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

Glutathione-S-transferase P promotes glycolysis in asthma in association with oxidation of pyruvate kinase M2

peer reviewedBackground: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease. © 202

Soil bacteria, nitrite and the skin

Little is known about the composition of the skin microbiome and its potential significance for health and disease in the context of the ‘hygiene hypothesis’. We here propose that mammals evolved with a dermal microflora that contributed to the regulation of body physiology by providing nitrite from commensal ammonia-oxidising bacteria in response to ammonia released during sweating. We further hypothesise that modern skin hygiene practices have led to a gradual loss of these bacteria from our skin. Together with other lifestyle-related changes associated with an insufficient bodily supply with nitrite and depletion of other nitric oxide(NO)-related species, a condition we here define as ‘nitropenia’, this has led to a perturbation of cellular redox signalling which manifests as dysregulated immunity and generalised inflammation. If proven correct, this scenario would provide an additional evolutionary rationale and a mechanistic basis for the simultaneous rises in prevalence of a number of seemingly unrelated chronic illnesses over the last 3–4 decades