Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells
Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells
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DOI：
10.4049/jimmunol.173.1.285
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发表时间：
2004-07-01
影响因子：
4.4
通讯作者：
Niitsu, Y
中科院分区：
文献类型：
--
作者：
Sato, T;Machida, T;Niitsu, Y
活性氧 (ROS) 的产生和 caspase 级联的激活在 Fas 介导的细胞凋亡信号传导中都是不可或缺的。尽管基于抗氧化剂抑制 caspase 的激活以及 ROS 本身的刺激激活 caspase 的发现，推测 ROS 会影响 caspase 级联的活性，但这些细胞事件整合到 Fas 信号传导中的机制目前尚不清楚。在这项研究中，我们使用人 T 细胞白血病 Jurkat 细胞以及体外重建系统，证明了凋亡体的形成需要 ROS。我们首先表明，源自线粒体通透性转变的 ROS 正向调节线粒体通透性转变下游的细胞凋亡事件。然后，我们通过免疫沉淀和尺寸排阻色谱方法发现，N-乙酰基-L-半胱氨酸和锰超氧化物歧化酶明显抑制 Fas 刺激的 Jurkat 细胞中凋亡体的形成。为了证实这些体内发现，我们接下来使用了体外重建系统，其中体外翻译的凋亡蛋白酶激活因子 1 (Apaf-1)、procaspase-9 和从人胎盘纯化的细胞色素 c 被 dATP 激活形成凋亡体； DTT 或还原型谷胱甘肽 (GSH) 等还原剂显着抑制凋亡体的形成，而过氧化氢则阻止这种抑制。我们还发现，GSH 预处理的 Apaf-1 显着损害了凋亡体的形成，但 GSH 预处理的 procaspase-9 或 GSH 预处理的细胞色素 c 则没有。总的来说，这些结果表明 ROS 通过氧化 Apaf-1 以及随后激活 caspase-9 和 -3 在凋亡体形成中发挥重要作用。
Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.