Biochemistry of Infectious Prions

描述（由申请人提供）：传染性朊病毒的生物化学“纯蛋白质”假说提出朊病毒是缺乏信息核酸的传染性蛋白质。最近在丝状真菌和酵母中的研究已经证明了这一假说的特定真菌蛋白质。哺乳动物朊病毒也在体外产生。然而，科赫的假设仍然没有实现的哺乳动物朊病毒，因为在体外朊病毒繁殖的野生型朊病毒的研究已经使用粗匀浆，而不是纯化的蛋白质;和截短的，合成的PrP分子折叠成淀粉样蛋白原纤维，迄今尚未引起疾病时，直接接种在非转基因动物。我们的实验室最近开发了一种体外系统，扩增PrPres，蛋白酶抗性蛋白构象与感染性哺乳动物朊病毒，仅使用纯化的PrPC和合成的核酸分子。重要的是，初步数据表明，这种纯化的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的“蛋白质”的假设，并在研究翻译后修饰的影响PrPC到PrPres的体外转化。目标1。在体外纯化系统中产生并增殖感染性哺乳动物朊病毒。目标2.目的：探讨纯化的PrP分子能否忠实地复制哺乳动物朊病毒的菌株特性。目标3.研究PrP翻译后修饰对朊病毒增殖效率和特异性的影响。总之，我们建议严格测试非正统的想法，如克雅氏病（CJD）的传染性病原体可以由一个单一的蛋白质。

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.