Redox regulation of proteins of the mitochondrial intermembrane space

线粒体膜间隙蛋白在细胞能量代谢、金属离子、代谢产物和蛋白质在线粒体膜之间的运输以及细胞凋亡的调控中发挥着重要作用。这些蛋白质中有许多含有二硫键，可能反映了细菌周质的膜间隙的进化起源。我们最近发现了一种机制(称为线粒体二硫化物继电器)，它利用蛋白质氧化来驱动新合成的蛋白质通过外膜转移。在之前的资助期间，我们表征了二硫键接力的中心参与者Erv1和Mia40蛋白在简单结构的螺旋-环-螺旋蛋白的折叠中的作用。在下一个资助期，我们希望研究包含保守半胱氨酸模式的膜间隙蛋白。到目前为止，一组特征很差的蛋白质由双胞胎Cx9C蛋白组成。利用蛋白质组学筛选，我们希望确定这些保守成分的稳定和瞬时相互作用伙伴，并探索它们在线粒体生物发生中的功能。此外，我们想要研究具有更复杂半胱氨酸模式和/或不是简单螺旋-环-螺旋结构的蛋白质的氧化。我们的模型蛋白之一是Atp23，它是膜间隙中的一种蛋白酶，它包含10个保守的半胱氨酸残基。在体内，Atp23在Mia40依赖的过程中被完全氧化。我们想了解Atp23中五个二硫键的正确连接性是如何达到的，以及这个隔间中二硫键的异构化是如何被催化的。在另一种方法中，我们希望解决在Mia40独立反应中输入到线粒体的蛋白质的氧化问题。我们的初步结果表明，半胱氨酸残基的氧化被用来调节膜间隙中的蛋白质活性。从这些项目中，我们期望对线粒体蛋白质的生物发生和氧化还原控制的分子机制有基本的了解。

Proteins of the mitochondrial intermembrane space play important roles in cellular energy metabolism, in the transport of metal ions, metabolites and proteins between both mitochondrial membranes and in the regulation of apoptosis. Many of these proteins contain disulfide bonds presumably reflecting the evolutionary origin of the intermembrane space from the periplasm of bacteria. We recently identified a machinery (called the mitochondrial disulfide relay) which uses protein oxidation to drive the translocation of newly synthesized proteins across the outer membrane. During the previous funding period we characterized the proteins Erv1 and Mia40, the central players of the disulfide relay, in the folding of simply structured helix-loop-helix proteins. In the next funding period we want to study intermembrane space proteins that contain conserved cysteine patterns. One group of proteins that is poorly characterized so far consists of Twin Cx9C proteins. Employing a proteomic screen we want to identify stable and transient interaction partners of these conserved components and explore their function in the biogenesis of mitochondria. Moreover, we want to investigate the oxidation of proteins that have more complex cysteine patterns and/or are not of simple helix-loop-helix structure. One of our model proteins is Atp23, a protease in the intermembrane space, which contains ten conserved cysteine residues. In vivo Atp23 is completely oxidized in a Mia40-dependent process. We want to understand how the correct connectivity of the five disulfide bonds in Atp23 is reached and how the isomerisation of disulfide bonds in this compartment is catalyzed. In a further approach, we want to address the oxidation of proteins that are imported into mitochondria in a Mia40-independent reaction. Our preliminary results suggest that oxidation of cysteine residues is used to regulate protein activity in the intermembrane space. From these projects we expect fundamental insight into the molecular mechanisms of the biogenesis and redox control of mitochondrial proteins.