Structure and assembly of the fungal prion HET-s

真菌朊病毒HET-s的结构和组装

• 批准号: 8516940
• 负责人: William N Wan
• 金额: $ 1.56万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516940
• 关键词: Agreement; Alzheimer' s Disease; Amino Acid Sequence; Amyloid; Amyloid Fibrils; Asparagine; Behavior; Biological Models; Bovine Spongiform Encephalopathy; Brain; Cattle; Characteristics; Chronic Wasting Disease; Complex; Creutzfeldt-Jakob Syndrome; Crystallography; Data; Deer; Development; Disease; Electron Microscopy; Elements; Fiber; Fluorescence Spectroscopy; Genetic Polymorphism; Goals; Higher Order Chromatin Structure; Human; Hybrids; In Vitro; Indium; Infectious Agent; Joints; Link; Measures; Methodology; Methods; Modeling; Molecular; Molecular Structure; Nature; Neurodegenerative Disorders; Parkinson Disease; Pathology; Peptides; Phenotype; Podospora anserina; Prion Diseases; Prions; Process; Property; Proteins; Public Health; Publishing; Recording of previous events; Resolution; Roentgen Rays; Running; Scrapie; Sheep; Site-Directed Mutagenesis; Sodium Chloride; Structural Models; Structure; Symptoms; System; Techniques; Therapeutic; Variant; Work; X-Ray Crystallography; amyloid formation; amyloid structure; analog; base; fungus; human disease; in vivo; insight; meetings; prion-like; protein aggregate; protein folding; solid state nuclear magnetic resonance; therapy design; tool; yeast prion

DESCRIPTION (provided by applicant): Prions are aberrantly folded proteins implicated in the invariably fatal transmissible spongiform encephalopathies (TSEs). TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and several forms of Creutzfeldt-Jakob disease (CJD) in humans. In addition to the TSEs, it has become apparent that other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, contain prion-like behavior in their pathologies. One common feature of these diseases is the aggregation of aberrantly folded proteins and peptides into amyloid fibrils. While the mechanism by which amyloid fibrils are involved in disease is unknown, differences in their molecular structures have been linked with differences in pathologies, a property called the strain phenomenon, which indicates that structure and pathology are interdependent. In order to understand the pathology of these diseases, it is imperative to determine the molecular structures of the implicated amyloids. However, this is a particularly difficult task owing to the heterogeneous and disordered nature of amyloid fibrils. In efforts to develop techniques, in addition to expanding our mechanistic understanding of amyloid formation, the proposed work aims to develop the prion forming domain of the functional fungal prion HET-s as a model system in studying the structure of pathological prions and prion-related amyloids. The HET-s prion forming domain, (218-289), is an ideal model system because infectious fibrils can be formed reproducibly and easily, but the domain still possesses a relatively complex fold comparable to those of pathologically relevant prions. The first aim will be to determine the structure of the infectious form of HET-s(218-289) using joint refinement methods between X-ray fiber diffraction and solid state NMR (ssNMR). The second aim will be to use site- directed mutagenesis to probe the necessity and redundancy of structural elements in correct folding of HET- s(218-289). The third aim will be to characterize infectious and several non-infectious species of HET-s(218- 289), which together can serve as an analog to the strain phenomenon. The methods of characterization will include X-ray fiber diffraction, fluorescence spectroscopy and electron microscopy. These aims will allow for a deeper understanding of the mechanism of prion folding as well as the critical elements necessary for correct folding. Characterization of HET-s(218-289) species, taken with the results of the first two aims, will provide insight into the interplay between environmental conditions and amino acid sequence that determine the final amyloid structure. While these studies cannot replace the need for studies of brain-derived prions, they provide a framework for understanding the mechanisms of prion folding and polymorphism. Preliminary results demonstrate the amenability of HET-s(218-289) to structure determination and biophysical characterization as well as its similarities to and subsequent extensibility in the study of pathological prions.

描述（由申请人提供）：朊病毒是一种异常折叠的蛋白质，与致死性传染性海绵状脑病（TSE）有关。TSE包括绵羊的瘙痒症、牛的牛海绵状脑病（BSE）、鹿和麋鹿的慢性消耗病（CWD）以及人类的几种形式的克雅氏病（CJD）。除了TSEs之外，很明显，其他神经退行性疾病，例如阿尔茨海默病和帕金森病，在其病理学中也含有朊病毒样行为。这些疾病的一个共同特征是异常折叠的蛋白质和肽聚集成淀粉样纤维。虽然淀粉样蛋白纤维参与疾病的机制尚不清楚，但其分子结构的差异与病理学的差异有关，这种特性称为应变现象，表明结构和病理学是相互依赖的。为了了解这些疾病的病理学，必须确定所涉及的淀粉样蛋白的分子结构。然而，由于淀粉样蛋白原纤维的异质性和无序性，这是一项特别困难的任务。在努力开发技术，除了扩大我们的淀粉样蛋白形成的机制的理解，拟议的工作旨在开发朊病毒形成域的功能性真菌朊病毒HET-s作为模型系统，在研究病理性朊病毒和朊病毒相关的淀粉样蛋白的结构。HET-s朊病毒形成结构域（218-289）是理想的模型系统，因为感染性原纤维可以可重复地且容易地形成，但该结构域仍然具有与病理相关朊病毒相当的相对复杂的折叠。第一个目标将是使用X射线纤维衍射和固态NMR（ssNMR）之间的联合细化方法来确定HET-s（218-289）的感染形式的结构。第二个目的是使用定点诱变来探测HET-1正确折叠中结构元件的必要性和冗余性（218-289）.第三个目标是表征HET-s（218- 289）的感染性和几种非感染性物种，它们一起可以作为菌株现象的模拟物。表征方法将包括X射线纤维衍射、荧光光谱和电子显微镜。这些目标将允许更深入地了解朊病毒折叠的机制以及正确折叠所需的关键要素。HET-s（218-289）种类的表征，与前两个目标的结果一起，将提供对环境条件和决定最终淀粉样蛋白结构的氨基酸序列之间相互作用的深入了解。虽然这些研究不能取代对脑源性朊病毒的研究，但它们为理解朊病毒折叠和多态性机制提供了一个框架。初步结果表明HET-s（218-289）对结构确定和生物物理表征的顺从性以及其与病理性朊病毒研究的相似性和随后的可扩展性。