利用基于GFP的FRET探针检测β-分泌酶的活性

The Detection of β-secretase Activity Using a GFP-based FRET Probe

β淀粉样肽(amyloidβ-peptide,Aβ)在阿尔茨海默病(Alzheimeir’s disease,AD)患者脑内的异常产生和积累在AD的发病机理中扮演着重要的角色。β-分泌酶对淀粉样前体蛋白的裂解是Aβ产生的关键步骤,因此抑制β-分泌酶的活性成为AD药物治疗的一个重要策略。为了检测β-分泌酶的活性,应用基因工程技术将β-分泌酶作用底物序列(NFEV)的两端分别与绿色荧光蛋白(green fluorescent protein,GFP)的颜色突变体--青色荧光蛋白(cyan fluorescent protein,CFP)和黄色荧光蛋白(yellow fluorescent protein,YFP)相连,构建了一个基于GFP的荧光能量共振转移(fluorescence resonance energy transfer,FRET)探针。荧光光谱分析结果显示,该荧光融合蛋白中CFP和YFP之间存在着较强的FRET。而当与β-分泌酶进行孵育后,FRET逐步降低,证明该探针能被β-分泌酶酶切。SDS-PAGE分析显示探针与β-分泌酶孵育后,荧光融合蛋白由60 kD大小逐渐变成30kD大小的蛋白,表明探针被β-分泌酶酶切成CFP和YFP分子,证实了荧光光谱的结果。这些结果表明,可通过检测探针的FRET效率方便地分析β-分泌酶的活性,为进一步筛选β-分泌酶的抑制剂提供了一个平台。

Abnormal production and accumulation of amyloid β-peptide （Aβ） plays a major role in the pathogenesis of Alzheimer＇ s disease （AD）. The cleavage of amyloid precursor protein （APP） by β-secretase（BACE） is the first step in the generation of Aβ, therefore, inhibiting the activity of β-secretase is believed to be a promising therapeutic target for the prevention and treatment of AD. Here we designed a genetically encoded fluoescence resonance energy transfer （FRET） biosensor for detecting β-secretase activity. The FRET sensor consists of a β-secretase substrate sites peptide （ APP‘NFEV＇ mutant） sandwiched between monomeric yellow and eyan mutants of green fluorescence protein （GFP）. Fluorescence spectroscopic analysis showed that a strong FRET signal was recorded, demonstrating energy transfer from CFP to yellow fluorescent protein（YFP） in integral FRET probe. When the probe was incubated with β-seeretase, a significant decrease of FRET was observed, suggesting the fluorescence probe was cleaved by β-secretase. The spectral result was confirmed by SDS-PAGE assay. These results indicate that the FRET probe is able to detect the β-secretase activity and provides a novel platform for β-secretase inhibitor screening.