Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.

Background Compositional differences in bronchial bacterial microbiota have been associated with asthma, but it remains unclear whether the findings are attributable to asthma, to aeroallergen sensitization or to inhaled corticosteroid treatment. Objectives To compare the bronchial bacterial microbiota in adults with steroid-naive atopic asthma (AA), with atopy but no asthma (ANA), and non-atopic healthy subjects (HC), and determine relationships of bronchial microbiota to phenotypic features of asthma. Methods Bacterial communities in protected bronchial brushings from 42 AA, 21 ANA, and 21 HC subjects were profiled by 16S rRNA gene sequencing. Bacterial composition and community-level functions inferred from sequence profiles were analyzed for between-group differences. Associations with clinical and inflammatory variables were examined, including markers of type 2-related inflammation and change in airway hyperresponsiveness following six weeks of fluticasone treatment. Results The bronchial microbiome differed significantly among the three groups. Asthmatic subjects were uniquely enriched in members of the Haemophilus, Neisseria., Fusobacterium, Porphyromonas and Sphingomonodaceae, and depleted in members of the Mogibacteriaceae and Lactobacillales. Asthma-associated differences in predicted bacterial functions included involvement of amino acid and short-chain fatty acid metabolism pathways. Subjects with type 2-high asthma harbored significantly lower bronchial bacterial burden. Distinct changes in specific microbiota members were seen following fluticasone treatment. Steroid-responsiveness was linked to differences in baseline compositional and functional features of the bacterial microbiome. Conclusion Even in mild steroid-naive asthma subjects, differences in the bronchial microbiome are associated with immunologic and clinical features of the disease. The specific differences identified suggest possible microbiome targets for future approaches to asthma treatment or prevention.

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.