Reshi Latif, Wang Hua-Ven, Hui Cho-Fat, Su Yu-Chin, Hong Jiann-Ruey
Lab of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng Kung University, No. 1 University Road, Tainan City 701, Taiwan, ROC; Department of Life Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, No. 1 University Road, Tainan City 701, Taiwan, ROC.
Department of Life Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, No. 1 University Road, Tainan City 701, Taiwan, ROC.
Fish Shellfish Immunol. 2017 Feb;61:120-129. doi: 10.1016/j.fsi.2016.12.026. Epub 2016 Dec 23.
Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability.
虽然已知丝氨酸/苏氨酸（ST）激酶可诱导GF-1细胞中的宿主细胞死亡，但ST激酶如何诱导线粒体功能丧失仍不清楚。在本研究中，我们通过确定Bcl-2家族成员Bcl-2和Bcl-xL是否能通过与促凋亡基因Bax相互作用来预防ST激酶诱导的细胞死亡活性，解决了线粒体功能丧失的问题。选择携带EGFP-Bal-xL和EGFP-Bcl-2融合基因的石斑鱼鳍细胞（GF-1），在细胞培养中建立，并用于检测Bcl-2和Bcl-xL过表达在保护GF-1细胞免受巨石斑鱼虹彩病毒（GSIV）ST激酶基因影响中的作用。使用TUNEL检测法，我们发现EGFP-Bcl-2和EGFP-Bcl-xL在转染后24小时和48小时均将GSIV ST激酶诱导的凋亡率降低至20%。此外，Bcl-2和Bcl-xL分别在转染后24小时和48小时显著降低了GSIV ST激酶诱导的线粒体膜电位（Δψps）丧失的细胞百分比，与载体对照相比，这种降低分别与转染后24小时和48小时宿主细胞活力提高30%和50%相关。此外，在转染后48小时ST激酶表达期间分析Bcl-2和Bcl-xL与靶向线粒体的Bax相互作用的效果发现，Bcl-2和Bcl-xL也与Bax相互作用以阻止细胞色素c释放。最后，Bcl-2和Bcl-xL过表达在转染后48小时导致ST激酶功能受阻，这与阻止半胱天冬酶-9和-3的切割和激活相关，从而阻断下游死亡信号事件。综上所述，我们的结果表明，ST激酶诱导的Bax/线粒体介导的细胞死亡途径可被Bcl-2和Bcl-xL与Bax的相互作用阻断，以在MMP丧失期间抑制细胞色素c释放。这种拯救活性也与抑制半胱天冬酶-9和-3的激活相关，从而提高细胞活力。
