S-nitrosylation Of P65 Research Articles

ENOS-dependent S-nitrosylation of the NF-\u03baB subunit p65 has neuroprotective effects

Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

C-reactive protein reduces protein S-nitrosylation in endothelial cells

C-reactive protein (CRP) emerges as an important mediator of cardiovascular lesions. In this study, we aimed to assess the role of CRP in the S-nitrosylation of proteins in endothelial cells and elucidate the potential mechanisms. Our results showed that CRP reduced protein S-nitrosylation in human umbilical vein endothelial cells (HUVECs). NO donor S-nitrosoglutathione antagonized CRP-mediated reduction of protein S-nitrosylation. Neutralizing antibody to Fcγ receptor II remarkably attenuated these changes. In addition, CRP increased NF-κB activation via the reduction of S-nitrosylation of p65, but not p50 in HUVECs, and induced the upregulation of NF-kB target gene vascular cell adhesion molecule-1. Furthermore, we confirmed that CRP reduced S-nitrosylated proteins in the rat aorta. Taken together, these data suggest that CRP-induced decline of protein S-nitrosylation by activating NF-κB via reduction of S-nitrosylation of p65, which may contribute to the endothelial dysfunction.

NOS2 Regulation of NF-κB by S-Nitrosylation of p65

Signal transduction in the NF-kappaB transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-kappaB p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-kappaB p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-kappaB-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-kappaB binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-kappaB activity and regulates gene expression in inflammation.

GSNO attenuates EAE disease by S‐nitrosylation‐mediated modulation of endothelial‐monocyte interactions

S-Nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier and recently, has been documented for its anti-inflammatory effects in rat model of cerebral ischemia (Khan et al. (2005) J Cereb Blood Flow Metab 25:177-192). Here, we explored the neuroprotective effects mediated by GSNO in Lewis rat model of EAE and its mechanism of action using in vitro model of monocyte-endothelial cell interaction. Oral administration of GSNO attenuated the clinical disease course in EAE animals by inhibiting the infiltration of vascular immune cells in the CNS that subsequently led to the reduction in the expression of proinflammatory cytokines and consequently limited demyelination. Based on the inhibition in infiltration of immune cells, we hypothesized that GSNO modulated endothelial cell activation that led to reduce cellular infiltration in the CNS. Using an in vitro model, we established that GSNO inhibited monocyte adhesion to the activated endothelial cell, which was mediated by down regulation of endothelial cell adhesion molecules (CAMs). The mechanism by which GSNO modulated CAMs expression appeared to be via S-nitrosylation of p65, which consequently inhibited nuclear factor kappa B (NF-kappaB) activation in endothelial cells. These observations suggest that GSNO exerts its protective effects in EAE by inhibition of cellular infiltration into the CNS by S-nitrosylation of p65, thereby modulating NF-kappaB-CAMs pathway in endothelial cells.