Conformational Conversions of Prion Protein

描述（由申请人提供）：我们研究的长期目标是了解传染性海绵状脑病（TSE）（也称为朊病毒疾病）致病过程的分子机制。尽管传染性疾病病原体的确切性质存在争议，但一种流行的模型是基于“仅蛋白质”假设。根据这一假设，致病过程中的关键事件是朊病毒蛋白从正常的ct螺旋型PrPC转化为构象改变的、耐蛋白酶的、富含β -sheet的PrPSc。本项目的主要目标是阐明朊病毒蛋白构象转化的机制，并深入了解异常的、富含β -sheet的构象的结构。拟议的研究包括重组人朊病毒蛋白以及从患病脑中分离的异常PrPSc亚型的实验。最近，我们已经证明，在适当的实验条件下，重组人朊病毒蛋白huprp9o - 231可以转化为具有类似于脑PrPS的物理化学性质的富含β -片的寡聚形式。第一个特定目的是表征重组prpsc样模型的构象结构。该蛋白的结构将通过荧光光谱方法（荧光猝灭，共振能量转移）进行探测，使用一系列带有基因工程单色氨酸残基的蛋白质变体和外源性荧光探针。第二个特异性目的是确定与疾病相关的c -截断的Y145Stop变异体（残基23-144）形成淀粉样蛋白的机制。与这种变异相对应的重组蛋白自发地经历了向淀粉样原纤维的自我繁殖转变，为研究朊病毒蛋白构象转换的机制方面提供了一个有吸引力的和实验上可获得的模型。最后的具体目的是确定与遗传性朊病毒疾病相关的突变对患病人脑真实PrPSC构象特性的影响。用于此目的的主要技术是傅里叶变换红外光谱。

DESCRIPTION (provided by applicant): The long-term objective of our research is to understand the molecular mechanisms of the pathogenic process in transmissible spongiform encephalopathies (TSE), also known as prion diseases. Although the precise nature of an infectious agent in TSEs is controversial, a prevailing model is based on the 'protein-only' hypothesis. According to this hypothesis, the key event in the pathogenic process is the conversion of the prion protein from its normal, ct-helical form, PrPC, to a conformationally altered, protease-resistant, beta-sheet rich form, PrPSc The major goal of this project is to elucidate the mechanism of the conformational conversion(s) of the prion protein and gain insight into the structure of the abnormal, beta-sheet-rich conformer. The proposed study involves experiments with the recombinant human prion protein as well as with the abnormal PrPSc isoform(s) isolated from diseased brain. Recently, we have shown that, under appropriate experimental conditions, the recombinant human prion protein huPrP9O-23 1 can be converted to an oligomeric beta-sheet-rich form with physicochemical properties similar to those of brain PrPS. The first Specific Aim is to characterize the conformational structure of this recombinant PrPSc-like model. The structure of the protein will be probed by fluorescence spectroscopic methods (fluorescence quenching, resonance energy transfer) using a series of protein variants with genetically engineered single tryptophan residues and extrinsic fluorescent probes. The second Specific Aim is to determine the mechanism of amyloid formation by the disease-associated, C-truncated Y145Stop variant (residues 23-144) of the human prion protein. The recombinant protein corresponding to this variant spontaneously undergoes a self-propagating transition to amyloid fibrils, providing an attractive and experimentally accessible model for studying mechanistic aspects of the conformational conversion(s) in the prion protein. The final Specific Aim is to determine the effect of mutations associated with inherited prion diseases on conformational properties of authentic PrPSC from diseased human brain. The main technique to be used for this purpose is Fourier-transform infrared spectroscopy.