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后果。“仅蛋白”假说假设异常朊蛋白构象（PrPSc）通过招募相同蛋白（PrPC）的正常异构体以自催化方式进行自我繁殖，因此充当疾病的传播因子。尽管经过多年的努力，在体外用合成成分重建PrPSc是很困难的。这些困难是由于缺乏可靠的朊病毒传染性的生化标记，以及我们对感染性所必需的物理特性的了解不足。在之前的资助期间，我们开发了第一个将全长PrP转化为自繁殖淀粉样原纤维的无细胞实验程序；我们描述了PrP聚合的几种途径；我们介绍了最全面的PrP转化机制；我们还建立了几种新的检测方法，包括用于探测单个PrP纤维或颗粒内构象的免疫构象检测。在目前的应用中，我们建议阐明PrPSc的超微结构，并建立感染性与朊病毒原纤维物理性质之间的联系。第一个具体目标是利用我们实验室开发的新型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。第二个特异性目的是阐明体外生成的朊病毒原纤维的超微结构，第三个特异性目的是测试体外生成的朊病毒原纤维的构象性质与其内在感染性之间的关系。这些知识应该为开发敏感的生前诊断和有效的治疗朊病毒疾病的方法奠定基础。

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.