INRES-Chemical Signalling, University of Bonn Bonn, Germany.
Interactions Arbres Microorganismes, IFR 110 EFABA, Faculté des sciences, Université de Lorraine, UMR 1136 Université de Lorraine/INRA Vandoeuvre lès-Nancy, France.
Front Plant Sci. 2013 Dec 16;4:506. doi: 10.3389/fpls.2013.00506. eCollection 2013.
Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (E GSH ) of more negative than -310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure E GSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between -280 and -290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of E GSH values that can be measured in vivo with roGFP2-based probes from about -320 mV for GRX1-roGFP2 down to about -210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an E GSH of about -260 mV in this mutant.
谷胱甘肽对于解毒、生化反应的辅助因子以及作为硫醇氧化还原缓冲剂非常重要。细胞质中的谷胱甘肽缓冲剂通常处于高度还原状态，谷胱甘肽氧化还原电位（E GSH ）比-310 mV 更负。通过谷胱甘肽还原酶（GR）不断还原谷胱甘肽二硫化物，可维持这种负氧化还原电位。谷胱甘肽氧化还原稳态的偏离被认为是通过关键硫醇残基的转换来改变氧化还原敏感蛋白活性的一种可能方式。为了更好地理解这种信号机制，必须能够在从高度负的氧化还原电位到已经表现出严重表型的突变体中发现的电位的广泛范围内测量 E GSH。随着氧化还原敏感 GFP（roGFP）的出现，基于硫醇的氧化还原缓冲系统的体内动力学变得可行。然而，原始的 roGFP 版本 roGFP1 和 roGFP2 的中点电位在-280 至-290 mV 之间，这使得它们在 ER 中完全氧化，在细胞质、质体、线粒体和过氧化物酶体中几乎完全还原。为了扩展合适探针的范围，我们设计了一个 roGFP2 衍生物 roGFP2-iL，其中点电位约为-238 mV。该值在报告的同源 roGFP1-iX 探针的氧化还原电位范围内，尽管激发特性不同。为了与谷胱甘肽池快速且特异性地平衡，生成并在体外对与人谷胱甘肽还原酶 1（GRX1）融合的构建体进行了表征。GRX1-roGFP2-iL 被证明适合于体内氧化还原电位测量，并扩展了基于 roGFP2 的探针在体内测量 E GSH 值的范围，从约-320 mV（GRX1-roGFP2）降低到约-210 mV（GRX1-roGFP2-iL）。在严重谷胱甘肽缺乏的 rml1 幼苗的细胞质中使用这两种探针，在该突变体中发现 E GSH 约为-260 mV。
