Kawai Hiroshi, Suzuki Takuo, Kobayashi Tetsu, Mizuguchi Hiroyuki, Hayakawa Takao, Kawanishi Toru
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. kawai.nihs.go.jp
Biochim Biophys Acta. 2004 Aug 23;1693(2):101-10. doi: 10.1016/j.bbamcr.2004.05.009.
A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.
半胱天冬酶家族作为一类半胱氨酸蛋白酶，在介导细胞死亡过程中发挥着核心作用。在本研究中，我们实时测定了起始半胱天冬酶和效应半胱天冬酶的激活情况，并通过生物成像研究了活细胞中半胱天冬酶活性与线粒体膜电位之间的关系。我们还设计并开发了一种基于荧光共振能量转移（FRET）的基因编码荧光指示剂，它由黄色荧光蛋白（YFP）、一段可被特定半胱天冬酶切割的肽序列以及青色荧光蛋白（CFP）组成。我们分别使用了两种可被起始半胱天冬酶和效应半胱天冬酶切割的肽序列。对用肿瘤坏死因子-α（TNF-alpha）和星形孢菌素处理的细胞进行半胱天冬酶活性和线粒体膜电位的同步实时测量发现，濒死细胞表现出半胱天冬酶激活和线粒体去极化，然而，这些事件之间并没有紧密的联系。尽管在添加细胞死亡诱导剂后，半胱天冬酶开始被激活需要1到10多个小时，但在导致细胞死亡的整个过程的最后阶段，起始半胱天冬酶和效应半胱天冬酶会在短时间内被激活。
