STRUCTURAL STUDIES ON PRION PROTEINS

大量的实验数据令人信服地证明了传染性的普恩 颗粒大部分(如果不是全部)是由瘙痒异构体组成的。 之前的蛋白质(PrPSc)。更多的调查表明， PrPSc是由细胞内的PrPc蛋白通过一种 翻译后过程，发生在PrPC到达细胞之后 由糖基磷脂酰肌醇(GPI)锚定的表面 半个月。目前还没有确定任何候选的化学修饰 到目前为止，可能会区分PrPSc和PrPC。这些实验结果 认为从PrPC到PrPSc的转换很可能涉及到 蛋白质的构象变化。在这里提出的研究中， 我们计划改进PrPc的纯化，并确定是否 PrPC的一级结构包括翻译后修饰 与已经为PrPSc确定的值相同。第二， PrPc和PrPSc的三元和四元结构及其合成 PRP多肽将使用光谱方法进行测定。这些 研究应有助于阐明该蛋白的构象变化。 不同类型的瘙痒病是由于细微的差异 PrPSc的翻译后修改，包括数组 多相天冬酰胺连接的寡糖和GPI锚定。我们 建议调查产生瘙痒病传染性的可能性 通过变性的PRSC的复性以及对其进行修饰 PrPc、重组PrP90-228和合成PrP多肽的折叠。我们 希望建立最小感染性的大小和成分 用锌离子或焦碳酸二乙酯(DEPC)微扰普里子粒子 可逆地，PrPSc的结构。锌离子和二乙基苯丙氨酸的测定结果 以及用合成的PrP多肽获得的与His111有关的 PrPSc的合成及其在羊瘙痒病毒中的繁殖 传染性。放射性标记的DEPC将被用来检验以下假设 当His111被甲乙氧基化时，瘙痒病的传染性就会降低。 对Pron病的分子机制的研究应 对该病的病因、发病机制和治疗提供新的见解 更普遍的神经退行性疾病，如阿尔茨海默病。

A wealth of experimental data argues persuasively that infectious prion particles are composed largely, if not entirely, of the scrapie isoform of the prior protein (PrPSc). Additional investigations have shown that PrPSc is formed from the cellular prion protein (PrPc) by a posttranslational process, which occurs after PrPC reaches the cell surface where it is anchored by a glycosyl phosphatidyl-inositol (GPI) moiety. No candidate chemical modifications have been identified that might distinguish PrPSc from PrPC, to date. These experimental results argue that it is likely that the conversion from PrPC into PrPSc involves a conformational change in the protein. In the studies proposed here, we plan to refine the purification of PrPc and establish whether the primary structure of PrPC including the posttranslational modifications is identical to that already determined for PrPSc. The secondary, tertiary and quaternary structures of PrPC and PrPSc as well as synthetic PrP peptides will be determined using spectroscopic methods. These studies should help elucidate the conformational changes involved in the various "strains" of scrapie are due to detailed differences in the posttranslational modifications of PrPSc including the arrays of heterogeneous asparagine-linked oligosaccharides and the GPI anchor. We propose to investigate the possibility of generating scrapie infectivity in vitro through refolding of denatured PrSc as well as modifying the folding of PrPc, recombinant PrP90-228 and synthetic PrP peptides. We hope to establish the size and composition of the smallest infectious prion particle using Zn2+ or diethylpyrocarbonate (DEPC) to perturb reversibly the structure of PrPSc. The results with Zn2+ ions and DEPC as well as those obtained with synthetic PrP peptides implicate His111 in the synthesis of PrPSc and the propagation of scrapie prion infectivity. Radiolabeled DEPC will be used to test the hypothesis that scrapie infectivity is diminished when His111 is carbethoxylated. Studies of the molecular mechanisms responsible for prion diseases should give new insights into the etiology, pathogenesis and treatment of the more prevalent neurodegenerative disorders such as Alzheimer's disease.