β-分泌酶活性及其二聚化的动态光学成像研究
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摘要
阿尔茨海默症(AD)是一种常见的中枢神经系统退行性疾病,临床表现为渐进的记忆丧失和认知障碍,是老年痴呆的一种主要类型。目前普遍认为β-淀粉样肽(Aβ)在AD患者脑内的产生、聚集和沉积在阿尔茨海默症的发病机理中扮演着重要的角色。而β-分泌酶(BACE)对淀粉样前体蛋白(APP)的酶解是Aβ产生的关键限速步骤,因此BACE成为AD药物治疗的一个重要靶标,发展检测BACE活性的技术和对BACE结构的了解对于药物的开发和筛选都具有重要意义。
然而传统的生化方法难以实现在活细胞生理条件下对BACE的酶活性和结构的实时动态研究。本文利用荧光能量共振转移(FRET)光学成像技术,结合基因工程技术合成的FRET探针和荧光标记蛋白,在活细胞水平检测了BACE的活性及其二聚化结构。主要的研究结果如下:
1)利用基因工程技术构建了五种基于绿色荧光蛋白(GFP)的可遗传编码的FRET探针。这些FRET探针由GFP的两个颜色突变体——青色荧光蛋白(CFP)和黄色荧光蛋白(YFP),及其之间的一段短肽构成。根据中间连接短肽序列的不同,获得了4种中间短肽为BACE识别底物位点(BACE substrate site,BSS)的FRET探针:YβCwt(野生型BSS)、YβCSweS(Sweden突变的短链BSS)、YβCSweL(Sweden突变的长链BSS)、YβCNFEV(‘NFEV’突变的BSS),和1种中间短肽为随机序列的对照探针YcC。利用荧光光谱扫描和SDS-PAGE凝胶电泳比较了五种探针的体外荧光特性和酶切效果。结果显示,五种荧光探针都具有较强的FRET效率;中间短肽为BSS的YβCwt、YβCSweS、YβCSweL、YβCNFEV的酶切效率依次增强,而对照探针YcC不能被BACE酶切。
2)由于BACE及其底物APP均为分泌的跨膜蛋白,为了在活细胞水平检测BACE的活性,将经体外验证的FRET探针构建到真核表达载体pDisplay中,获得真核表达的FRET探针dYβC(displayed YβC)和对照探针dYcC(displayed YcC)。共聚焦荧光成像结果显示,在BACE阴性的HeLa细胞内,表达的dYβC和dYcC能够进入分泌途径,展示在细胞膜上,并显现出很高的FRET效率。当共转染BACE到HeLa细胞内时,dYβC的FRET效率明显降低,而对照探针dYcC的FRET效率始终保持不变。此结果表明dYβC可在活细胞中被BACE有效酶切,可用于动态检测活细胞内的BACE活性,为BACE抑制剂的开发提供了一个活细胞筛选的平台。此外,利用该探针分析了BACE在分泌途径中具备酶活性的起始位点,证实BACE早在内质网中就具有酶活性,能够对其底物进行酶切。
3)为了研究BACE在活细胞中的存在形式,构建了CFP和YFP标记的全长BACE(BACE-FL)和BACE活性部分(BACE-NT)。共聚焦荧光成像和受体漂白FRET实验结果显示,BACE-FL能够定位到高尔基体、质膜、内涵体等细胞器中,而BACE-NT则被滞留在内质网里;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-末端对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,而不由酶的活性位点决定。
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in memory loss, global cognitive dysfunction, and functional impairments. It is one of the most common forms of dementia. Generation, aggregation, and deposition of amyloidβ-peptide (Aβ) in brains of AD patients are a prominent pathological feature of this devastating neurodegenerative disease, and play an important role in the AD pathogenesis. Cleavage of the amyloid precursor protein (APP) by the aspartyl proteaseβ-site APP-cleaving enzyme (BACE) is the first step in the generation of the Aβ. Therefore, BACE plays an important role in the generation of Aβand is believed to be a promising therapeutic target for the prevention and treatment of AD. It is crucial to exploit the monitoring technology of BACE activity and understand the structure of BACE.
However, the traditional methods can not be used to monitor the activity and structure of BACE in the living cells under physiological conditions. Here, using the fluorescence resonance energy transfer ( FRET) technology , combining with the genetically encoded FRET probes and fluorescent proteins, we monitored the activity and structure of BACE in the living cells. The major results of this study are showed as following:
1) Five genetically encoded FRET probes that based on green fluorescent protein (GFP) were constructed using bioengineering technique. The FRET sensors consist of a peptide linker sandwiched between monomeric yellow and cyan mutants of GFP. According to the sequence of the peptide linker, the FRET probes are used as a control probe (YcC) or four FRET probes for detecting the BACE activity: YβCwt, YβCSweS, YβCSweL, YβCNFEV, in those the peptide linkers are wild type BACE substrate site (BSS), shorter Sweden mutant BSS, longer Sweden mutant BSS, and‘NFEV’mutant BSS, respectively. Fluorescence spectroscopic analysis showed that a strong FRET signal was recorded, demonstrating energy transfer from CFP to YFP in integral FRET probes. The fluorescence spectroscopic and SDS-PAGE analysis results showed that the control probe YcC can not be cleaved by BACE, and the other four probes that peptide linker are BSS can be cleaved by BACE, and the cleavage efficiency of YβCwt、YβCSweS、YβCSweL and YβCNFEV was orderly increased.
2) Since BACE and APP are both secretory transmembrane proteins, the FRET probe is chosen to be constructed into a mammalian expression vector pDisplay, creating a FRET probe dYβC (displayed YβC) for detecting BACE activity and a control probe dYcC (displayed YcC). Confocal imaging showed that dYβC and dYcC could be directed into the secretory pathway and displayed on the cell surface, giving it the chance of being cleaved by BACE. The dYβC and dYcC in the living cells showed high FRET efficiency. The FRET efficiency of dYβC decreased significantly in present of BACE, and the FRET efficiency of dYcC was unchanged. The results indicate the FRET probe can be cleaved by BACE efficiency in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo. Furthermore, the activity of BACE in secretory pathway was detected using this FRET probe. The results suggest thatβ-secretase cleavage was initiated in the secretory pathway, as early as in the ER.
3) The dimerization of BACE in intact living cells were monitered using confocal microscopy and acceptor photobleaching FRET. We constructed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) tagged BACE-FL and BACE-NT, respectively. The expression and location of BACE-FL and BACE-NT are observed by confocal microscopy and FRET between CFP- and YFP- tagged BACE was detected by acceptor photobleaching method. The results showed that the BACE-FL can be transported to the Golgi apparatus, plasma membrane and endosomes, however, the BACE-NT is retained in the ER. BACE-FL exists as a dimmer in living cells, but the BACE-NT is monomer. This results suggesting the transmembrane and C-terminus region is important for normal transport and location, and is necessary for the dimerization of BACE.
然而传统的生化方法难以实现在活细胞生理条件下对BACE的酶活性和结构的实时动态研究。本文利用荧光能量共振转移(FRET)光学成像技术,结合基因工程技术合成的FRET探针和荧光标记蛋白,在活细胞水平检测了BACE的活性及其二聚化结构。主要的研究结果如下:
1)利用基因工程技术构建了五种基于绿色荧光蛋白(GFP)的可遗传编码的FRET探针。这些FRET探针由GFP的两个颜色突变体——青色荧光蛋白(CFP)和黄色荧光蛋白(YFP),及其之间的一段短肽构成。根据中间连接短肽序列的不同,获得了4种中间短肽为BACE识别底物位点(BACE substrate site,BSS)的FRET探针:YβCwt(野生型BSS)、YβCSweS(Sweden突变的短链BSS)、YβCSweL(Sweden突变的长链BSS)、YβCNFEV(‘NFEV’突变的BSS),和1种中间短肽为随机序列的对照探针YcC。利用荧光光谱扫描和SDS-PAGE凝胶电泳比较了五种探针的体外荧光特性和酶切效果。结果显示,五种荧光探针都具有较强的FRET效率;中间短肽为BSS的YβCwt、YβCSweS、YβCSweL、YβCNFEV的酶切效率依次增强,而对照探针YcC不能被BACE酶切。
2)由于BACE及其底物APP均为分泌的跨膜蛋白,为了在活细胞水平检测BACE的活性,将经体外验证的FRET探针构建到真核表达载体pDisplay中,获得真核表达的FRET探针dYβC(displayed YβC)和对照探针dYcC(displayed YcC)。共聚焦荧光成像结果显示,在BACE阴性的HeLa细胞内,表达的dYβC和dYcC能够进入分泌途径,展示在细胞膜上,并显现出很高的FRET效率。当共转染BACE到HeLa细胞内时,dYβC的FRET效率明显降低,而对照探针dYcC的FRET效率始终保持不变。此结果表明dYβC可在活细胞中被BACE有效酶切,可用于动态检测活细胞内的BACE活性,为BACE抑制剂的开发提供了一个活细胞筛选的平台。此外,利用该探针分析了BACE在分泌途径中具备酶活性的起始位点,证实BACE早在内质网中就具有酶活性,能够对其底物进行酶切。
3)为了研究BACE在活细胞中的存在形式,构建了CFP和YFP标记的全长BACE(BACE-FL)和BACE活性部分(BACE-NT)。共聚焦荧光成像和受体漂白FRET实验结果显示,BACE-FL能够定位到高尔基体、质膜、内涵体等细胞器中,而BACE-NT则被滞留在内质网里;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-末端对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,而不由酶的活性位点决定。
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in memory loss, global cognitive dysfunction, and functional impairments. It is one of the most common forms of dementia. Generation, aggregation, and deposition of amyloidβ-peptide (Aβ) in brains of AD patients are a prominent pathological feature of this devastating neurodegenerative disease, and play an important role in the AD pathogenesis. Cleavage of the amyloid precursor protein (APP) by the aspartyl proteaseβ-site APP-cleaving enzyme (BACE) is the first step in the generation of the Aβ. Therefore, BACE plays an important role in the generation of Aβand is believed to be a promising therapeutic target for the prevention and treatment of AD. It is crucial to exploit the monitoring technology of BACE activity and understand the structure of BACE.
However, the traditional methods can not be used to monitor the activity and structure of BACE in the living cells under physiological conditions. Here, using the fluorescence resonance energy transfer ( FRET) technology , combining with the genetically encoded FRET probes and fluorescent proteins, we monitored the activity and structure of BACE in the living cells. The major results of this study are showed as following:
1) Five genetically encoded FRET probes that based on green fluorescent protein (GFP) were constructed using bioengineering technique. The FRET sensors consist of a peptide linker sandwiched between monomeric yellow and cyan mutants of GFP. According to the sequence of the peptide linker, the FRET probes are used as a control probe (YcC) or four FRET probes for detecting the BACE activity: YβCwt, YβCSweS, YβCSweL, YβCNFEV, in those the peptide linkers are wild type BACE substrate site (BSS), shorter Sweden mutant BSS, longer Sweden mutant BSS, and‘NFEV’mutant BSS, respectively. Fluorescence spectroscopic analysis showed that a strong FRET signal was recorded, demonstrating energy transfer from CFP to YFP in integral FRET probes. The fluorescence spectroscopic and SDS-PAGE analysis results showed that the control probe YcC can not be cleaved by BACE, and the other four probes that peptide linker are BSS can be cleaved by BACE, and the cleavage efficiency of YβCwt、YβCSweS、YβCSweL and YβCNFEV was orderly increased.
2) Since BACE and APP are both secretory transmembrane proteins, the FRET probe is chosen to be constructed into a mammalian expression vector pDisplay, creating a FRET probe dYβC (displayed YβC) for detecting BACE activity and a control probe dYcC (displayed YcC). Confocal imaging showed that dYβC and dYcC could be directed into the secretory pathway and displayed on the cell surface, giving it the chance of being cleaved by BACE. The dYβC and dYcC in the living cells showed high FRET efficiency. The FRET efficiency of dYβC decreased significantly in present of BACE, and the FRET efficiency of dYcC was unchanged. The results indicate the FRET probe can be cleaved by BACE efficiency in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo. Furthermore, the activity of BACE in secretory pathway was detected using this FRET probe. The results suggest thatβ-secretase cleavage was initiated in the secretory pathway, as early as in the ER.
3) The dimerization of BACE in intact living cells were monitered using confocal microscopy and acceptor photobleaching FRET. We constructed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) tagged BACE-FL and BACE-NT, respectively. The expression and location of BACE-FL and BACE-NT are observed by confocal microscopy and FRET between CFP- and YFP- tagged BACE was detected by acceptor photobleaching method. The results showed that the BACE-FL can be transported to the Golgi apparatus, plasma membrane and endosomes, however, the BACE-NT is retained in the ER. BACE-FL exists as a dimmer in living cells, but the BACE-NT is monomer. This results suggesting the transmembrane and C-terminus region is important for normal transport and location, and is necessary for the dimerization of BACE.
引文
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