In situ detection of S-glutathionylated proteins following glutaredoxin-1 catalyzed cysteine derivatization

S-glutathionylation is rapidly emerging as an important post-translational modification, responsible for transducing oxidant signals. However, few approaches are available that allow visualization of glutathione mixed disulfides in intact cells. We describe here a glutaredoxin1-dependent cysteine derivatization and labeling approach, in order to visualize S-glutathionylation patterns in situ. Using this new method, marked S-glutathionylation was observed in epithelial cells, which was predominant at membrane ruffles. As expected, the labeling intensity was further enhanced in response to bolus oxidant treatments, or in cells overexpressing Nox1 plus its coactivators. In addition, manipulation of endogenous levels of glutaredoxin-1 via RNAi, or overexpression resulted in altered sensitivity to H2O2 induced formation of glutathione mixed disulfides. Overall, the derivatization approach described here preferentially detects S-glutathionylation and provides an important means to visualize this post-translational modification in sub-cellular compartments and to investigate its relation to normal physiology as well as pathology

Cigarette Smoke Targets Glutaredoxin 1, Increasing S-glutathionylation and Epithelial Cell Death

It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death

Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease

Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired reepithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor Fas, which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with profibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, such as S-glutathionylation (PSSG), is known to act as a regulatory event that affects protein function. This manuscript will discuss evidence that S-glutathionylation regulates death receptor induced apoptosis, and the potential implications for cysteine oxidations in the pathogenesis of in fibrotic lung disease

Activation of the glutaredoxin-1 gene by nuclear factor B enhances signaling.

The transcription factor nuclear factor kappaB (NF-kappaB) is a critical regulator of inflammation and immunity and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyzes deglutathionylation and enhances NF-kappaB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-kappaB in regulating activation of Glrx1. Transgenic mice that express a doxycycline-inducible constitutively active version of inhibitory kappaB kinase-beta (CA-IKKbeta) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKbeta also resulted in significant induction of Grx1. A 2-kb region of the Glrx1 promoter that contains two putative NF-kappaB binding sites was activated by CA-IKKbeta, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression and time-dependent increases in S-glutathionylation of IKKbeta. Overexpression of Grx1 decreased S-glutathionylation of IKKbeta, prolonged NF-kappaB activation, and increased levels of proinflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-kappaB and suggests a feed-forward mechanism to promote NF-kappaB signaling by decreasing S-glutathionylation