Molecluar mechanism of propagation of abnormal prion protein in the cells

在本研究中，我们通过分析抑制PrP^<Sc>形成的化合物的作用，以及鉴定参与朊病毒繁殖的宿主因子和微环境，研究了朊病毒在细胞内繁殖的分子机制。在持续感染朊病毒的细胞中，四种不同的抗PrP抗体与细胞表面的PrP^C反应，抑制PrP^<Sc>的形成。细胞表面的抗体- prp ^C复合物没有被有效地内化，倾向于保留在细胞表面。这些结果表明，抗PrP单抗通过干扰常规的PrP^C降解途径来拮抗PrP^<Sc>的形成。此外，我们筛选了抑制PrP^<Sc>形成的合成磺化糖苷，发现4-磺基- n-乙酰氨基葡萄糖和6-磺基- n-乙酰氨基葡萄糖抑制PrPSc在朊病毒感染细胞中的形成。这些磺化糖苷加速了PrP^C的内吞作用，减少了PrP^C的总量，而磺化糖苷不抑制PrP^<Sc>的形成并没有减少PrP^C的总量。这些结果表明，硫酸盐糖苷和糖胺聚糖通过促进PrP^C的降解来抑制PrP^<Sc>的形成。我们建立了神经2a （N2a）小鼠神经母细胞瘤细胞亚克隆，并区分了朊病毒易感和非易感亚克隆。一个非易感亚克隆N2a-1表达PrP^C的水平与亲本N2a和其他朊病毒易感亚克隆相同。PrP^<Sc>在易感亚克隆和非易感亚克隆上的结合没有差异。表达PrP^C但对朊病毒繁殖具有抗性的N2a-1的存在表明，PrP^C以外的宿主因子参与了朊病毒的繁殖。为了确定这些宿主因子，利用DNA芯片分析了N2a亚克隆之间的基因表达谱。我们选择了36个和18个分别在朊病毒易感亚克隆N2a-5和耐朊病毒亚克隆N2a-1中表达2倍以上的基因。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、Al和C5基因的siRNA抑制了朊病毒感染的N2a-5细胞中的朊病毒繁殖。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