Molecular Mechanisms of Prion and Amyloid Propagation

描述（由申请人提供）：朊病毒的概念，根据该概念，蛋白质单独可以是感染性的，代表了生物学中的一种新范式。这种仅蛋白质的传染性病原体被认为是引起传染性海绵状脑病的原因，传染性海绵状脑病是一组致命的神经退行性疾病，包括人类的克雅氏病和牛的牛海绵状脑病。哺乳动物朊病毒通过一种机制繁殖，该机制涉及主要是α-螺旋细胞朊病毒蛋白PrPC向错误折叠的富含淀粉样蛋白的聚集体PrPSc的构象转化。最近，朊病毒假说被进一步扩展到包括酵母和其他真菌中基于蛋白质构象的遗传现象。这种构象感染性和遗传性的特别令人困惑的特征是存在与同一宿主物种内的离散疾病表型相关的所谓朊病毒株，以及它们在传播性障碍中的作用，这些障碍经常阻止朊病毒在不同物种之间的有效传播。虽然最近的研究表明，朊病毒株起源于蛋白质的能力，错误折叠成多个不同的构象形成自我繁殖的淀粉样蛋白聚集体，这些现象的分子水平的理解已受到阻碍的缺乏高分辨率的结构数据。在这个项目中，我们的目标是通过提供详细的见解淀粉样蛋白的传播和非传染性Y145 Stop PrP变体（PrP 23 -144）的传播障碍的分子机制和结构基础，推进对蛋白质构象为基础的遗传和感染性的基本方面的理解。这种C-截短的PrP突变体与人类遗传性脑淀粉样血管病相关，并在体外表现出PrP传播的一些最基本的方面，包括朊病毒株和物种障碍的现象。具体目标集中于确定和验证人PrP 23 -144淀粉样蛋白聚集体的详细结构模型，并阐明与具有独特传播特征的不同PrP 23 -144淀粉样蛋白菌株的出现相关的结构差异（即，接种特异性）与PrP 23 -144的C-末端附近的两个关键氨基酸残基的突变相关。现代多维固态核磁共振光谱将是主要的实验结构技术在研究中使用。此外，我们将使用氢/氘交换溶液NMR方法，原子力显微镜和倾斜束透射电子显微镜。

DESCRIPTION (provided by applicant): The prion concept, according to which proteins alone can be infectious, represents a new paradigm in biology. Such protein-only infectious agents are believed to be responsible for transmissible spongiform encephalopathies, a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Mammalian prions propagate by a mechanism involving the conformational conversion of a largely a-helical cellular prion protein, PrPC, to misfolded, ¿-rich amyloid-like aggregates, PrPSc. More recently, the prion hypothesis has been extended further to include the phenomenon of protein conformation-based inheritance in yeast and other fungi. Particularly puzzling features of such conformational infectivity and inheritance are the existence of so-called prion strains linked to discrete disease phenotypes within the same host species, and their role in transmissibility barriers which frequently prevent efficient prion transmission between different species. While recent studies indicate that prion strains originate from the ability of proteins to misfold into multiple distinct conformers in forming self-propagating amyloid aggregates, molecular-level understanding of these phenomena has been hampered by the paucity of high-resolution structural data. In this project we aim to advance the understanding of fundamental aspects of protein conformation-based inheritance and infectivity by providing detailed insights into the molecular mechanisms and structural basis of amyloid propagation and transmissibility barriers for a non-infectious Y145Stop PrP variant (PrP23-144). This C-truncated PrP mutant is associated with hereditary cerebral amyloid angiopathy in humans and exhibits in vitro some of the most fundamental aspects of PrP propagation including the phenomena of prion strains and species barriers. The specific aims focus on the determination and validation of a detailed structural model for human PrP23-144 amyloid aggregates, and elucidation of the structural differences related to the emergence of distinct PrP23-144 amyloid strains with unique transmission characteristics (i.e., seeding specificities) linked to mutations of two critical amino acid residues near the C-terminus of PrP23-144. Modern multidimensional solid-state NMR spectroscopy will be the primary experimental structural technique employed in the study. In addition, we will use hydrogen/deuterium exchange solution NMR methods, atomic force microscopy and tilted-beam transmission electron microscopy.