Stegh A H, Herrmann H, Lampel S, Weisenberger D, Andrä K, Seper M, Wiche G, Krammer P H, Peter M E
Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany.
Mol Cell Biol. 2000 Aug;20(15):5665-79. doi: 10.1128/MCB.20.15.5665-5679.2000.
Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.
半胱天冬酶8在死亡受体介导的细胞凋亡执行过程中发挥着至关重要的作用。为了确定内源性半胱天冬酶8的定位，我们在共聚焦免疫荧光显微镜中使用了一组亚基特异性抗半胱天冬酶8单克隆抗体。在人乳腺癌细胞系MCF7中，半胱天冬酶8主要与线粒体共定位并结合在线粒体上。通过CD95或肿瘤坏死因子受体I诱导细胞凋亡后，活性半胱天冬酶8转移至网蛋白（一种三种主要细胞质细丝系统的主要交联蛋白），而半胱天冬酶8前结构域仍与线粒体结合。在所有测试细胞中，网蛋白在分子中心的天冬氨酸2395处被半胱天冬酶8定量切割。网蛋白的切割明显早于其他半胱天冬酶底物（如聚（ADP - 核糖）聚合酶、凝溶胶蛋白、细胞角蛋白或核纤层蛋白B）的切割。在来自网蛋白缺陷小鼠的原代成纤维细胞中，如在野生型细胞中所见的凋亡诱导的肌动蛋白细胞骨架重组严重受损，这表明在细胞凋亡过程中，网蛋白是微丝系统重组所必需的。
