Self-Propagating Mechanism of Prion Diseases

描述(由申请人提供)：Prion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。“纯蛋白质”假说认为，异常蛋白构象(PrPSc)通过招募相同蛋白质的正常异构体(PrPC)，以一种自我催化的方式自我繁殖，因此，它是疾病的传播者。尽管多年来的努力，PrPSc在体外从合成成分中重建一直是困难的。这些困难归因于缺乏可靠的Pron感染性生化标记物，以及我们对感染性所必需的物理性质的了解不足。在之前的资助期间，我们开发了第一个将全长PrP转化为自蔓延淀粉样纤维的实验程序；我们描述了PrP聚合的几种途径；我们介绍了PrP转化最全面的机制；我们还建立了几种新的分析方法，包括用于探测单个PrP纤维或颗粒中的构象的免疫构象分析。在目前的应用中，我们建议阐明PrPSc的超微结构，并建立PrPSc的感染性和物理性质之间的联系。第一个具体目标是使用我们实验室开发的新的免疫构象分析结合高分辨率原子力显微镜来阐明PrPSc的亚结构。第二个特定目标将阐明体外产生的普恩纤维的超微结构，第三个特定目标旨在测试体外产生的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灵敏的生前诊断方法和治疗普恩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.