Structural Investigations of the Prion Protein het-s

描述(申请人提供)：“纯蛋白质假说”指出，普恩疾病，如绵羊瘙痒病、牛海绵状脑病(BSE)和人类的克雅氏病，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变化有关，而核酸对感染源的传播不是必需的。因此，Prion是一种感染性蛋白质，它通过将正常形式的蛋白质转化为富含β-折叠的改变的构象来传播。在低等真核生物，即酵母和丝状真菌Podospora anserina中也发现了Pron蛋白。到目前为止，只有四种不同的蛋白质可能处于Pron状态，并且只有Podospora anserina中的Het-S Prion系统被令人信服地证明Het-S Prion蛋白确实是侵染体。此外，Het-S蛋白是已知的唯一一种Prion蛋白，其Prion状态Phet-S是正常细胞功能的一部分，即细胞融合不亲和性，这是丝状真菌的共同特征。这项建议中所描述的项目的目的是利用溶液状态核磁共振光谱、其他生物物理技术、诱变和体内研究来深入了解丝状真菌Podospora anserina的het-S Prion系统的组成。我们将测定Het-S蛋白的非病毒形式和一个非病毒类似物的三维结构，并伴随着突变，以阐明对感染性产生重要的空间区和残基。此外，还将启动对het-S蛋白的普恩形式的结构研究，以深入了解与感染性相关的het-S蛋白的构象转变。因此，对单个组分的结构知识和与Prion表型产生相关的构象转变的详细分析将扩大我们对Het-S系统的理解，也将扩大我们对Prion的机制及其一般传染性来源的理解。

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.