【摘要】 研究转运膜蛋白21(TMP21)在小鼠成纤维细胞中超表达状态下,对γ分泌酶活性的影响.利用TMP21 cDNA转染敲除淀粉样前体蛋白表达基因的小鼠胚胎成纤维细胞系(MEF(KO)),以梯度浓度G418筛选TMP21表达量最高的细胞.处理超表达组(T)和对照组(C)细胞,并收集纯化含γ分泌酶复合物的样本.用Western blotting检测TMP21蛋白表达量,选用饱和浓度的γ分泌酶底物C99,运用ELISA检测A-βpeptide40和A-βpeptide42的生成总量.结果表明,样本T1、T2及IPT2所含TMP21的蛋白表达量较相应对照组样本C1、C2和IPC2分别上升11.5%(P=0.08),28.1%(P<0.01),69.1%(P<0.001),提示经TMP21 cDNA转染后MEF(KO)细胞中TMP21蛋白表达明显提高.同时,对照样本C1、C2及IPC2,样本T1、T2及IPT2中所含γ分泌酶活性分别降低33.4%(P=0.01),71.9%(P<0.001),96.5%(P<0.001).转染组样本中,样本IPT2所含γ分泌酶活性最低,提示经处理γ分泌酶得到明显纯化分离,与Western blotting所得结果一致.因此,高表达状态下的TMP21蛋白,对MEF(KO)细胞中γ分泌酶的活性具有明显的降低作用,表明TMP21为γ分泌酶的负调控因子之一.更多还原

【Abstract】 Objective γ-secretase is one of the crucial enzymes which involved in the pathogenic mechanism of Alzheimer’s disease.The impacts of the transmembrane protein 21(TMP21) on γ-secretase activity were studied in APP knockout mouse embryonic fibroblasts(MEF(KO)).Methods MEF(KO) was transfectted with TMP21 cDNA to induce protein expression.During the purification of γ-secretase,samples comprising γ-secretase complex were collected in transfection(T) or control(C) group at three phases.The samples were respectively marked as follow: T1 and C1 which included the whole cell membrane proteins;T2 and C2 which were the lysates by CHAPSO;IPT2 and IPC2 which were prepared after co-immunoprecipitation by presenilin-1 antibody.The protein levels of TMP21 were detected by Western blotting.The γ-secretase activity against artificial substrate C99 was measured by ELISA.Results The protein levels of TMP21 in T1,T2 and IPT2 were,compared with C1,C2 and IPC2,increased by 11.5%(P=0.08),28.1%(P<0.01) and 69.1%(P<0.001) respectively.The amounts of Aβ-peptide40 and Aβ-peptide42 from C99 catalyzed by γ-secretase in T1,T2 and IPT2 were reduced by 33.4%(P=0.01),71.9%(P<0.001),96.5%(P<0.001) when respectively compared with C1,C2 and IPC2.Conclusions TMP21 can be one of the negative regulators of γ-secretase as the component of γ-secretase complex.The activity of γ-secretase was reduced in MEF(KO) by the induction of TMP21 protein expression via cDNA transfection.更多还原