In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors
In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors
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DOI：
10.1016/s0301-472x(02)00886-x
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发表时间：
2002-09-01
影响因子：
2.6
通讯作者：
Darzynkiewicz, Z
中科院分区：
文献类型：
--
作者：
Grabarek, J;Darzynkiewicz, Z
半胱天冬酶的激活是凋亡的关键事件。为了原位检测这一事件，我们使用荧光标记的半胱天蛋白酶抑制剂（FLICA）作为这些酶活性中心的亲和标记。FLICA是荧光素或磺胺偶联肽-氟甲基酮，以1:1的化学计量，共价结合到半胱天冬酶的酶中心，其特异性由氨基酸的肽序列提供。同样，我们使用丝氨酸蛋白酶荧光抑制剂（FLISP）检测后一种酶的活性位点。活细胞暴露于FLISP的FLICA导致这些配体被摄取并与活化的半胱天蛋白酶或丝氨酸蛋白酶的活性位点结合；用细胞冲洗除去未结合的试剂A。只有发生凋亡的细胞用FLISP或FLICA标记。FLICA的细胞内结合位点与已知的caspases定位一致。FLICA或FLISP的共价结合使我们能够通过免疫印迹法鉴定标记蛋白：与FLICA或FLISP结合的蛋白分子量在17至22 kDa之间，对应于caspases的大亚基；与FLISP反应的两种蛋白分别约为57和60 kDa，表明它们是新型酶。检测半胱天冬酶或丝氨酸蛋白酶活化可以结合其他凋亡或细胞周期标记物，通过流式或激光扫描细胞术进行多参数分析。作为caspase抑制剂，FLICA可以阻止细胞凋亡过程，防止细胞解体。因此，研究人员开发了稳态凋亡测定法，以获得长时间内的累积凋亡指数，并估计细胞群的细胞进入凋亡的速率。(C) 2002国际实验血液学学会。Elsevier Science出版。
Activation of caspases is the key event of apoptosis. To detect this event in situ we applied fluorochrome-labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA are fluorescein- or sulforhodamine-conjugated peptide-fluoromethyl ketones that covalently, with 1:1 stoichiometry, bind to enzymatic centers of caspases, the specificity is provided by the peptide sequence of amino acids. Similarly, we applied fluorescent inhibitors of serine proteases (FLISP) to detect active sites of the latter enzymes. Exposure of live cells to FLICA of FLISP led to uptake of these ligands and their binding to activated caspases or active sites of serine proteases; the unbound reagents A ere removed by cell rinse. Only cells undergoing apoptosis were labeled with FLISP or FLICA. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA or FLISP allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases; two proteins reacting with FLISP were about 57 and 60 kDa, which suggests that they are novel enzymes. Detection of caspases or serine proteases activation can be combined with other markers of apoptosis or cell cycle for multiparametric analysis by flow or laser scanning cytometry. Being caspase inhibitors, FLICA arrest the process of apoptosis and prevent cell disintegration. The stathmo-apoptotic assay, was developed, therefore, to obtain cumulative apoptotic index over a long period of time and estimate a rate of cell entry into apoptosis for cell populations. (C) 2002 International Society of Experimental Hematology. Published by Elsevier Science.