Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.

凋亡诱导因子（AIF）是一种黄素蛋白，是线粒体膜间空间导入机制中CHCHD4氧化还原蛋白的重要伴侣。哺乳动物AIF具有三个半胱氨酸残基，很少受到关注。先前的报道已证明AIF与硫氧还蛋白1（Trx1）之间的氧化还原相互作用，尤其是在氧化剂条件下。因此，我们询问人AIF的半胱氨酸残基是否可以被氧化。我们的数据表明内源性AIF可以被氧化成二硫键连接的共轭物（DLC）。HEK293T细胞中过表达的WT AIF以及重组WT AIF形成了DLC。C256S，C317S或C441S AIF突变体的表达严重抑制了暴露于氧化剂的细胞中DLC的形成。体外，DLC的形成被C256S和C441S AIF突变体完全排除，而被C317S突变体部分抑制。研究表明，DLC可增强细胞对星形孢菌素诱导的细胞凋亡的敏感性，这可能是通过阻止AIF维持线粒体的氧化磷酸化来实现的。Trx1表达降低的细胞比正常Trx1水平的细胞产生更多的AIF DLC，在体外， Trx1能够减少AIF DLC的数量。最后，共聚焦分析以及线粒体级分的免疫印迹表明，线粒体中存在一部分Trx1。总体而言，这些数据提供了证据，表明AIF的所有三个半胱氨酸残基都可以被氧化成DLC，而DLC可以被线粒体Trx1破坏。