Structural Investigations of the Prion Protein het-s

描述（申请人提供）：“纯蛋白质假说”指出，朊病毒疾病，如羊瘙痒症、牛海绵状脑病（BSE）和人类克雅氏病，与细菌、病毒或类病毒引起的传染病不同，因为疾病的起源与普遍存在的蛋白质的构象改变有关，并且核酸对于病毒的传播不是必需的。传染源因此，朊病毒是感染性蛋白质，其通过将蛋白质的正常形式转化为改变的富含β折叠的构象来繁殖。朊病毒蛋白也已在低等真核生物中被鉴定，即酵母和丝状真菌Podospora anserina。迄今为止，总共只有四种不同的蛋白质可能采取朊病毒状态，并且只有在鹅柄孢霉中的het-s朊病毒系统中，才令人信服地表明het-s朊病毒蛋白确实是感染因子。此外，het-s蛋白是唯一已知的朊病毒蛋白，其朊病毒状态pHET-s是正常细胞功能的一部分，即细胞融合不相容性，这是丝状真菌的共同特征。本提案中所述项目的目的是利用溶液状态核磁共振光谱（NMR）、其他生物物理技术、诱变和体内研究，对丝状真菌Podospora anserina的het-s朊病毒系统的组成部分进行结构分析。het-s蛋白的非朊病毒形式和朊病毒无能力类似物的三维结构将被确定，并伴随诱变作用以阐明对感染性产生重要的空间区域和残基。此外，朊病毒形式的het-s蛋白的结构研究将被启动，以了解het-s蛋白的构象转变，这已与感染性。单个组分的结构知识和与朊病毒表型的产生相关的构象转变的详细分析，因此将扩展我们对het-s系统的理解，特别是对朊病毒的机制及其感染性起源的理解。

DESCRIPTION (provided by applicant): The "protein-only hypothesis" states that prion diseases such as scrapie in sheep, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease in human are distinct from infectious diseases caused by bacteria, viruses, or viroids, in that the origin of the disease is related to conformational alterations of an ubiquitous protein and that nucleic acids are not essential for the propagation of the infectious agent. Thus, prions are infectious proteins which propagate by converting the normal form of the protein into an altered beta-sheet-rich conformation. Prion proteins have also been identified in lower eukaryotes, namely yeast and the filamentous fungus Podospora anserina. Totally, there are only four different proteins known so far which may adopt a prion state, and only for the het-s prion system in Podospora anserina has it been shown convincingly that the het-s prion protein is indeed the infectious agent. Furthermore, the het-s protein is the only known prion protein of which the prion state, pHET-s, is part of a normal cellular function, namely cell fusion incompatibility, a common feature in filamentous fungi. The aim of the project described in this proposal is to get structural insights into the components of the het-s prion system of the filamentous fungus Podospora anserina using solution-state nuclear magnetic resonance spectroscopy (NMR), other biophysical techniques, mutagenesis and in vivo studies. The three-dimensional structures of the non-prion form of the het-s protein and a prion-incompetent analog will be determined and accompanied with mutagnesis to elucidate the spatial regions and the residues important for the generation of infectivity. Furthermore, structural studies of the prion form of the het-s protein will be initiated to get insights into the conformational transition of the het-s protein which has been assoicated with infectivity. The structural knowledge of the individual components and detailed analysis of the conformational transition which is associated with the generation of a prion phenotype will therefore extend our understanding of the het-s system in particular, and also of the mechanism of prions and their origin of infectivity in general.