Biochemistry of Infectious Prions

描述(申请人提供)：传染性普恩的生物化学“纯蛋白质”假说认为普恩是感染性蛋白质，缺乏信息性的核酸。最近对丝状真菌和酵母的研究证实了这一假说适用于特定的真菌蛋白。哺乳动物的Prion也是在体外产生的。然而，科赫的假设对于哺乳动物的普恩来说仍然没有实现，因为野生型普恩的体外繁殖研究使用了粗匀浆而不是纯化的蛋白质；截断的、折叠成淀粉样纤维的合成PrP分子到目前为止还没有直接接种到非转基因动物中导致疾病。我们实验室最近开发了一种体外系统，仅使用纯化的PrPC和合成的核酸分子就可以扩增PrPres，PrPres是一种与感染性哺乳动物Prion相关的抗蛋白酶蛋白构象。值得注意的是，初步数据表明，这个纯化的系统可以在体外自动催化形成PrPres分子。我们的具体目标将集中在严格测试“纯蛋白质”假说，并调查翻译后修饰对PrPC到PrPres体外转化的影响。目的1.在体外纯化系统中产生和繁殖具有感染性的哺乳动物普恩病毒。目的2.确定纯化的PrP分子能否在体外忠实地繁殖哺乳动物PrP的株系特性。目的3.研究PrP翻译后修饰对PrP扩增效率和特异性的影响。总而言之，我们建议严格测试非正统的想法，即克雅病(CJD)等疾病的感染源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.