Aim32p; a novel multi-faceted protein in mitochondrial biogenesis

目标32p；

• 批准号: 9812708
• 负责人: Deepa Vinay Dabir
• 金额: $ 32.38万
• 依托单位: LOYOLA MARYMOUNT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812708
• 关键词: Affect; Anaerobic Bacteria; Binding; Biochemical; Biogenesis; Bioinformatics; C-terminal; Cells; Cellular Stress Response; Complex; Coupled; Cysteine; Cytosol; DNA Damage; Defect; Disease; Disulfide Linkage; Disulfides; Electron Transport; Electrons; Equilibrium; Experimental Models; Ferredoxin; Fumarates; Future; Goals; Health; Homeostasis; Human; Huntington Disease; Hypoxia; In Vitro; Inner Cell Mass; Institution; Iron; Iron-Binding Proteins; Knowledge; Lead; Learning; Lipids; Malignant Neoplasms; Mammalian Cell; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Molecular; Molecular Chaperones; Myopathy; Neuropathy; Organelles; Oxidants; Oxidation-Reduction; Oxidoreductase; Oxygen; Parkinson Disease; Pathology; Pathway interactions; Physiological; Physiological Processes; Physiology; Play; Process; Protein Import; Protein translocation; Proteins; Proteome; Public Health; Reaction; Recombinant Delta Chemokine; Reporting; Research; Respiratory Chain; Role; Saccharomyces cerevisiae; Saccharomycetales; Seminal; Series; Specific qualifier value; Structure; Students; Sulfhydryl Compounds; System; TXN gene; Translating; Twin Multiple Birth; Work; Yeasts; antioxidant enzyme; biological adaptation to stress; blastocyst; career; cytochrome c; defense response; experience; glutaredoxin 2; hands on research; human embryonic stem cell; hydroxyurea; in vivo; insight; mitochondrial dysfunction; non-Native; novel; oxidation; peroxiredoxin 2; prevent; protein complex; receptor; reproductive tract; sulfhydryl oxidase; undergraduate student; vector

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.

项目摘要 线粒体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是输入的 在胞质溶胶中合成后通过蛋白质输入途径。线粒体膜间隙（IMS） 特异性地将蛋白质导入IMS的组装（MIA）途径是独特的，因为氧化折叠驱动 导入和折叠靶蛋白。具体来说，一系列研究充分的硫醇-二硫化物交换反应 由MIA途径的两个主要组成部分，即Erv 1和Mia 40决定载体进行 易位到线粒体IMS中。研究表明，几种非经典底物， 不具有双CX 3C或CX 9 C基序，利用这一途径，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途径在以下方面高度相关： 例如肌病、神经病、亨廷顿氏病、ALS和 癌然而，仍有几个问题没有得到解答。随着这一技术的底物范围的不断扩大， 因此，有必要了解潜在的分子机制。MIA途径必须适应 通过与IMS中参与还原反应的抗氧化酶（硫氧还蛋白）的相互作用而发生氧化还原变化 1、过氧化物氧还蛋白和谷氧还蛋白2）。然而，这些氧化还原平衡系统与MIA的作用 机制并不为人所知，更值得注意的是，IMS中可能存在其他还原机制。最后， 因为MIA途径在厌氧条件下是可操作的，所以必须有额外的电子受体。 这个由本科生提出的建议的目标是调查新发现的 Erv 1相互作用蛋白，Aim 32 p在实验模型中，芽殖酵母酿酒酵母。 初步研究强烈表明，Aim 32 p对于蛋白质在多个细胞中的易位是重要的。 translocons，稳定几种天然蛋白质复合物，属于一类蛋白质，称为 硫氧还蛋白样铁氧还蛋白（Fds）;其功能未知，但范围从电子穿梭到氧化还原 传感。由于其在IMS中的独特位置，必须检查Aim 32 p 可以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 在氧化还原中。利用生物化学和生物信息学方法相结合的三个具体建议目标 在目标1中，将探索Aim 32 p在MIA途径中的作用。在目标2中，生物化学 验证Aim 32 p是否是Fe-S蛋白的研究、关键半胱氨酸残基的鉴定以及对Aim 32 p至关重要的途径 它的细胞应激反应，将被执行。最后，目标3将阐明Aim 32 p相互作用网络。 成功完成后，这项工作将提供令人兴奋的新信息的功能，多- 多面线粒体蛋白和推进我们的蛋白质易位过程的基础知识。 这项研究将对公共卫生产生广泛的影响，因为这些机制研究将提供关键的 深入了解线粒体生物合成缺陷如何导致疾病。