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.