siRNA干扰跨膜转运蛋白21的表达对KO小鼠胚胎成纤维细胞γ分泌酶活性的影响

Effect of siRNA-interfered transmembrane protein 21 expression on γ-secretase activity in embryonic fibroblast cells of KO mice

目的探讨跨膜转运蛋白21(TMP21)对γ分泌酶活性的影响。方法将淀粉样前体蛋白基因缺陷型KO小鼠胚胎成纤维细胞[MEF(KO)]分为转染组(T)和对照组(C),转染组转染载有TMP21小分子干扰RNA(siRNA)的质粒,对照组转染空质粒。每组均制备包含细胞全膜蛋白的样品(T1、C1),经CHAPSO进一步溶解后超速离心制备所得纯化全膜蛋白样品(T2、C2),用γ分泌酶组分早老蛋白1(PS-1)特异性抗体免疫沉降C2或T2后获得的γ分泌酶样品(IPT2、IPC2)。蛋白质印迹法检测各类样品中γ分泌酶重要组分TMP21、PS-1和Nicastrin(NCT)蛋白表达量;应用ELISA法检测β淀粉样肽40和β淀粉样肽42的生成总量。结果蛋白质印迹法检测显示,TMP21蛋白表达量在转染组样品IPT2中为5294±247ng/ml,在对照组样品IPC2中为19110±579ng/ml,组间差异有统计学意义(P<0.05);各类样品中PS-1与NCT蛋白表达量无明显变化;TMP21可与PS-1共沉降。ELISA法检测显示,转染组样品IPT2与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为348±18pg/ml,对照组样品IPC2为与饱和浓度底物C99反应生成的β淀粉样肽40和β淀粉样肽42总量为342±18pg/ml,组间差异有统计学意义(P<0.01)。结论TMP21可能为γ分泌酶的组分之一。在TMP21蛋白低表达状态下γ分泌酶活性升高,提示TMP21为γ分泌酶的负调控因子之一。

Objective To investigate the effect of transmembrane protein 21 （TMP 21） on the activity of γ-secretase. Methods Embryonic fibroblast cells obtained from KO mice [MEF （KO）] with disrupted app gene were divided into transfection （T） and control （C） groups. The cells in the T group were transfected with the plasmids carrying the TMP 21 interfered by siRNA, and the control groups were transfected with blank plasmids. In each group, and samples of γ-secretase cell samples were prepared as follow： T1 and C1： the whole cell membrane proteins; T2 and C2： purified samples of whole membrane protein, which were the centrifugated lysates of CHAPSO, and IPT2 and IPC2： prepared by co-immunoprecipitation of by presenilin-1 （PS-1） antibody and C2 or T2. The expression levels of TMP 21, PS- 1 and Nicastrin （NCT）, were detected by Western blotting. The levels of Aβ-peptide 40 and Aβ-peptide 42 were measured by ELISA. Results Western blotting showed that the level of TMP 21 was 5294±247 ng/ml in IPT2, and was 19110±579 ng/ml in IPC2. The difference in the TMP 21 level among the groups was significant （P 〈 0.05）. No significant difference was found in the levels of PS-1 and NCT among the groups. TMP 21 and PS-1 were co-sedimentated. ELISA showed that the total amounts of Aβpeptide 40 and Aβpeptide 42 from C99 catalyzed by γ-secretase was 348±18 pg/ml in IPT2, and was 342±18 pg/ml in IPC2. The difference was significant among the groups （P 〈 0.05）. Conclusions TMP 21 could be one of the components of γ-secretase complex. The activity of γ-secretase increases when TMP 21 expresses at a low level, indicating that TMP 21 is one of the negative modulators of γ-secretase.