STRUCTURAL STUDIES ON PRION PROTEINS

大量的实验数据令人信服地表明，传染性朊病毒 颗粒即使不是全部，也是大部分由羊瘙痒病同种型组成 蛋白质（PrPSc）。 进一步的调查表明， PrPSc由细胞朊病毒蛋白（PrPc）通过 翻译后过程，发生在PrPC到达细胞后 通过糖基磷脂酰肌醇（GPI）锚定的表面 部分。 尚未确定候选化学修饰， 可以区分PrPSc和PrPC。 这些实验结果 我认为，从PrPC到PrPSc的转换可能涉及 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 PrPC的一级结构，包括翻译后修饰 与已测定的PrPSc相同。 第二， PrPC和PrPSc的三级和四级结构以及合成的 PrP肽将使用光谱法测定。 这些 研究应该有助于阐明参与的构象变化， 瘙痒病的各种“菌株”是由于在 PrPSc的翻译后修饰，包括 异质天冬酰胺连接的寡糖和GPI锚。 我们 建议研究产生羊瘙痒病传染性的可能性 在体外通过变性PrSc的重折叠以及修饰 PrPc、重组PrP 90 -228和合成PrP肽的折叠。 我们 希望能确定最小传染源的大小和组成 使用Zn 2+或焦碳酸二乙酯（DEPC）干扰朊病毒颗粒 PrPSc的结构。 用Zn 2+离子和DEPC的结果 以及用合成PrP肽获得的那些涉及His 111 PrPSc的合成和羊瘙痒症朊病毒的繁殖 传染性 放射性标记DEPC将用于检验以下假设： 当His 111被羧乙氧羰基化时，羊瘙痒病的传染性降低。 对朊病毒疾病分子机制的研究应该 为研究糖尿病的病因、发病机制和治疗提供了新的见解。 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.