In Vitro studies on the formation of prion amyloid

本研究的最终目标是使用小鼠PrP^C体外形成感染性朊病毒淀粉样蛋白，使用少量小鼠朊病毒进行PrP^C的体外结构转化。为了消除未知小鼠蛋白污染PrP^C的影响，使用在成熟PrP^C的N-末端具有组氨酸标签的小鼠重组PrP ^C。在pH5.2和室温下，在千分之一量的小鼠瘙痒症朊病毒存在下，重组PrP^C转化为蛋白酶K（PK）抗性形式，14天后，PK抗性增加更多。在PK抗性的重组PrP^C中，通过CD光谱估计的α-螺旋含量减少，通过刚果红结合估计的β-折叠含量增加，而在没有小鼠朊病毒的情况下孵育14天的重组PrP^C显示出PK抗性的轻微增加，而α-螺旋和β-折叠含量没有变化。这些事实表明，在小鼠朊病毒存在的情况下，重组PrP^C发生了结构转换。加入百分之一量的PK抗性重组PrP^C也能诱导转化，但加入40 mM对应于小鼠朊病毒密码子113-141的合成肽可抑制转化。聚丙烯酰胺凝胶电泳结果显示，抗PK重组PrP^C蛋白经PK处理后分子量无明显变化，而朊病毒多肽经PK处理后分子量下降。这表明抗PK重组PrP^C的结构不同于朊病毒。除了PrP^C和朊病毒外，朊病毒淀粉样蛋白的形成可能还需要一些未知的因子。与这一主题有关的其他研究也已完成。

As a final goal of this study is in vitro formation of infectious prion amyloid using mouse PrP^C, in vitro structural conversion of PrP^C using a small amount of mouse prion was carried out. To eliminate the effects of unknown mouse protein contaminating in PrP^C, mouse recombinant PrP^C which possessed a histidine tag at N-terminus of mature PrP^C was used. The recombinant PrP^C converted to a proteinase K (PK) resistant form in the presence of one-thousandth amounts of mouse scrapie prion at pH 5.2 and room temperature for one day and after 14 days the PK-resistance increased more. A decrease of alpha-helix contents estimated by CD spectrum and an increase of beta-sheet contents estimated by Congo red binding were observed in the PK-resistant recombinant PrP^C, while the recombinant PrP^C incubated for 14 days without mouse prion showed a slight increase of PK-resistance and no change in alpha-helix and beta-sheet contents. These facts indicate that structural conversion occurred in the recombinant PrP^C in the presence of mouse prion. The conversion was also induced by adding one-hundredth amounts of the PK-resistant recombinant PrP^C, but was inhibited by adding 40 mM of a synthetic peptide corresponding to mouse prion codons 113-141. The molecular weight of the PK-resistant recombinant PrP^C did not change after PK-treatment but that of prion polypeptide decreases after PK-treatment in polyacrylamide gel electrophoresis. This indicates that the structure of the PK-resistant recombinant PrP^C differed from that of prion. In addition to PrP^C and prion, some unknown factors may be required to form prion amyloid. Other studies in relation to this theme also have been done.