Department of Chemistry, University of Houston, Houston, TX 77204, USA.
College of Optometry, University of Houston, Houston, TX 77204, USA.
Metallomics. 2022 Nov 24;14(11). doi: 10.1093/mtomcs/mfac087.
Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.
细胞内过量的铜会扰乱细胞的氧化还原平衡，从而导致疾病。然而，细胞氧化还原状态与铜稳态之间的关系，以及这种相互作用如何在细胞区室中协调，尚未得到很好的确定。本研究使用细胞器特异性氧化还原传感器 Grx1-roGFP2 和非靶向蛋白质组学的联合方法，研究了活 HEK293 细胞中线粒体和细胞质中铜依赖性抗氧化防御的实时变化。铜依赖性实时成像实验表明，CuCl2 处理导致细胞质和线粒体中的氧化应激增加。相比之下，随后用 bathocuproine sulfonate（一种铜螯合剂）去除多余的铜会降低线粒体中的氧化应激，但会导致细胞质中的氧化应激更高。蛋白质组学数据显示，在 Cu 处理下，几种线粒体蛋白而非细胞质蛋白的丰度发生显著变化。蛋白质组学分析还表明，发生显著变化的蛋白质与线粒体氧化磷酸化和谷胱甘肽合成有关。不同细胞区室中氧化还原行为和蛋白质谱的差异揭示了铜诱导的氧化应激下线粒体和细胞质的不同反应机制。这些发现深入了解了在亚细胞水平上通过调节特定蛋白质表达来实现氧化还原和铜稳态相互作用的机制，为理解铜诱导的氧化还原失调对疾病的影响提供了线索。
