MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg A H
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9.
J Exp Med. 1999 Jan 4;189(1):131-44. doi: 10.1084/jem.189.1.131.
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 (Deltapsi). 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 Deltapsi 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, Deltapsi 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, Deltapsi 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.
颗粒酶B（GraB）是细胞毒性T淋巴细胞和自然杀伤细胞有效激活凋亡所必需的。我们发现GraB和穿孔素会诱导严重的线粒体扰动，细胞色素c释放到细胞质以及跨膜电位（Δψ）的抑制就证明了这一点。最早的线粒体事件是细胞色素c的释放，它与半胱天冬酶3的加工同时发生，并且始终在凋亡激活之前。颗粒酶K/穿孔素或穿孔素处理，这两种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，不会诱导细胞色素c快速释放。然而，它们抑制了Δψ并增加了活性氧的产生，表明线粒体功能障碍也与这种非凋亡性细胞死亡有关。用肽半胱天冬酶抑制剂zVAD-FMK或YVAD-CHO预处理可防止GraB诱导的凋亡和细胞色素c释放，而DEVD-CHO可阻断凋亡但不能阻止细胞色素c释放，表明半胱天冬酶在线粒体的上游和下游均起作用。另外有趣的是，由GraK或GraB和穿孔素介导的Δψ抑制不受zVAD-FMK影响，因此是不依赖半胱天冬酶的。Bcl-2和Bcl-XL的过表达抑制了半胱天冬酶激活、线粒体细胞色素c释放、Δψ抑制以及由GraB、GraK或穿孔素诱导的凋亡和细胞死亡。在体外无细胞系统中，高剂量的GraB在S-100细胞质中激活核凋亡，然而添加线粒体可使GraB活性放大15倍以上。在存在线粒体的情况下，GraB诱导的S-100细胞质中半胱天冬酶3加工成p17仅增加了三倍，这表明另一种半胱天冬酶参与了GraB凋亡的线粒体放大过程。我们得出结论，GraB诱导的凋亡以半胱天冬酶依赖性方式被线粒体高度放大，但GraB也可以在没有线粒体的情况下启动半胱天冬酶3加工和凋亡。
