) can be composed of a single protein.