蛋白酶体抑制剂处理神经细胞系后Nicastrin的表达变化及其与Aβ的关系

Expression change of Nicastrin in the neuronal cells and its relationship with Aβ generation after proteasomal inhibitor treatment

目的探讨蛋白酶体抑制剂处理后神经细胞内Nicastrin(NCT)的表达变化,及其与γ-分泌酶活性和Aβ生成的关系。方法运用亚细胞器分级分离技术、免疫共沉淀、Western blot和ELISA等检测神经细胞内NCT的表达及Aβ水平。结果正常情况下NCT主要分布于内质网和高尔基复合体,极少量分布于溶酶体,蛋白酶体抑制剂Lac-tacystin处理后NCT水平升高(P<0.001),且细胞内增多的NCT也主要聚集在内质网和高尔基复合体;NCT与泛素在细胞内共存;NCT的蛋白降解不受PS的影响;NCT降解受阻可引起细胞内γ-分泌酶的底物C99、C83显著减少,而γ-分泌酶的产物Aβ40、Aβ42的生成显著增多(P<0.01)。结论 NCT的降解可通过泛素-蛋白酶体途径实现,蛋白酶体抑制剂处理后神经细胞内NCT水平升高,且增多的NCT可促进APP的代谢及Aβ的生成。

Objective To explore whether Nicastrin undergoes ubiquitination before proteasomal degradation,as well as the relationship between Nicastrin protein level and Aβ generation.Methods Cell fractionation,Western blot,immunoprecipitation as well as ELISA were used to check the expression of NCT and Aβ level.Results NCT distributes primarily in ER and Golgi apparatus but less in lysosome.Increased NCT accumulates was found in the ER and Golgi apparatus after proteasomal inhibition.NCT and ubiquitin colocalized and interacted with each other in cells.The degradation of NCT was not affected by PS.Overexpression of NCT by transient NCT plasmid transfection or inhibition of NCT proteasomal degradation can decrease substrate of γ-secretase,C99 and C83,and increase production of γ-secretase,Aβ40 and Aβ42（P0.01）.Conclusion The degradation of NCT is resulted from ubiquitin-proteasome pathway.The expression of NCT is increased following proteasomal inhibition,and over-expression of NCT facilitates APP processing and Aβ generation.