Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro
Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro
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DOI：
10.1080/13506120410001689634
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发表时间：
2004-03-01
影响因子：
5.5
通讯作者：
Kaneko, K
中科院分区：
文献类型：
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作者：
Kishida, H;Sakasegawa, Y;Kaneko, K
已知密码子 218 (Q218K) 处具有赖氨酸突变的显性阴性小鼠朊病毒蛋白 (PrP) 可抑制朊病毒复制。为了进一步了解这种显性失活抑制的机制，研究了非糖基磷脂酰肌醇 (GPI) 锚定的带有 Q218K 的重组 PrP (rPrP-Q218K)。当应用于痒病感染的小鼠神经母细胞瘤 (ScN2a) 细胞时，rPrP-Q218K（而非野生型 rPrP (rPrP-WT））专门抑制异常蛋白酶抗性致病亚型 (PrPSc) 复制，而不降低细胞的活力。它甚至比奎纳克林更有效，奎纳克林已经被用于治疗散发性克雅氏病（CJD）患者。 50%有效浓度(EC50)=0.20μM，99%有效浓度(EC99)=0.86μM对比EC50=0.45μM，EC99=1.5μM此外，在浓度高达4.3μM(100μM/ml)时没有观察到明显的细胞损伤。在与0.43μM（10μg/ml）的rPrP-Q218K联合处理时，奎纳克林的EC99从1.5μM降低到0.5μM，并且细胞活力从50％恢复到90％以上，与奎纳克林的浓度成反比。这种组合可以通过降低奎纳克林的有效浓度来减轻奎纳克林的副作用，而不改变甚至加速抑制功效。由于可以轻松地从大肠杆菌中大量制备同质、高质量的 rPrP，因此 rPrP-Q218K 是朊病毒复制拮抗剂的良好候选者。
Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 muM, 99% effective concentration (EC99) = 0.86 muM vs. EC50 = 0.45 muM, EC99 = 1.5 muM Besides, no apparent cell damage was observed at the concentration of up to 4.3 muM (100 mug/ml). In combination treatment with 0.43 muM (10 mug/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 muM to 0.5 muM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.