Molecular Mechanisms of Prion and Amyloid Propagation

描述（由申请人提供）：朊病毒的概念，根据蛋白质单独可以具有传染性，代表了生物学的新范式。这种仅含蛋白质的传染性病原体被认为是传染性海绵状脑病的罪魁祸首。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进行繁殖，这种机制涉及将大部分呈螺旋状的细胞朊蛋白PrPC转化为错误折叠的富含¿的淀粉样蛋白聚集体PrPSc。最近，朊病毒假说已经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。这种构象传染性和遗传的特别令人困惑的特征是，在同一宿主物种中存在与离散疾病表型相关的所谓朊病毒株，以及它们在传播障碍中的作用，这些障碍经常阻止不同物种之间的有效朊病毒传播。虽然最近的研究表明，朊病毒菌株起源于蛋白质在形成自我繁殖的淀粉样蛋白聚集体时错误折叠成多个不同的构象的能力，但由于缺乏高分辨率的结构数据，对这些现象的分子水平理解受到阻碍。在这个项目中，我们的目标是通过对非传染性Y145Stop PrP变体（PrP23-144）的淀粉样蛋白增殖的分子机制和结构基础以及传播障碍提供详细的见解，促进对蛋白质构象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有关，并在体外表现出PrP繁殖的一些最基本方面，包括朊病毒株和物种屏障现象。具体目标集中在确定和验证人类PrP23-144淀粉样蛋白聚集体的详细结构模型，并阐明与不同PrP23-144淀粉样蛋白菌株出现相关的结构差异，这些菌株具有独特的传播特征（即播种特异性），与PrP23-144 c端附近两个关键氨基酸残基突变相关。现代多维固体核磁共振波谱将是研究中主要的实验结构技术。此外，我们将使用氢/氘交换溶液核磁共振方法，原子力显微镜和倾斜光束透射电镜。

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. PUBLIC HEALTH RELEVANCE: Prions are infectious proteins thought to cause transmissible spongiform encephalopathies (TSEs), a group of fatal neurological conditions that include Creutzfeldt-Jakob disease in humans, "mad cow" disease in cattle, chronic wasting disease in deer and elk, and scrapie in sheep. The phenomena of prion strains and transmission barriers, both related to the structural characteristics of prion aggregates, are two critical features underlying the propagation mechanism of prions and, indeed, other amyloid-like protein aggregates. Understanding these phenomena with atomic level detail will provide novel insights about the molecular basis of prion and amyloid disorders and is of fundamental importance for public health.