利用受体漂白荧光共振能量转移技术研究β-分泌酶在活细胞内的二聚化

Detection of β-Secretase Dimerization in Living Cells Using Acceptor Photobleaching FRET

β淀粉样肽(Aβ)在阿尔茨海默病(AD)患者脑内的产生、聚集和沉积是AD的一个固有病理特征,也被普遍认为是AD的重要发病机理之一.β-分泌酶(BACE)对淀粉样前体蛋白(APP)的裂解是Aβ产生的关键步骤,β-分泌酶抑制剂是AD药物治疗的一个重要发展方向,因此对BACE结构的了解具有重要意义.利用受体漂白荧光共振能量转移(FRET)技术首次在活细胞内研究BACE的二聚化结构.通过基因工程技术分别构建了青色荧光蛋白(CFP)和黄色荧光蛋白(YFP)标记的全长BACE(BACE-FL)和BACE活性部分(BACE-NT),应用共聚焦荧光显微镜和受体漂白FRET技术研究了BACE-FL和BACE-NT在细胞中的表达和定位,以及它们在活细胞内的存在形式.实验结果表明:a.BACE-FL在内质网至细胞膜均有表达,主要分布在高尔基体、细胞膜和内体等细胞器中,而BACE-NT则被滞留在内质网里.b.BACE-NT是单体结构,而BACE-FL在活细胞内则以二聚体的形式存在.证明,BACE的跨膜序列和C端对BACE的转运和定位具有重要作用,其二聚体结构由这些序列决定,而不由其活性位点决定.

Generation, aggregation, and deposition of amyloid β-peptide （Aβ） in the brains of Alzheimer＇s disease （AD） patients are inherent pathological features of AD. Cleavage of the amyloid precursor protein （APP） by the β-secretase （BACE） is the first step in the generation of Aβ. Inhibition of BACE activity is a promising therapeutic strategy for the treatment of AD. Therefore, understanding the structure of BACE is very important. Using blue native gel electrophoresis it has been found that BACE exists as a homodimer. However, it is currently unknown whether in living cells BACE forms homodimer. To address this issue, the dimerization of BACE in intact living cells was monitored by using confocal microscopy and acceptor photobleaching FRET. Using bioengineering technique, fluorescence proteins （FPs）-tagged BACEs （BACE/FPs） was constructed. They are cyan fluorescent protein （CFP） or yellow fluorescent protein （YFP） tagged full length BACE （BACE-FL） and truncated BACE （BACE-NT）. The CFP- and YFP- tagged BACE-FL （BACE-FL/FPs） or CFP- and YFP- tagged BACE-NT （BACE-NT/FPs） plasmids were transfected into HeLa cells, and the expression and location of BACE-FL and BACE-NT were observed by confocal microscopy. The dimerization of BACE was evaluated by the FRET between CFP- and YFP- tagged BACE which was detected by acceptor photobleaching method. The results show that the BACE-FL can be transported to the Golgi apparus, plasma membrane and endosomes. However, the BACE-NT is found in the ER 24 hours after transfection. The FRET efficiency of BACE-FL/FPs group is （12.90± 0.73）%, significantly higher than that of control group （3.32 ±0.55% ） （P 〈 0.05）. The FRET efficiency of BACE-NT/FPs group is （3.80±0.74）%, which is not statistically different from that of control group. These results indicat that the BACE-FL exists as a dimmer in living cells, but the BACE-NT exists as a monomer, suggesting the transmembrane and C-terminus regions are important for normal transport and localization, and is necessary for the dimerization of BACE.