BIOPHYSICAL MECHANISMS OF PRION PROTEIN PATHOGENICITY

朊病毒蛋白致病性的生物物理机制

• 批准号: 6188123
• 负责人: WITOLD K SUREWICZ
• 金额: $ 26万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6188123
• 关键词: calorimetry; cell free system; chemical stability; circular dichroism; 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传染性神经退行性疾病（称为海绵状脑病或朊病毒病）传播的分子机制。我们在“仅蛋白质”假说的背景下讨论了这种组织，该假说假定致病过程中的关键事件是细胞朊病毒蛋白PrP/c转化为构象改变的蛋白酶抗性形式，PrP/本项目的主要目标是提供重组朊病毒蛋白在溶液和膜中的生物物理和构象特性的综合表征（2）确定致病性突变对重组人朊病毒蛋白的三维结构的影响;（3）确定致病性突变对重组朊蛋白的聚集特性和无细胞转化效率的影响;（4）表征膜环境对朊蛋白及其家族突变体的生物物理特性的影响。该项目的实验设计结合了生物物理、光谱和生物化学方法。含有对应于朊病毒疾病遗传形式的突变的重组朊病毒蛋白变体将在E.杆菌将使用光谱技术（圆二色谱、傅里叶变换红外光谱、NMR、荧光光谱）研究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FTIR光谱、刚果红结合试验、电子显微镜和无细胞转化试验表征差异突变体。