Self-Propagating Mechanism of Prion Diseases

描述（由申请人提供）：朊病毒蛋白（PrP）是一系列疾病的基础，没有既定的治疗方法，对人类和经济造成毁灭性的后果。“仅蛋白质”假说假定异常朊病毒蛋白构象（PrPSc）通过募集相同蛋白质的正常同种型（PrPC）以自催化的方式自我繁殖，因此充当疾病的传播因子。尽管多年的努力，PrPSc在体外从合成组分的重建一直难以实现。这些困难归因于缺乏朊病毒感染性的可靠生化标志物，以及我们对感染性所必需的物理性质的理解不足。在上一个资助期间，我们开发了第一个实验程序，用于将全长PrP无细胞转化为自繁殖淀粉样蛋白原纤维;我们描述了PrP聚合的几种途径;我们介绍了PrP转化的最全面机制;我们还建立了几种新的检测方法，包括用于探测单个PrP原纤维或颗粒内构象的免疫构象检测。在本申请中，我们建议阐明PrPSc超微结构，并建立感染性和朊病毒原纤维的物理性质之间的联系。第一个具体的目标将阐明的PrPSc的亚结构，使用新的免疫构象分析结合高分辨率原子力显微镜在我们的实验室开发。第二个具体目标将阐明体外产生的朊病毒原纤维的超微结构，第三个具体目标旨在测试体外产生的原纤维的构象特性与其内在感染性之间的关系。这些知识应该为开发敏感的死前诊断和治疗朊病毒疾病的有效疗法奠定基础。

DESCRIPTION (provided by applicant): Prion protein (PrP) underlies a spectrum of diseases with no established treatment and devastating human and economic consequences. The "protein-only" hypothesis postulates that an abnormal prion protein conformation (PrPSc) propagates itself in an autocatalytic manner by recruiting normal isoform of the same protein (PrPC) and, therefore, acts as a transmissible agent of disease. The reconstitution of PrPSc in vitro from synthetic components has been difficult to achieve despite many years of effort. These difficulties are attributed to the lack of reliable biochemical markers of prion infectivity and to our poor understanding of the physical properties that are essential for infectivity. During the previous funding period, we developed the first experimental procedure for cell-free conversion of full-length PrP into self-propagating amyloid fibrils; we described several pathways of PrP polymerization; we introduced the most comprehensive mechanism of PrP conversion; we also established several novel assays including an immunoconformational assay for probing conformation within a single PrP fibril or particle. In the present application, we propose to elucidate PrPSc ultrastructure and to establish a link between infectivity and physical property of prion fibrils. The first specific aim will elucidate the substructure of PrPSc using novel immunoconformational assay developed in our laboratory combined with high resolution Atomic Force Microscopy. The second specific aim will elucidate the ultrastructure of prion fibrils generated in vitro, and the third specific aim is designed to test a relationship between conformational properties of the in vitro generated fibrils and their intrinsic infectivity. Such knowledge should lay the foundation for development of sensitive antemortem diagnostics and efficient therapeutics for treating prion diseases.