Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.
Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.
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DOI：
10.3892/ijo.20.2.225
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发表时间：
2002-02
影响因子：
5.2
通讯作者：
J. Grabarek;M. Dragan;Brian W. Lee;G. Johnson;Z. Darżynkiewicz
中科院分区：
文献类型：
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作者：
J. Grabarek;M. Dragan;Brian W. Lee;G. Johnson;Z. Darżynkiewicz
文献中有证据表明，丝氨酸蛋白酶和半胱氨酸氨基转移酶一样，在细胞凋亡过程中被激活。然而，人们对单个丝氨酸蛋白水解酶及其激活机制知之甚少。在本研究中，我们使用一种新型的细胞检测试剂来检测诱导凋亡的人白血病HL-60细胞中凝乳酶样蛋白水解酶的激活。该试剂5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl酮(FFCK)是N-对甲苯磺酰-L-苯丙氨酸氯甲基酮(TPCK)的荧光标记类似物，TPCK是已知的特异性和共价结合到类糜蛋白酶活性中心的抑制剂。在DNA拓扑异构酶I抑制剂喜树碱(CPT)或肿瘤坏死因子(TNFpha)处理的培养中，出现了具有结合FFCK能力的细胞群。荧光显微镜和激光扫描细胞仪(LSC)证实大多数FFCK结合细胞为正在发生凋亡的细胞。细胞结合FFCK的频率与激活caspase的细胞频率密切相关(在CPT处理的细胞中r=0.98，在TNFα处理的培养中r=0.99)。观察到的FFCK结合的诱导，我们解释为代表着一种类糜蛋白酶样的凋亡丝氨酸蛋白酶(S)的激活。用多聚半胱氨酸天冬氨酸氨基转移酶抑制剂Z-VAD-FMK预处理细胞，可阻止这些丝氨酸蛋白水解酶的激活(S)。然而，TPCK预处理对caspase激活的抑制作用不那么明显，尽管明显且可重现。因此，这些数据表明，caspase的激活是丝氨酸蛋白酶激活所需的上游事件(S)。然而，后者的激活似乎以级联方式额外放大了caspase的激活。不同颜色的荧光标记使我们能够在相同的细胞内区分激活的半胱氨酸天冬氨酸酶和丝氨酸蛋白酶(S)。尽管有一定程度的共定位，但caspase-vs.Ser-Protease(S)的细胞内和细胞内模式是不同的。我们的方法使得同时监测被诱导凋亡的同一活细胞中caspase和Ser蛋白酶的激活成为可能。
There is evidence in the literature that serine (Ser) proteases, like caspases, are activated during apoptosis. Little is known, however, about individual Ser proteases and the mechanism of their activation. In the present study, we employed a new type of cell permeant reagent to detect activation of chymotrypsin-like proteases in human leukemic HL-60 cells induced to undergo apoptosis. The reagent, 5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl ketone (FFCK), is a fluorochrome-labeled analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the inhibitor known to specifically and covalently bind to the active center of chymotrypsin-like enzymes. In cultures treated with the DNA topoisomerase I inhibitor, camptothecin (CPT), or tumor necrosis factor (TNFalpha), populations of cells appeared that had the capability to bind FFCK. Most FFCK-binding cells were identified by fluorescence microscopy and laser scanning cytometry (LSC) as the cells undergoing apoptosis. Frequency of cells binding FFCK strongly correlated with frequency of cells having activated caspases (r=0.98 in CPT-treated, and r=0.99 in TNFalpha-treated cultures). The observed induction of FFCK binding we interpret as representing the activation of a chymotrypsin-like apoptotic Ser protease(s). Pretreatment of cells with the poly-caspase inhibitor, Z-VAD-FMK, prevented the activation of these Ser protease(s). Pretreatment with TPCK, however, had a less pronounced, although distinct and reproducible suppressive effect, on caspase activation. The data, thus, suggest that activation of caspases is an upstream event required for activation of Ser protease(s). Activation of the latter, however, appears to additionally amplify, in a cascade-like mode, caspases activation. Differential color fluorochrome-labeling allowed us to discriminate, within the same cells, between the activation of active caspases and Ser protease(s). Despite a certain degree of co-localization, the inter- and intra-cellular pattern of caspase- vs. Ser-protease(s) was different. Our approach makes it possible to simultaneously monitor activation of caspases and Ser proteases in the same live cells that are induced to apoptosis.