蛋白酶体抑制剂对神经细胞nicastrin表达的影响

目的:探讨蛋白酶体抑制剂能否引起神经细胞内nicastrin(NCT)蛋白表达及分布的变化,以明确NCT的蛋白降解是否与蛋白酶体有关。方法:在用人神经母细胞瘤细胞(SH-SY5Y)建立稳定表达NCT细胞株的基础上,应用蛋白酶体抑制剂处理NCT细胞株,并结合Western blotting、放射性同位素脉冲示踪技术、免疫荧光双标技术,检测蛋白酶体抑制剂处理后神经细胞内NCT的蛋白表达变化。结果:Western blotting结果显示,特异性蛋白酶体抑制剂处理后,神经细胞内源性和外源性转染的NCT的表达显著增强,高度特异的蛋白酶体抑制剂lacta-cystin对NCT蛋白的增强效应呈剂量依赖性和时间依赖性;放射性同位素脉冲示踪结果显示,蛋白酶体抑制剂可显著增强细胞内新合成的NCT蛋白的表达水平,其增强作用是通过阻止35S]-NCT的蛋白降解来实现的;免疫荧光双标结果提示,NCT与泛素在细胞内共存。结论:神经细胞内NCT的降解由蛋白酶体途径介导;NCT在降解之前经泛素化修饰。

Effect of proteasomal inhibitors on nicastrin expression in neuronal cells

AIM: To explore the possibility that proteasome is involved in nicastrin (NCT) degradation and NCT is ubiquitinated before degradation. METHODS: Following the generation of NCT stable cell lines, the methods of Western blotting, pulse-chase metabolic labeling technique, double immunofluorescent staining, combined with proteasomal inhibition were used to investigate the NCT expression in NCT stable cell line. RESULTS: Treatment of the cells with proteasomal inhibitors significantly increased both endogenous NCT (produced by the cell itself) and exogenous NCT (produced by the gene transfection) in SH-SY5Y cells. The effect of specific proteasomal inhibitor lactacystin on NCT expression was in time- and dose-dependent manners. Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radio-labeled nicastrin protein was blocked by lactacystin. The results of double immunofluorescent staining showed that NCT and ubiquitin were co-located in the cells. CONCLUSION: The proteasome is involved in the degradation of NCT in neuronal cells, and NCT is ubiquitinated before degradation.