Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells
Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells
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DOI：
10.1006/bbrc.2001.4896
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发表时间：
2001-05-25
影响因子：
3.1
通讯作者：
Chang, DC
中科院分区：
文献类型：
--
作者：
Luo, KQ;Yu, VC;Chang, DC
caspase-3的激活是细胞凋亡的中心事件。我们已经开发了一种基于GFP的FRET（荧光共振能量转移）探针，该探针对完整活细胞中caspase-3的激活高度敏感。该探针通过将CFP（青色荧光蛋白）和YFP（黄色荧光蛋白）与含有胱天蛋白酶-3切割序列的专门接头DEVD融合而构建。优化接头设计以产生大的FRET效应。使用纯化的蛋白质，我们观察到当探针被caspase-3切割时荧光发射率的五倍变化。为了证明这种方法的有用性，我们通过瞬时和稳定转染将这种FRET探针引入HeLa细胞。我们观察到，在紫外线诱导的细胞凋亡过程中，caspase-3的激活在不同的细胞之间有显着差异;但一旦caspase被激活，细胞内的酶在几分钟内变得完全活跃。这种技术将是非常有用的相关的caspase-3激活与其他凋亡事件和快速筛选潜在的药物，可能靶向凋亡过程。(C)北京：科学出版社.
Activation of caspase-3 is a central event in apoptosis. We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3, To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process. (C) 2001 Academic Press.