Molecular Mechanisms of Prion and Amyloid Propagation

描述(由申请人提供)：普里恩概念，根据该概念，仅蛋白质就可以感染，这代表了生物学中的一种新范式。这种仅含蛋白质的感染物被认为是传染性海绵状脑病的罪魁祸首，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。哺乳动物的Prion通过一种机制进行繁殖，该机制涉及到一个主要是a-螺旋的细胞内Prion蛋白PrPC向错误折叠的富含淀粉样蛋白聚集体PrPSc的构象转换。最近，Prion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。这种构象感染性和遗传的特别令人费解的特征是，在同一寄主物种中存在与离散的疾病表型相关联的所谓普恩菌株，以及它们在传播性障碍中的作用，这些障碍经常阻止不同物种之间有效的普恩传播。虽然最近的研究表明，Pron菌株起源于蛋白质在形成自传播的淀粉样聚集体时错误折叠成多个不同构象的能力，但由于缺乏高分辨率结构数据，分子水平上对这些现象的理解一直受到阻碍。在这个项目中，我们的目标是通过详细了解淀粉样蛋白传播的分子机制和结构基础，以及非传染性Y145Stop PrP变异体(PrP23-144)的传递性障碍，促进对基于蛋白质构象的遗传和传染性的基本方面的理解。这个C-截短的PrP突变体与人类遗传性脑淀粉样血管病变有关，并在体外表现出PrP繁殖的一些最基本的方面，包括PrP菌株和物种障碍的现象。具体目标集中于确定和验证人类PrP23-144淀粉样蛋白聚集体的详细结构模型，并阐明与PrP23-144 C末端附近两个关键氨基酸残基突变有关的具有独特传递特征(即种子特异性)的不同PrP23-144淀粉样蛋白菌株的结构差异。现代多维固体核磁共振波谱将是这项研究中采用的主要实验结构技术。此外，我们还将使用氢/氚交换溶液的核磁共振方法、原子力显微镜和倾斜光束透射电子显微镜。 与公共卫生相关：Prion是一种传染性蛋白质，被认为会导致传染性海绵状脑病(TSE)，这是一组致命的神经系统疾病，包括人类的克雅氏病、牛的“疯牛”病、鹿和麋鹿的慢性消耗性疾病以及绵羊的瘙痒病。Prion菌株和传递障碍的现象都与Prion聚集体的结构特征有关，是Prion和其他类淀粉样蛋白聚集体传播机制的两个关键特征。了解这些现象的原子水平的细节将提供新的见解的分子基础的Pron和淀粉样蛋白疾病，对公众健康是至关重要的。

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.