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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response to hydrogen peroxide, leading to rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable to stimulate the oxidative pentose phosphate pathway and enhance their reductive capacity. Specifically, we find that anchorage-independent growth of cells and spheroids is limited by an elevation of endogenous peroxide levels and is largely dependent on a functional GAPDH redox switch. Likewise, tumour growth in vivo is limited by peroxide stress and suppressed when the GAPDH redox switch is disabled in tumour cells. The induction of additional intratumoural oxidative stress by chemo- or radiotherapy synergized with the deactivation of the GAPDH redox switch. Mice lacking the GAPDH redox switch exhibit altered fatty acid metabolism in kidney and heart, apparently in compensation for the lack of the redox switch. Together, our findings demonstrate the physiological and pathophysiological relevance of oxidative GAPDH inactivation in mammals.

Early genetic studies hinted the role of airway epithelial cells in the development of idiopathic pulmonary fibrosis (IPF), while recent single-cell RNA sequencing (scRNA-seq) atlases utilized explant IPF lungs and therefore represent late-stage disease. Here, we used air liquid interface (ALI) cultures of primary cells taken from the subsegmental bronchi of newly diagnosed IPF patients, reflecting early-stage fibrosis, to interrogate the transcriptional landscape of the airway mucosa. Profiling of 129,986 cells identified a shared proinflammatory state in epithelial cells and an early activation state of fibroblasts. Moreover, IPF basal cells initiated awry repair mechanisms and primed the airway mucosa for TGF-β activation. Treatment with nintedanib, pirfenidone, both established antifibrotic drugs, and saracatinib, an Src kinase inhibitor that can limit IPF progression, only significantly affected certain IPF signatures. This study provides insight into the early disease mechanisms of IPF and may serve as a resource to further investigate pharmacological inhibition effects.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance that can contribute to mitochondrial dysfunction. Mitochondria are critical for cellular homeostasis and when dysfunctional they can contribute to epithelial cell death. Recently, SRC family kinases (SFK) have been associated with IPF pathology although their exact role and activation in this disease are still unknown. In this study, we investigated whether SFK are activated in human bronchial epithelial (HBE) cells as well as in primary HBE cells derived from IPF patients and if SFK modulation interferes with molecular pathways controlling mitochondrial function. Here, we demonstrate that TGF-β1 can rapidly activate the SRC kinase FYN in HBE cells, which subsequently induces mitochondrial ROS (mtROS) production. Moreover, TGF-β1-induced FYN activation involves initial activation of NOX4 and direct oxidation of FYN. At the same time, both FYN and mtROS contribute to TGF-β-induced induction of NOX4. Interestingly, MitoQ pretreatment attenuated the TGF-β-induced increase in the mRNA expression of NOX4 and the profibrotic genes VIM and FN1 but not of COL1A1. In HBE cells derived from IPF patients, we found an increased SFK activation compared to that in control HBE cells which was reduced upon treatment with the SFK-inhibitor AZD. Additionally, this SFK inhibition in HBE cells from IPF patients increased the mitochondrial biogenesis markers NRF2, PGC-1β and ERRα and the mitophagy marker PINK1 which were originally decreased in HBE cells from IPF patients compared to control HBE cells. Collectively, these results indicate complex and reciprocal interactions between NOX4 and mtROS in TGF-β-induced profibrotic responses suggesting a role for SFK in mitochondrial dysfunction in IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-beta (TGF-beta) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-beta signaling is not known. Here, we demonstrate that TGF-beta 1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker 7H2AX, and increased expression of profibrotic genes. Moreover, TGF-beta 1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-beta 1-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-beta 1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.

The SARS‐CoV‐2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID‐19 pathogenesis, especially host factors facilitating virus infection and replication. SARS‐CoV‐2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS‐CoV‐2 infection. Our data provide a rich resource for future investigations of COVID‐19 infection and pathogenesis., A single‐cell RNA‐seq resource identifies expression of coronavirus entry‐linked host cofactors ACE2,TMPRSS2 and FURIN primarily in bronchial cells in cells transitioning from secretory to ciliated identity.

SUMMARYThe SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigateACE2andTMPRSS2expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. WhileTMPRSS2is expressed in both tissues, in the subsegmental bronchial branchesACE2is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated., The activity of several protein kinases is increased upon cellular production of reactive oxygen species, which can cause cysteine oxidation. Here the authors show that sulfenylation of specific cysteine residues within Src induce local structural changes that directly impact its activation.

The NADPH oxidase homolog dual oxidase 1 (DUOX1) plays an important role in innate airway epithelial responses to infection or injury, but the precise molecular mechanisms are incompletely understood and the cellular redox-sensitive targets for DUOX1-derived H2O2 have not been identified. The aim of the present study was to survey the involvement of DUOX1 in cellular redox signaling by protein S-glutathionylation, a major mode of reversible redox signaling. Using human airway epithelial H292 cells and stable transfection with DUOX1-targeted shRNA as well as primary tracheal epithelial cells from either wild-type or DUOX1-deficient mice, DUOX1 was found to be critical in ATP-stimulated transient production of H2O2 and increased protein S-glutathionylation. Using cell pre-labeling with biotin-tagged GSH and analysis of avidin-purified proteins by global proteomics, 61 S-glutathionylated proteins were identified in ATP-stimulated cells compared to 19 in untreated cells. Based on a previously established role of DUOX1 in cell migration, various redox-sensitive proteins with established roles in cytoskeletal dynamics and/or cell migration were evaluated for S-glutathionylation, indicating a critical role for DUOX1 in ATP-stimulated S-glutathionylation of β-actin, peroxiredoxin 1, the non-receptor tyrosine kinase Src, and MAPK phosphatase 1. Overall, our studies demonstrate the importance of DUOX1 in epithelial redox signaling through reversible S-glutathionylation of a range of proteins, including proteins involved in cytoskeletal regulation and MAPK signaling pathways involved in cell migration., Graphical abstract, Highlights • ATP-mediated activation of DUOX1 results in increased protein S-glutathionylation. • ATP stimulation promotes S-glutathionylation of a number of diverse proteins. • DUOX1-dependent S-glutathionylation affects proteins involved in cell migration.

Background: The IL-1 family member IL-33 plays a critical role in type 2 innate immune responses to allergens and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood.Objective: Based on previous findings indicating involvement of the NADPH oxidase dual oxidase 1 (DUOX1) in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of asthmatic patients.Methods: Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite, and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored by using small interfering RNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy subjects or asthmatic patients were evaluated for DUOX1 expression and allergen-induced responses.Results: In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which depended critically on DUOX1-mediated activation of epithelial epidermal growth factor receptor and the protease calpain-2 through a redox-dependent mechanism involving cysteine oxidation within epidermal growth factor receptor and the tyrosine kinase Src. Primary nasal epithelial cells from patients with allergic asthma were found to express increased DUOX1 and IL-33 levels and demonstrated enhanced IL-33 secretion in response to allergen challenge compared with values seen in nasal epithelial cells from nonasthmatic subjects.Conclusion: Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens and indicate that enhanced DUOX1 expression and IL-33 secretion might present important contributing features of allergic asthma.