Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA.
J Virol. 2011 Aug;85(16):7965-75. doi: 10.1128/JVI.02515-10. Epub 2011 Jun 8.
Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.
Casp8p41 是一种新型蛋白，当 HIV-1 蛋白酶切割半胱天冬酶 8 时会产生 Casp8p41，它可独立引起 NF-κB 激活、促炎细胞因子产生和细胞死亡。在此，我们研究了 Casp8p41 诱导细胞死亡的机制。免疫金染色和电子显微镜显示，Casp8p41 定位于激活的原代 CD4 T 细胞的线粒体，提示线粒体参与其中。因此，我们评估了 Casp8p41 诱导的死亡对 Bax/Bak 和半胱天冬酶 9 的依赖性。在野生型 (WT) 小鼠胚胎成纤维细胞 (MEF) 细胞中，Casp8p41 导致线粒体迅速去极化 (P < 0.001)，但 Bax/Bak 双敲除 (DKO) MEF 细胞中 Casp8p41 的表达则不会。同样，表达 Casp8p41 的 caspase 9 缺陷型 T 细胞 (JMR 细胞) 仅发生最小程度的细胞死亡，而用 caspase 9 重建这些细胞 (F9 细胞) 则恢复 Casp8p41 的细胞毒性 (P < 0.01)。用绿色荧光蛋白 (GFP) HIV-1 报告病毒感染 caspase 9 缺陷型细胞，导致 32%感染 GFP 阳性细胞发生细胞死亡，而这些细胞中 caspase 9 表达的恢复则将感染细胞杀伤恢复至 68% (P < 0.05)，两种感染之间的病毒复制水平相似。我们的数据表明，Casp8p41 需要 Bax/Bak 诱导线粒体去极化，这导致 Casp8p41 表达或 HIV-1 感染后 caspase 9 的激活。这种理解允许设计中断 HIV-1 感染细胞这种形式死亡的策略。
