MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK.
School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.
Cell Chem Biol. 2019 Mar 21;26(3):449-461.e8. doi: 10.1016/j.chembiol.2018.12.002. Epub 2019 Jan 31.
Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.
线粒体谷胱甘肽（GSH）和硫氧还蛋白（Trx）系统独立于细胞的其他部分发挥作用。虽然维持线粒体硫醇氧化还原状态被认为对细胞存活至关重要，但由于难以独立于其他细胞区室的硫醇系统来操纵细胞器的硫醇系统，因此这一点无法进行测试。为了克服这一限制，我们通过与线粒体靶向三苯基膦阳离子缀合来修饰谷胱甘肽 S-转移酶底物和硫氧还蛋白还原酶（TrxR）抑制剂 1-氯-2,4-二硝基苯（CDNB）。其产物，MitoCDNB，被线粒体摄取，其中它通过谷胱甘肽 S-转移酶选择性耗尽线粒体 GSH 池，并直接抑制线粒体 TrxR2 和过氧化物酶 3，一种过氧化物酶。重要的是，MitoCDNB 使分离细胞和体内的线粒体硫醇氧化还原稳态失活，而不影响细胞质的稳态。因此，MitoCDNB 能够评估线粒体硫醇稳态在活性氧产生、细胞器动力学、氧化还原信号转导和细胞死亡中的生物医学重要性。
