In Vitro studies on the formation of prion amyloid

本研究的最终目的是利用小鼠PrP^C在体外形成具有感染性的Prion淀粉样蛋白，利用小鼠PrP^C进行了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，采用在成熟PrP^C的N端带有组氨酸标签的小鼠重组PrP^C。重组PrP^C在室温、pH值5.2的条件下，在百分之一的小鼠瘙痒病毒存在下1天转化为抗蛋白酶K(PK)的形式，14天后抗PK能力进一步增强。PK抗性重组PrP^C的CD谱显示α-螺旋含量减少，刚果红结合估计β-折叠含量增加，而在无小鼠PrP^C孵育14天后，重组PrP^C对PK的抗性略有增加，而α-螺旋和β-折叠含量无明显变化。这些事实表明，重组PrP^C在小鼠PrP^C存在下发生了结构转换。加入百分之一的PK抗性重组PrP^C也可以诱导转化，但加入40 mM的对应于小鼠PrP密码子113-141的合成肽可以抑制转化。在聚丙烯酰胺凝胶电泳法中，PK处理后重组PrP^C的相对分子质量没有变化，而PK处理后PrP^C的相对分子质量有所下降。这表明，PK抗性重组PrP^C的结构与PrP^C的结构不同。除了PrP^C和Prion外，可能还需要一些未知因素才能形成Prion淀粉样蛋白。与这一主题相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.