激活泛素-蛋白酶体系对脊髓小脑共济失调7型细胞模型中共济失调蛋白-7的作用研究

Effect of Activating Ubiquitin Proteasome System on Mutant Protein Ataxin-7 in Spinocerebellar Ataxia Type 7 Cell Model

目的探索在脊髓小脑共济失调7型(SCA7)细胞模型中,1-[1-(4-氟苯基)-2,5-二甲基-1H-吡咯-3-基]-2-(1-吡咯烷基)乙酮(IU-1)激活泛素-蛋白酶体系(UPS)后对多聚谷氨酰胺(PolyQ)共济失调蛋白-7(ATXN7)的作用。方法在稳定表达ATXN7突变蛋白及非突变蛋白的ATXN7-Q65和ATXN7-Q10神经细胞模型中,利用5×10^(4)μmol/L IU-1和0.2μmol/L环氧甲酮四肽分别增强和抑制UPS的降解作用,进而观察ATXN7突变蛋白对细胞的毒性作用。利用蛋白质印迹技术和斑点过滤杂交法检测ATXN7突变蛋白的表达,2-(4-磺苯)-3-(4-硝基苯)-5-(2,4-二磺基苯)-2H-四氮唑钠盐(WST-1)法分析细胞存活率。结果在构建的两组神经细胞模型中,ATXN7-Q65细胞表达的ATXN7蛋白及其聚合体随着诱导时间的延长而明显增加,且与ATXN7-Q10细胞相比,在诱导12 d后ATXN7-Q65细胞的存活率明显下降,细胞凋亡率增加(P<0.05);当加入Dox抑制剂后,与ATXN7-Q10细胞相比,ATXN7-Q65细胞可溶性ATXN7蛋白在24 h内降解缓慢,ATXN7聚合体水平不变。在ATXN7-Q65细胞中,加入IU-1增强UPS活性,12 d后细胞毒性降低,存活率升高;而用环氧甲酮四肽抑制UPS活性,12 d后细胞毒性增高,存活率降低,差异有统计学意义(P<0.05)。结论UPS可有效清除突变蛋白ATXN7,IU-1激活UPS活性能增强突变蛋白ATXN7的清除并降低细胞毒性。

Objective This study aimed to investigate the degradation effect of ubiquitin proteasome system(UPS)on polyglutamine(PolyQ)expanded Ataxin-7(ATXN7)when UPS was activated by 1-[1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl]-2-pyrrolidin-1-yle-thanone(IU-1)in the cell model of spinocerebellar ataxia type 7(SCA7)disease.Methods In the ATXN7-Q65 and ATXN7-Q10 nerve cell models stably expressing ATXN7 mutant protein and non-mutant protein,the toxic effect of mutant protein ATXN7 was evaluated under the treatment of either 5×10^(4)μmol/L IU-1 or 0.2μmol/L epoxomycin to stimulate or inhibit UPS activity.The expression of mutant protein ATXN7 was detected by Western Blot and the aggregated species were analyzed by Filter Trap assay.2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt(WST-1)was applied to test the cell viability.Results In the two groups of neural cell models constructed,the expression of ATXN7 protein and its aggregates in ATXN7-Q65 cells increased significantly with the extension of induction time.Compared with ATXN7-Q10 cells,ATXN7-Q65 cells had decreased cell viability and increased apoptosis rate after 12 days of induction(P<0.05).Compared with ATXN7-Q10 cells,ATXN7-Q65 cells had slower degradation of soluble ATXN7 mutant protein within 24 hours and unchanged polymer level when the expression of ATXN7 protein was inhibited by Dox.In ATXN7-Q65 cells,the cytotoxicity was reduced and the cell viability was increased after enhancing UPS activity by IU-1 for 12 days,while the cytotoxicity was increased and the survival rate was decreased after inhibiting UPS activity by Epoxomycin for 12 days(P<0.05).Conclusion UPS effectively eliminates mutant ATXN7-Q65,and the stimulation of UPS activity by IU-1 significantly enhanced the clearance of mutant protein ATXN7 and counteracted to the cytotoxicity induced by mutant ATXN7-Q65 expression.