Redox regulation of proteins of the mitochondrial intermembrane space

线粒体膜间空间的蛋白质在细胞能量代谢、金属离子、代谢物和蛋白质在线粒体膜间的转运以及细胞凋亡的调控中发挥重要作用。许多这些蛋白质含有二硫键，推测反映了膜间空间从细菌的周质进化起源。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在外膜上的易位。在之前的资助期间，我们对蛋白Erv1和Mia40进行了表征，它们是二硫化物接力的核心参与者，在简单结构的螺旋-环-螺旋蛋白的折叠中。在下一个资助期内，我们希望研究含有保守半胱氨酸模式的膜间空间蛋白。到目前为止，Twin Cx9C蛋白是一组特征不明确的蛋白质。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，我们想研究具有更复杂的半胱氨酸模式和/或不是简单的螺旋-环-螺旋结构的蛋白质的氧化。我们的模型蛋白之一是Atp23，它是膜间空间中的一种蛋白酶，含有十个保守的半胱氨酸残基。在体内，Atp23在一个依赖mia40的过程中被完全氧化。我们想了解Atp23中5个二硫键的正确连通性是如何实现的，以及这个隔室中二硫键的异构化是如何被催化的。在进一步的方法中，我们希望解决在不依赖mia40的反应中输入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空间的蛋白质活性。从这些项目中，我们期望对线粒体蛋白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.