Ben-Hail Danya, Shoshan-Barmatz Varda
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Biochim Biophys Acta. 2016 Jul;1863(7 Pt A):1612-23. doi: 10.1016/j.bbamcr.2016.04.002. Epub 2016 Apr 8.
Mitochondria-mediated apoptosis involves pro-apoptotic protein release from the mitochondria to the cytosol, triggering apoptosis. However, the mechanisms by which apoptotic initiators cross the outer mitochondrial membrane (OMM) remain unclear. The voltage-dependent anion channel 1 (VDAC1), an OMM protein, is central to mitochondria-mediated apoptosis. In previous work, we demonstrated that apoptosis induction is associated with VDAC1 oligomerization, forming a mega-pore that mediates pro-apoptotic protein release. Here, we demonstrated that several known anion transport inhibitors, DIDS, SITS, H(2)DIDS, DNDS, and DPC, all interact with VDAC1, as revealed by micro-scale thermophoresis and decreased conductance of bilayer-reconstituted VDAC1. These compounds inhibited apoptosis stimuli-induced release of mitochondrial pro-apoptotic proteins, apoptosis and VDAC1 oligomerization, as monitored by chemical cross-linking or in living cells by BRET2. Moreover, the compounds inhibited VDAC1 oligomerization in isolated mitochondria and as induced by VDAC1 over-expression, suggesting that the inhibitory effect of the tested compounds involved VDAC1. Finally, the compounds also inhibited apoptosis-associated increases in intracellular Ca(2+), ([Ca(2+)]i), ROS production, mitochondria membrane potential dissipation and the increase in VDAC1 expression levels. The results presented here explored a new mechanism of action for DIDS and its analogs. All inhibited apoptosis via direct interaction with VDAC1 to inhibit its oligomerization and subsequent Cyto c release and apoptosis. Such results may allow the development of a VDAC1-specific inhibitor that would offer substantial insight into the function of VDAC1 in controlling metabolism, energy production, cholesterol transport and apoptosis. Finally, inhibitors of apoptosis could serve in pathological conditions where enhanced apoptosis is found, such as neurodegenerative diseases.
线粒体介导的细胞凋亡涉及促凋亡蛋白从线粒体释放到细胞质中，从而引发细胞凋亡。然而，凋亡启动子穿过线粒体外膜（OMM）的机制仍不清楚。电压依赖性阴离子通道1（VDAC1）是一种线粒体外膜蛋白，在介导线粒体凋亡中起核心作用。在之前的研究中，我们证明细胞凋亡诱导与VDAC1寡聚化有关，形成一个介导促凋亡蛋白释放的大孔。在此，我们证明几种已知的阴离子转运抑制剂，如DIDS、SITS、H(2)DIDS、DNDS和DPC，都与VDAC1相互作用，这通过微量热泳分析以及双层重构VDAC1的电导率降低得以揭示。这些化合物抑制了凋亡刺激诱导的线粒体促凋亡蛋白释放、细胞凋亡以及VDAC1寡聚化，这通过化学交联监测或在活细胞中利用BRET2进行监测。此外，这些化合物在分离的线粒体中以及由VDAC1过表达诱导的情况下抑制VDAC1寡聚化，表明所测试化合物的抑制作用涉及VDAC1。最后，这些化合物还抑制了与细胞凋亡相关的细胞内Ca(2+)（[Ca(2+)]i）增加、活性氧生成、线粒体膜电位耗散以及VDAC1表达水平的升高。此处呈现的结果探索了DIDS及其类似物的一种新作用机制。所有这些化合物都通过与VDAC1直接相互作用来抑制其寡聚化以及随后的细胞色素c释放和细胞凋亡，从而抑制细胞凋亡。这样的结果可能有助于开发一种VDAC1特异性抑制剂，这将为深入了解VDAC1在控制代谢、能量产生、胆固醇转运和细胞凋亡中的功能提供重要见解。最后，凋亡抑制剂可用于存在增强细胞凋亡的病理状况，如神经退行性疾病。
