Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.
Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.
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DOI：
10.1084/jem.189.1.131
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发表时间：
1999-01-04
期刊：
影响因子：
--
通讯作者：
Greenberg AH
中科院分区：
文献类型：
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作者：
MacDonald G;Shi L;Vande Velde C;Lieberman J;Greenberg AH
颗粒酶B （Granzyme B, GraB）是细胞毒性T淋巴细胞和自然杀伤细胞有效激活细胞凋亡所必需的。我们发现，通过细胞色素c释放到细胞质中并抑制跨膜电位，GraB和穿孔素可诱导严重的线粒体扰动（Δψ）。最早的线粒体事件是细胞色素c的释放，与caspase 3加工同时发生，并始终在细胞凋亡激活之前发生。颗粒酶K/穿孔素或穿孔素处理都能有效杀死靶细胞，但在短期试验中是较差的凋亡激活剂，不能诱导细胞色素c的快速释放。然而，它们抑制Δψ并增加活性氧的产生，表明线粒体功能障碍也与这种非凋亡性细胞死亡有关。用多肽半胱天酶抑制剂zVAD-FMK或YVAD-CHO预处理可阻止GraB细胞凋亡和细胞色素c的释放，而DEVD-CHO可阻断细胞凋亡，但不阻止细胞色素c的释放，这表明半胱天酶在线粒体的上游和下游都起作用。另外有趣的是，GraK或GraB和perforin介导的Δψ抑制不受zVAD-FMK的影响，因此与caspase无关。Bcl-2和Bcl-XL的过表达抑制了caspase的激活、线粒体细胞色素c的释放、Δψ的抑制以及GraB、GraK或perforin诱导的细胞凋亡和细胞死亡。在体外无细胞系统中，高剂量的GraB激活S-100细胞质中的核凋亡，然而，线粒体的加入使GraB的活性增加了15倍以上。在线粒体存在的情况下，s -100细胞质中GraB诱导的caspase 3对p17的加工仅增加了3倍，这表明另一种caspase(s)参与了GraB细胞凋亡的线粒体扩增。我们得出结论，GraB诱导的细胞凋亡以caspase依赖的方式被线粒体高度扩增，但在没有线粒体的情况下，GraB也可以启动caspase 3加工和细胞凋亡。
Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Δψ). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Δψ and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Δψ suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Δψ suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.