蛋白酶体抑制剂对神经细胞nicastrin表达的影响

Effect of proteasomal inhibitors on nicastrin expression in neuronal cells

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。

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.