BIOPHYSICAL MECHANISMS OF PRION PROTEIN PATHOGENICITY

朊病毒蛋白致病性的生物物理机制

• 批准号: 6639568
• 负责人: WITOLD K SUREWICZ
• 金额: $ 28.41万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2005-01-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6639568
• 关键词: calorimetry; cell free system; chemical stability; circular dichroism; gene mutation; infrared spectrometry; membrane activity; membrane lipids; nuclear magnetic resonance spectroscopy; pathologic process; prions; protein denaturation; protein folding; protein structure function; recombinant proteins; stop flow technique; thermodynamics; ultraviolet spectrometry

The long term objective of this research is to understand the molecular mechanism underlying propagation of transmissible neurodegenerative disorders known as spongiform encephalopathies or prion diseases. We address this tissue within the context of the 'protein-only' hypothesis which postulates that the key event in the pathogenic process is the conversion of the cellular prion protein, PrP/c, to a conformationally altered, protease-resistant form, PrP/res. The major goal of this project is to provide comprehensive characterization of the biophysical and conformation properties of the recombinant prion protein in solution and in a membrane environment, and to determine how these properties are affected by the effect of pathogenic mutations on the thermodynamic stability and the folding pathway on the recombinant stability and the folding pathway on the recombinant human prion protein; (2) to determine the effect of pathogenic mutations on the three dimensional structure of the recombinant human prion protein; (3) To determine the effect of pathogenic mutations on the aggregation properties and the efficiency of cell-free conversion of the recombinant prion protein; (4) To characterize the effect of a membrane environment on the biophysical properties of prion protein and familial mutants thereof. The experimental design of this project constitutes a combination of biophysical, spectroscopic and biochemical approaches. Recombinant prion protein variants containing mutations corresponding to hereditary forms of prion disease will be expressed in E. coli. The conformational properties, thermodynamic stability and the folding pathway of these proteins in solution and a membrane environment will be studied using spectroscopic techniques (circular dichroism, Fourier-transform infrared spectroscopy, NMR, fluorescence spectroscopy), differential mutants will be characterized using FTIR spectroscopy, Congo red binding assay, electron microscopy, and the cell-free conversion assay.

这项研究的长期目标是了解被称为海绵状脑病或朊病毒疾病的传染性神经退行性疾病传播的分子机制。我们在“仅蛋白”假说的背景下处理这种组织，该假说假设致病过程中的关键事件是细胞朊蛋白PrP/c转化为构象改变的蛋白酶抗性形式PrP/res。本项目的主要目标是全面表征重组朊病毒蛋白在溶液和膜环境中的生物物理和构象特性，并确定致病突变对重组朊病毒蛋白热力学稳定性和折叠途径对重组朊病毒蛋白稳定性和折叠途径的影响对这些特性的影响；(2)确定致病突变对重组人朊病毒蛋白三维结构的影响；(3)确定致病突变对重组朊病毒蛋白聚集特性和无细胞转化效率的影响；(4)表征膜环境对朊病毒蛋白及其家族突变体生物物理特性的影响。本项目的实验设计结合了生物物理、光谱学和生化方法。重组朊病毒蛋白变异包含相应的遗传形式的朊病毒疾病的突变将在大肠杆菌中表达。这些蛋白质在溶液和膜环境中的构象性质、热力学稳定性和折叠途径将使用光谱技术（圆二色性、傅里叶变换红外光谱、核磁共振、荧光光谱）进行研究，差异突变体将使用FTIR光谱、刚果红结合试验、电子显微镜和无细胞转化试验进行表征。