Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis
Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis
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DOI：
10.1074/jbc.271.49.31075
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发表时间：
1996-12-06
影响因子：
4.8
通讯作者：
Orrenius, S
中科院分区：
文献类型：
--
作者：
Vanags, DM;PornAres, MI;Orrenius, S
对细胞凋亡的三个核外终点（磷脂酰丝氨酸暴露、α-胞嘧啶降解和质膜起泡）进行了详细的动力学分析，并与抗 Fas 刺激的 Jurkat T 淋巴细胞中的核碎裂和白介素-1 β 转换酶 (ICE) 样蛋白酶的激活进行了比较 单克隆抗体（抗 Fas mAb）以及肿瘤坏死因子 (TNF) 和放线菌酮刺激的单核 U937 细胞。通过血浆凝固时间以及膜联蛋白 V-异硫氰酸荧光素结合来定量磷脂酰丝氨酸暴露，并通过特定荧光肽底物 Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin 的裂解来检查类 ICE 蛋白酶活性。 VAD-氯甲基酮 (VAD-cmk) 是一种 ICE 样蛋白酶抑制剂，可有效抑制所研究的两种细胞类型中的 ICE 样活性，而钙蛋白酶抑制剂 calpeptin 则无效。 VAD-cmk 还有效抑制抗 Fas 单克隆抗体刺激的 Jurkat 细胞中的所有三种核外事件以及核碎裂，表明 ICE 样蛋白酶在该凋亡系统的调节中发挥重要作用，而 Calpain 抑制剂在此系统中无效。 U937 细胞中 TNF 诱导的核外和核变化受到 calpeptin 的抑制，但 VAD-cmk 的抑制效果不如 Jurkat 细胞那样有效。这表明 ICE 样酶在抗 Fas 单克隆抗体刺激的 Jurkat 细胞中占主导地位，而受钙蛋白酶抑制剂影响的蛋白酶以及 ICE 样酶参与 TNF 诱导的 U937 细胞中细胞凋亡事件的信号传导。重要的是，这两种细胞凋亡系统似乎受不同的蛋白酶调节。
A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the intertleukin-1 beta-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system, Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells, Importantly, the two apoptotic systems seem to be regulated by different proteases.