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Architecture Mapping of the Inner Mitochondrial Membrane Proteome by Chemical Tools in Live Cells

机译:内细胞线粒体膜蛋白质组学的化学工具在活细胞中的架构图。

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摘要

The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.
机译:内线粒体膜(IMM)蛋白质组在维持线粒体生理和细胞代谢中起着核心作用。 IMM蛋白质组学进行了各种重要的生化反应,例如氧化磷酸化,代谢产物的产生和线粒体的生物发生,并且针对IMM蛋白开发了针对线粒体的疗法,该疗法与包括癌症和神经退行性疾病在内的各种人类代谢性疾病密切相关。然而,由于缺乏在活细胞中以高通量的方式对其进行评估的方法,IMM蛋白质组的膜拓扑结构仍然很大程度上不清楚。在本文中,我们使用具有基因靶向过氧化物酶(APEX)的原位产生的自由基探针揭示了135个IMM蛋白的体内拓扑学方向。由于线粒体靶向APEX原位产生的苯氧基自由基的寿命短,以及IMM对小分子的不渗透性,IMM蛋白的基质和膜间空间(IMS)侧都暴露于溶剂的酪氨酸残基被专门标记为分别通过Matrix-APEX和IMS-APEX对活细胞中的自由基探针进行质谱鉴定。通过该分析,我们确定了58种IMM蛋白拓扑结构,并且可以确定77种IMM蛋白的拓扑结构方向,这些蛋白的IMM拓扑结构尚未完全表征。我们还发现了几种IMM蛋白(例如LETM1和OXA1),其拓扑信息应根据我们的结果进行修改。总体而言,我们对线粒体内膜蛋白质组的结构信息的鉴定可以为活细胞中IMM蛋白质组的结构和连接组提供有价值的见解。

著录项

  • 来源
    《Journal of the American Chemical Society》 |2017年第10期|3651-3662|共12页
  • 作者单位

    Department of Chemistry and Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea;

    Department of Chemistry and Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea;

    Center for RNA Research, Institute of Basic Science (IBS), Seoul 08826, Korea, School of Biological Sciences, Seoul National University, Seoul 08826, Korea;

    Department of Chemistry and Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea;

    Department of Biomedical Engineering,Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea;

    UNIST Central Research Facilities (UCRF), Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea;

    Center for RNA Research, Institute of Basic Science (IBS), Seoul 08826, Korea, School of Biological Sciences, Seoul National University, Seoul 08826, Korea;

    Center for RNA Research, Institute of Basic Science (IBS), Seoul 08826, Korea;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
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  • 入库时间 2022-08-18 03:07:56

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