Structure and assembly of the fungal prion HET-s

描述（由申请人提供）：朊病毒是一种异常折叠的蛋白质，与致命的传染性海绵状脑病（tse）有关。tse包括羊的痒病，牛的牛海绵状脑病（BSE），鹿和麋鹿的慢性消耗性疾病（CWD），以及人类几种形式的克雅氏病（CJD）。除了tse之外，其他神经退行性疾病，如阿尔茨海默病和帕金森病，在其病理中也含有类似朊病毒的行为，这一点已经变得很明显。这些疾病的一个共同特征是异常折叠的蛋白质和肽聚集成淀粉样蛋白原纤维。虽然淀粉样蛋白原纤维参与疾病的机制尚不清楚，但它们分子结构的差异与病理的差异有关，这种特性被称为应变现象，表明结构和病理是相互依存的。为了了解这些疾病的病理，必须确定相关淀粉样蛋白的分子结构。然而，由于淀粉样蛋白原纤维的异质性和无序性，这是一项特别困难的任务。在开发技术的努力中，除了扩大我们对淀粉样蛋白形成机制的理解外，本研究还旨在开发功能性真菌朊病毒het - 5的朊病毒形成域，作为研究病理性朊病毒和朊病毒相关淀粉样蛋白结构的模型系统。HET-s朊病毒形成结构域（218-289）是一个理想的模型系统，因为感染性原纤维的形成具有可重复性和易感性，但该结构域仍具有相对复杂的折叠，可与病理相关的朊病毒相比较。第一个目标将是利用x射线纤维衍射和固态核磁共振（ssNMR）的联合细化方法确定et -s（218-289）的感染形式的结构。第二个目标将是使用位点定向诱变来探索HET- s正确折叠中结构元件的必要性和冗余性（218-289）。第三个目标将是表征传染性和几种非传染性的het - 5（218- 289）物种，它们共同可以作为菌株现象的类比。表征方法包括x射线纤维衍射、荧光光谱和电子显微镜。这些目标将允许更深入地了解朊病毒折叠的机制以及正确折叠所需的关键要素。根据前两个目标的结果，对het - 5（218-289）物种进行表征，将有助于深入了解环境条件和决定最终淀粉样蛋白结构的氨基酸序列之间的相互作用。虽然这些研究不能取代对脑源性朊病毒的研究，但它们为理解朊病毒折叠和多态性的机制提供了一个框架。初步结果表明，HET-s（218-289）可用于结构测定和生物物理表征，并与病理性朊病毒具有相似性和可扩展性。

DESCRIPTION (provided by applicant): Prions are aberrantly folded proteins implicated in the invariably fatal transmissible spongiform encephalopathies (TSEs). TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and several forms of Creutzfeldt-Jakob disease (CJD) in humans. In addition to the TSEs, it has become apparent that other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, contain prion-like behavior in their pathologies. One common feature of these diseases is the aggregation of aberrantly folded proteins and peptides into amyloid fibrils. While the mechanism by which amyloid fibrils are involved in disease is unknown, differences in their molecular structures have been linked with differences in pathologies, a property called the strain phenomenon, which indicates that structure and pathology are interdependent. In order to understand the pathology of these diseases, it is imperative to determine the molecular structures of the implicated amyloids. However, this is a particularly difficult task owing to the heterogeneous and disordered nature of amyloid fibrils. In efforts to develop techniques, in addition to expanding our mechanistic understanding of amyloid formation, the proposed work aims to develop the prion forming domain of the functional fungal prion HET-s as a model system in studying the structure of pathological prions and prion-related amyloids. The HET-s prion forming domain, (218-289), is an ideal model system because infectious fibrils can be formed reproducibly and easily, but the domain still possesses a relatively complex fold comparable to those of pathologically relevant prions. The first aim will be to determine the structure of the infectious form of HET-s(218-289) using joint refinement methods between X-ray fiber diffraction and solid state NMR (ssNMR). The second aim will be to use site- directed mutagenesis to probe the necessity and redundancy of structural elements in correct folding of HET- s(218-289). The third aim will be to characterize infectious and several non-infectious species of HET-s(218- 289), which together can serve as an analog to the strain phenomenon. The methods of characterization will include X-ray fiber diffraction, fluorescence spectroscopy and electron microscopy. These aims will allow for a deeper understanding of the mechanism of prion folding as well as the critical elements necessary for correct folding. Characterization of HET-s(218-289) species, taken with the results of the first two aims, will provide insight into the interplay between environmental conditions and amino acid sequence that determine the final amyloid structure. While these studies cannot replace the need for studies of brain-derived prions, they provide a framework for understanding the mechanisms of prion folding and polymorphism. Preliminary results demonstrate the amenability of HET-s(218-289) to structure determination and biophysical characterization as well as its similarities to and subsequent extensibility in the study of pathological prions. PUBLIC HEALTH RELEVANCE: Prion diseases such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) as well as prion-related diseases such as Alzheimer's and Parkinson's diseases are a continually growing public health concern. The pathologies of these diseases are intricately associated with the molecular structures of aberrantly folded protein aggregates, termed amyloid, that are implicated in these diseases. The proposed studies aim to establish the fungal prion HET-s(218-289) as a model system in efforts to obtain useful structural and mechanistic information that will further advance efforts in the study of disease-associated amyloids.