BIOPHYSICAL MECHANISMS OF PRION PROTEIN PATHOGENICITY

描述（由申请人提供）：本项目的长期目标是阐明 传染性海绵状脑病（一组折磨人类和动物的神经退行性疾病）致病过程的分子基础。据信，感染性病原体是朊病毒蛋白PrP的错误折叠形式，其通过与正常朊病毒蛋白结合并催化其转化为致病形式而自我繁殖。下一个资助期的第一个具体目标是深入了解朊病毒繁殖中氨基酸序列和菌株依赖性障碍的构象和生物物理基础。这些研究将使用最近开发的疾病相关朊病毒蛋白变体PrP 23 -144的有核构象转换体外模型进行。第二个具体目标是阐明重组朊病毒蛋白PrP 90 -231的构象转换的替代途径和机制。我们还将确定选定的疾病相关突变对这些转换途径的影响，并探讨使用重组PrP 90 -231作为模型研究朊病毒传播障碍的分子基础的可能性。最终的具体目标是确定稳定正常形式的人朊病毒蛋白的突变对人源化转基因小鼠感染朊病毒剂的易感性的影响，以及对体外人朊病毒蛋白的生物物理性质的影响。最近，我们已经建立了表达“超稳定”人类PrP变体的转基因小鼠系。我们将用来自CJD病例的朊病毒剂感染这些动物，假设它们应该比表达野生型人PrP的小鼠更能抵抗感染。我们还将确定这种朊病毒蛋白变异体的高度稳定性的生物物理和结构基础。该跨学科项目将采用各种实验方法，从生物物理技术（FTIR，CD和荧光光谱，氢交换，纤维X射线衍射，NMR，自旋标记）到转基因动物研究。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular basis of the pathogenic process in transmissible spongiform encephalopathies, a group of neurodegenerative diseases that afflict humans and animals. It is believed that the infectious pathogen is a misfolded form of the prion protein, PrP, which self propagates by binding to normal prion protein and catalyzing its conversion to the pathogenic form. The first specific aim for the next funding period is to gain insight into the conformational and biophysical basis of the amino acid sequence and strain dependent barriers in prion propagation. These studies will be performed using the recently developed in vitro model of nucleated conformational conversion of a disease-associated prion protein variant PrP23-144. The second specific aim is to elucidate alternative pathways and mechanisms of conformational conversion of the recombinant prion protein PrP90-231. We will also determine the effect of selected disease-associated mutations on these conversion pathways, and explore the possibility of using recombinant PrP90-231 as a model for studying the molecular basis of barriers in prion propagation. The final specific aim is to determine the effect of a mutation that stabilizes the normal form of human prion protein on the susceptibility of humanized transgenic mice to infection with the prion agent, and on biophysical properties of human prion protein in vitro. Recently, we have created lines of transgenic mice that express the 'superstable' human PrP variant. We will inoculate these animals with the prion agent from CJD cases, hypothesizing that they should be more resistant to infection than mice expressing the wild-type human PrP. We will also determine the biophysical and structural basis of the remarkably high stability of this prion protein variant. This interdisciplinary project will employ a variety of experimental approaches, ranging from biophysical techniques (FTIR, CD and fluorescence spectroscopy, hydrogen exchange, fiber x-ray diffraction, NMR, spin labeling) to transgenic animal studies.