Molecluar mechanism of propagation of abnormal prion protein in the cells

本研究通过分析抑制PrP^C形成的化合物的作用，<Sc>以及鉴定与朊病毒增殖有关的宿主因子和微环境，研究朊病毒在细胞内增殖的分子机制，发现4种不同的抗PrP抗体与细胞表面的PrP^C反应，抑制朊<Sc>病毒持续感染细胞中PrP^C的形成。细胞表面的抗体-PrP ^C复合物不能有效地内化，并且倾向于保留在细胞表面。这些结果表明，抗PrP mAb<Sc>通过干扰常规PrP^C降解途径来拮抗PrP^的形成。此外，我们筛选了合成的硫酸化糖苷用于抑制PrPSc形成<Sc>，并且发现4-磺基-N-乙酰基葡糖胺和6-磺基-N-乙酰基葡糖胺抑制朊病毒感染的细胞中的PrPSc形成。这些硫酸化糖苷加速了PrP^C的内吞作用，减少了PrP^C的总量，而硫酸化Gly ...更多信息 不抑制PrP ^C形成的糖苷<Sc>不能减少PrP^C的总量。这些结果表明硫酸化糖苷和糖胺聚糖<Sc>通过促进PrP^C的降解来抑制PrP^C的形成。我们建立了Neuro 2a（N2 a）小鼠神经母细胞瘤细胞的亚克隆，并区分朊病毒敏感和不敏感的亚克隆。一个不敏感的亚克隆N2 a-1表达的PrP^C水平与亲本N2 a和其他朊病毒敏感的亚克隆相同。PrP^<Sc>与敏感和非敏感亚克隆的结合没有差异。N2 a-1表达PrP^C，但对朊病毒增殖具有抗性，其存在表明朊病毒增殖中除PrP^C外还涉及宿主因子。为了鉴定这些宿主因子，通过DNA微阵列分析N2 a亚克隆之间的基因表达谱。我们选择了36个和18个基因，它们分别在朊病毒易感亚克隆N2 a-5和朊病毒抗性亚克隆N2 a-1中表达超过2倍。我们通过siRNA技术降低基因表达来评估这些基因对朊病毒易感性的影响，发现针对F2、A1和C5基因的siRNA抑制朊病毒感染的N2 a-5细胞中朊病毒的增殖。少

In this research, we studied on the molecular mechanism of prion propagation in the cells through analyses of the effect of compounds that inhibit PrP^<Sc> formation and identification of host factor(s) and microenvironment that are involved in prion propagation.Four different anti-PrP antibodies that react with PrP^C on the cell surface inhibited PrP^<Sc> formation in cells persistently infected with prion. The antibody-PrP^C complex on the cell surface was not internalized efficiently and tended to retain on the cell surface. These results suggest that anti-PrP mAb antagonized PrP^<Sc> formation by interfering with the regular PrP^C degradation pathway. In addition, we screened synthesized sulfated glycosides for the inhibition of PrP^<Sc> formation and found 4-sulfo-N-acetylglucosamie and 6-sulfo-N-acetylglucosamine inhibited PrPSc formation in prion-infected cells. These sulfated glycosides accelerated the endocytosis of PrP^C and reduced a total amount of PrP^C, while sulfated gly … More cosides that were not inhibited PrP^<Sc> formation did not reduce the total amount of PrP^C. These results indicated that sulfated glycosides and glycosaminoglycans inhibited PrP^<Sc> formation by facilitating the degradation of PrP^C.We established subclones of Neuro2a (N2a) mouse neuroblastoma cells and distinguished prion-susceptible and non-susceptible subclones. One non-susceptible subclone, N2a-1, expressed PrP^C as the same level as parental N2a and other prion-susceptible subclones. There was no difference in the binding of PrP^<Sc> to the susceptible and non-susceptible subclones. Presence of N2a-1, which expresses PrP^C but is resistance to prion propagation, indicated the involvement of host factors other than PrP^C in prion propagation. To identify such host factors, the gene expression profiles between N2a subclones were analyzed by DNA microarray. We selected 36 and 18 genes, which expressed more than two-fold in prion susceptible subclone N2a-5 and prion resistant subclone N2a-1, respectively. We assessed the influence of these genes on prion susceptibility by reducing the gene expression by siRNA technique and found that siRNA against F2, Al, and C5 genes inhibited prion propagation in prion-infected N2a-5 cells. Less