Gutscher Marcus, Pauleau Anne-Laure, Marty Laurent, Brach Thorsten, Wabnitz Guido H, Samstag Yvonne, Meyer Andreas J, Dick Tobias P
Redox Regulation Research Group, German Cancer Research Center (DKFZ/A160), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Nat Methods. 2008 Jun;5(6):553-9. doi: 10.1038/nmeth.1212. Epub 2008 May 11.
Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E(GSH)) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.
目前，由于缺乏合适的氧化还原生物传感器，活细胞中基于氧化还原过程的动态分析受到限制。传统的氧化还原敏感型绿色荧光蛋白（roGFP）存在特异性不明确以及对氧化还原电位变化响应缓慢的问题。在本研究中，我们证明了人谷氧还蛋白-1（Grx1）与roGFP2融合能够促进传感器蛋白与谷胱甘肽氧化还原对之间的特异性实时平衡。Grx1-roGFP2融合蛋白能够对不同细胞区室中的谷胱甘肽氧化还原电位（E(GSH)）进行高灵敏度和高时间分辨率的动态活细胞成像。该生物传感器在数秒到数分钟的时间尺度上，能在毫摩尔级的还原型谷胱甘肽（GSH）背景下检测到纳摩尔级的氧化型谷胱甘肽（GSSG）变化。它有助于观察与生长因子可用性、细胞密度、线粒体去极化、呼吸爆发活性和免疫受体刺激相关的氧化还原变化。
