Perforation of the outer mitochondrial membrane triggered by BAX and facilitated by its main activator cBID is a fundamental process in cell apoptosis. Here, we employ a newly designed correlative approach based on a combination of a fluorescence cross correlation binding with a calcein permeabilization assay to understand the involvement of BAX in pore formation under oxidative stress conditions. To mimic the oxidative stress, we enriched liposomal membranes by phosphatidylcholines with truncated sn-2 acyl chains terminated by a carboxyl or aldehyde moiety. Our observations revealed that oxidative stress enhances proapoptotic conditions involving accelerated pore-opening kinetics. This enhancement is achieved through increased recruitment of BAX to the membrane and facilitation of BAX membrane insertion. Despite these effects, the fundamental mechanism of pore formation remained unchanged, suggesting an all-or-none mechanism. In line with this mechanism, we demonstrated that the minimal number of BAX molecules at the membrane necessary for pore formation remains constant regardless of BAX activation by cBID or the presence of oxidized lipids. Overall, our findings give a comprehensive picture of the molecular mechanisms underlying apoptotic pore formation and highlight the selective amplifying role of oxidized lipids in triggering formation of membrane pores.

中文翻译：

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BAX 诱导的孔隙在暴露于氧化应激时开孔的最低要求


由 BAX 触发并由其主要激活剂 cBID 促进的线粒体外膜穿孔是细胞凋亡的基本过程。在这里，我们采用了一种新设计的相关方法，该方法基于荧光互相关结合与钙黄绿素透化测定相结合，以了解 BAX 在氧化应激条件下参与孔形成。为了模拟氧化应激，我们通过磷脂酰胆碱富集脂质体膜，磷脂酰胆碱具有截短的 sn-2 酰基链，以羧基或醛部分终止。我们的观察表明，氧化应激增强了涉及加速开孔动力学的促凋亡条件。这种增强是通过增加 BAX 向膜的募集和促进 BAX 膜插入来实现的。尽管有这些影响，但孔形成的基本机制保持不变，表明存在全有或全无机制。根据这种机制，我们证明，无论 cBID 是否激活 BAX 或存在氧化脂质，膜上形成孔所需的最小数量的 BAX 分子都保持不变。总体而言，我们的研究结果全面介绍了凋亡孔形成的分子机制，并强调了氧化脂质在触发膜孔形成中的选择性扩增作用。