Aim32p; a novel multi-faceted protein in mitochondrial biogenesis

项目总结 线粒体的生物发生依赖于有效的蛋白质输入，因为大多数线粒体蛋白质都是输入的 在胞浆中合成后通过蛋白质输入途径。线粒体膜间隙(IMS) 专门将蛋白质导入IMS的组装(MIA)途径是独特的，因为氧化折叠驱动 目的蛋白的导入和折叠。具体地说，一系列研究得很好的硫醇-二硫键交换反应 由MIA途径的两个主要组成部分，即Erv1和Mia40决定载体执行 移位到线粒体IMS。研究表明，几种非经典底物，它们确实 不具有双CX3C或CX9C基序，利用这条途径，重要的是，将MIA途径与 IMS中的其他重要过程，与氧化折叠无关。因此，MIA途径在 一系列疾病的病理学，如肌病、神经病、亨廷顿病、肌萎缩侧索硬化症和 癌症。然而，仍有几个问题没有得到解答。随着底物光谱的不断增长， 因此，有必要了解其潜在的分子机制。MIA途径必须适应 氧化还原通过与IMS(硫氧还蛋白)中参与还原反应的抗氧化酶的相互作用而改变 1、过氧化还蛋白和谷氧还蛋白2)。然而，这些氧化还原平衡系统在MIA中的作用 机械并不为人所熟知，更值得注意的是，在IMS中可能存在其他还原机制。最后， 因为MIA途径是在厌氧条件下运行的，所以必须有额外的电子受体。 这项由本科生主导的提案的目标是调查新发现的 Erv1相互作用蛋白，Aim32p在实验模型中，芽生酵母酿酒酵母。 初步研究有力地表明，Aim32p对蛋白质跨 转运蛋白，稳定几个天然的蛋白质复合体，属于一类蛋白质，被称为 硫氧还蛋白样铁还蛋白(FDS)；其功能未知，但范围从电子穿梭到氧化还原 感官。由于Aim32p在IMS中的独特位置，因此有必要研究Aim32p 可影响多个重要的线粒体过程的输入、电子传递，并具有调节作用 在氧化还原过程中。利用生化和生物信息学方法相结合的三个具体提案目标 将进行：在目标1中，将探索Aim32p在MIA途径中的作用。在目标2中，生化 验证Aim32P是否是铁-S蛋白的研究，鉴定关键的半胱氨酸残基和关键的信号转导途径 它的细胞应激反应，将被执行。最后，Aim 3将阐明Aim32p相互作用网络。 在成功完成后，这项工作将提供令人兴奋的关于多个- 对线粒体蛋白质进行刻面分析，并提高我们对蛋白质转位过程的基础知识。 这项研究将对公共健康产生广泛的影响，因为这些机械性的研究将为 对线粒体生物发生缺陷如何导致疾病的见解。

PROJECT SUMMARY Mitochondrial biogenesis relies on efficient protein import as most mitochondrial proteins are imported via protein import pathways after synthesis in the cytosol. The mitochondrial intermembrane space (IMS) assembly (MIA) pathway that specifically imports proteins into the IMS is unique in that oxidative folding drives import and folding of target proteins. Specifically, a series of well-studied thiol-disulfide exchange reactions carried out by the two main components of the MIA pathway, namely Erv1 and Mia40 dictate vectorial translocation into the mitochondrial IMS. Studies have shown that several non-classical substrates, which do not possess the twin CX3C or CX9C motifs, utilize this pathway and importantly, connect the MIA pathway with other vital processes in the IMS, unrelated to oxidative folding. Hence, the MIA pathway is highly relevant in pathology of a spectrum of diseases such as, myopathies, neuropathies, Huntington’s disease, ALS and cancer. However, several unanswered questions remain. With the growing spectrum of substrates of this pathway, there is a need to understand the underlying molecular mechanisms. The MIA pathway must adapt to redox changes via interactions with antioxidant enzymes involved in reductive reactions in the IMS (thioredoxin 1, peroxiredoxin, and glutaredoxin 2). However, the role of these redox-balancing systems with the MIA machinery is not well known, and more notably, other reductive mechanisms may exist in the IMS. Finally, because MIA pathway is operational under anaerobic conditions, there must be additional electron acceptors. The goal of this undergraduate-driven proposal is to investigate the function of the newly identified Erv1-interacting protein, Aim32p in the experimental model, the budding yeast Saccharomyces cerevisiae. Preliminary studies strongly suggest that Aim32p is important for protein translocation across multiple translocons, stabilizes several native protein complexes, and belongs to a class of proteins, termed as thioredoxin-like ferredoxins (Fds); functions of which are unknown but range from electron shuttling to redox sensing. Because of its unique placement in the IMS, it is imperative to examine mechanisms by which Aim32p could affect multiple important mitochondrial processes of import, electron transfer, and have a regulatory role in redox. Three specific proposal aims that utilize a combination of biochemical and bioinformatic approaches will be undertaken: In Aim 1 role of Aim32p within the MIA pathway will be explored. In Aim 2, biochemical studies to validate if Aim32p is a Fe-S protein, identification of key cysteine residues, and pathways crucial for its cellular stress response, will be performed. Finally, Aim 3 will elucidate the Aim32p interaction network. Upon successful completion, this work will provide exciting new information on the function of a multi- faceted mitochondrial protein and advance our fundamental knowledge of the process of protein translocation. This research will have a broad impact on public health because these mechanistic studies will provide key insights into how defects in mitochondrial biogenesis lead to disease.