BIOPHYSICAL MECHANISMS OF PRION PROTEIN PATHOGENICITY

这项研究的长期目标是了解传播性神经退行性疾病(海绵状脑病或普恩疾病)传播的分子机制。我们在“纯蛋白质”假说的背景下讨论这一组织，该假说假设致病过程中的关键事件是细胞内的PrP/c蛋白转化为构象改变的、对蛋白酶具有抵抗性的形式PrP/RES。本项目的主要目的是全面表征重组普鲁恩蛋白在溶液和膜环境中的生物物理和构象性质，并确定致病突变对重组人普鲁恩蛋白热力学稳定性和折叠途径的影响：(2)确定致病突变对重组人普鲁恩蛋白三维结构的影响；(3)确定致病突变对重组普鲁恩蛋白聚集性质和无细胞转化效率的影响；(4)研究膜环境对蛋白及其家族突变体生物物理性质的影响。该项目的实验设计结合了生物物理、光谱和生物化学方法。含有与Pron病遗传型相对应的突变的重组Prion蛋白变体将在大肠杆菌中表达。利用光谱技术(圆二色谱、傅立叶变换红外光谱、核磁共振、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，利用FTIR光谱、刚果红结合实验、电子显微镜和无细胞转化实验对差异突变体进行表征。

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.