Alteration of NF-κB p50 DNA Binding Kinetics by S-Nitrosylation

doi:10.1006/bbrc.1997.7279

Biochemical and Biophysical Research Communications

Volume 238, Issue 3, 29 September 1997, Pages 703-706

https://doi.org/10.1006/bbrc.1997.7279 Get rights and content

Abstract

Nitric oxide (NO) regulates a wide variety of cellular functions, in part, by formation of S–NO bonds at critical active site thiol groups within proteins, including transcription factors. Previous studies have qualitatively demonstrated that S-nitrosothiol formation can alter transcription factor binding to the DNA recognition site. To more precisely define the effect of S-nitrosylation on transcription factor binding, the equilibrium binding constant was derived for S-nitrosylated NF-κB p50 (S-NO-p50) in a cell free system utilizing gel shift assays. Binding of NF-κB p50 subjected to the nitrosylation conditions in the absence of NaNO₂(C-p50-2) was not different from that of wild type NF-κB (C-p50-1). The extent of S-NO-p50 binding to its DNA target sequence was significantly decreased in comparison to that noted with C-p50-1 and C-p50-2. The binding constant was derived for each of the NF-κB variants: C-p50-1 = 1.01 × 10¹⁰M⁻¹; C-p50-2 = 0.92 × 10¹⁰M⁻¹; and S-NO-p50 = 0.28 × 10¹⁰M⁻¹. These data indicate that S-nitrosylation of p50 decreases its affinity for the target DNA sequence by four-fold.

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Tumor cells can develop resistance to cell death which is divided into necrosis and programmed cell death (PCD). PCD, including apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis. Ferroptosis and pyroptosis, two new forms of cell death, have gradually been of interest to researchers. Boosting ferroptosis and pyroptosis of tumor cells could be a potential cancer therapy. Nitric oxide (NO) is a ubiquitous, lipophilic, highly diffusible, free-radical signaling molecule that plays various roles in tumorigenesis. In addition, NO also has regulatory mechanisms through S-nitrosylation that do not depend on the classic NO/sGC/cGMP signaling. The current tumor treatment strategy for NO is to promote cell death through promoting S-nitrosylation-induced apoptosis while multiple drawbacks dampen this tumor therapy. However, numerous studies have suggested that suppression of NO is perceived to active ferroptosis and pyroptosis, which could be a better anti-tumor treatment. In this review, ferroptosis and pyroptosis are described in detail. We summarize that NO influences ferroptosis and pyroptosis and infer that S-nitrosylation mediates ferroptosis- and pyroptosis-related signaling pathways. It could be a potential cancer therapy different from NO-induced apoptosis of tumor cells. Finally, the information shows the drugs that manipulate endogenous production and exogenous delivery of NO to modulate the levels of S-nitrosylation.
The role of hypernitrosylation in the pathogenesis and pathophysiology of neuroprogressive diseases
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There is a wealth of data indicating that de novo protein S-nitrosylation in general and protein transnitrosylation in particular mediates the bulk of nitric oxide signalling. These processes enable redox sensing and facilitate homeostatic regulation of redox dependent protein signalling, function, stability and trafficking. Increased S-nitrosylation in an environment of increasing oxidative and nitrosative stress (O&NS) is initially a protective mechanism aimed at maintaining protein structure and function. When O&NS becomes severe, mechanisms governing denitrosylation and transnitrosylation break down leading to the pathological state referred to as hypernitrosylation (HN). Such a state has been implicated in the pathogenesis and pathophysiology of several neuropsychiatric and neurodegenerative diseases and we investigate its potential role in the development and maintenance of neuroprogressive disorders. In this paper, we propose a model whereby the hypernitrosylation of a range of functional proteins and enzymes lead to changes in activity which conspire to produce at least some of the core abnormalities contributing to the development and maintenance of pathology in these illnesses.
S-nitrosoglutathione-mediated STAT3 regulation in efficacy of radiotherapy and cisplatin therapy in head and neck squamous cell carcinoma
2015, Redox Biology
Citation Excerpt :
In turn, this low and sustained expression of iNOS could participate in promotion of cancer cell survival via O2•−/ONOO− induced NF-κB activation. GSNO is known to inhibit activation of NF-κB, a prerequisite for iNOS expression, via S-nitrosylation dependent mechanisms [10,11,48,70,71]. Accordingly, we observed that GSNO treatment attenuated sustained low-level activation of NF-κB (Fig. 4A) and iNOS expression (Fig. 4B-i).
S-nitrosoglutathione (GSNO) is an endogenous nitric oxide (NO) carrier that plays a critical role in redox based NO signaling. Recent studies have reported that GSNO regulates the activities of STAT3 and NF-κB via S-nitrosylation dependent mechanisms. Since STAT3 and NF-κB are key transcription factors involved in tumor progression, chemoresistance, and metastasis of head and neck cancer, we investigated the effect of GSNO in cell culture and mouse xenograft models of head and neck squamous cell carcinoma (HNSCC). For the cell culture studies, three HNSCC cell lines were tested (SCC1, SCC14a and SCC22a). All three cell lines had constitutively activated (phosphorylated) STAT3 (Tyr⁷⁰⁵). GSNO treatment of these cell lines reversibly decreased the STAT3 phosphorylation in a concentration dependent manner. GSNO treatment also decreased the basal and cytokine-stimulated activation of NF-κB in SCC14a cells and reduced the basal low degree of nitrotyrosine by inhibition of inducible NO synthase (iNOS) expression. The reduced STAT3/NF-κB activity by GSNO treatment was correlated with the decreased cell proliferation and increased apoptosis of HNSCC cells. In HNSCC mouse xenograft model, the tumor growth was reduced by systemic treatment with GSNO and was further reduced when the treatment was combined with radiation and cisplatin. Accordingly, GSNO treatment also resulted in decreased levels of phosphorylated STAT3. In summary, these studies demonstrate that GSNO treatment blocks the NF-κB and STAT3 pathways which are responsible for cell survival, proliferation and that GSNO mediated mechanisms complement cispaltin and radiation therapy, and thus could potentiate the therapeutic effect in HNSCC.
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2014, Pharmacological Reports
There is an interaction between many cell types involved in the pathophysiology of ischemic acute renal failure. Nitric oxide (NO) precursors, especially l-arginine, may have protective effects on tissue ischemia/reperfusion injury (IRI); however, their molecular mechanisms are unclear. In the present study, the interaction between l-arginine, cyclo-oxygenase (COX)-2 and reactive oxygen species (ROS) in the pathogenesis of ischemic acute renal failure was investigated.
Ischemia/reperfusion injury model in rats was used and various biochemical parameters examined. The rat isolated aortic rings served as model for hypoxia/reoxygenation where endothelium dependent and independent relaxations were exerted.
Pre-treatment of rats subjected to IRI with l-arginine (125 mg/kg) significantly reduced kidney MDA levels, elevated kidney SOD activity, GSH level and total NO levels at 24 and 48 h after reperfusion. Kidney COX-2 level was only different in the l-arginine-treated group 48 h after reperfusion compared to the IRI group. Pre-treatment with l-arginine (10⁻² M) alone or in combination with celecoxib significantly potentiated the acetylcholine (Ach)-induced relaxations in control and hypoxic rings. The effect of the combination was synergistic only in hypoxic rings. Addition of ascorbic acid to the celecoxib–arginine combination did not produce further potentiation. Sodium nitroprusside-induced relaxations in control and hypoxic rings were potentiated by l-arginine or celecoxib–arginine combination but not by ascorbic acid.
The protective effect of l-arginine may result from the interaction between NO and ROS and increased NO bioavailability. The protective effects of combined celecoxib and l-arginine against IRI could be attributed to their antioxidant activity which exceeded that of ascorbic acid.
Mechanisms and targets of the modulatory action of S-nitrosoglutathione (GSNO) on inflammatory cytokines expression
2014, Archives of Biochemistry and Biophysics
A number of experimental studies has documented that S-nitrosoglutathione (GSNO), the main endogenous low-molecular-weight S-nitrosothiol, can exert modulatory effects on inflammatory processes, thus supporting its potential employment in medicine for the treatment of important disease conditions. At molecular level, GSNO effects have been shown to modulate the activity of a series of transcription factors (notably NF-κB, AP-1, CREB and others) as well as other components of signal transduction chains (e.g. IKK-β, caspase 1, calpain and others), resulting in the modulation of several cytokines and chemokines expression (TNFα, IL-1β, IFN-γ, IL-4, IL-8, RANTES, MCP-1 and others). Results reported to date are however not univocal, and a single main mechanism of action for the observed anti-inflammatory effects of GSNO has not been identified. Conflicting observations can be explained by differences among the various cell types studies as to the relative abundance of enzymes in charge of GSNO metabolism (GSNO reductase, γ-glutamyltransferase, protein disulfide isomerase and others), as well as by variables associated with the individual experimental models employed. Altogether, anti-inflammatory properties of GSNO seem however to prevail, and exploration of the therapeutic potential of GSNO and analogues appears therefore warranted.
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The role of nitric oxide (NO) in cerebral ischemia/reperfusion (IR) has been intensively investigated. In general NO is regarded as a mediator of ischemia-associated neuronal damage, as inhibitors of NO synthesis ameliorate neuronal injury during permanent focal cerebral ischemia, however the exact role of NO in ischemia remains controversial. It has been previously shown that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins and strong evidence supports that activation of NF-κB contributes to ischemia-induced neuronal injury. In this study, we have investigated whether NO production by nNOS, eNOS and iNOS modulates IkB-alpha expression in cerebral ischemia, by using various inhibitors of NOS, in rats subjected to transient (1 h) middle cerebral artery occlusion (tMCAo). Male Wistar rats were treated intraperitoneally (i.p.) with 3 mg/kg of NG-nitro-l-arginine methyl ester (l-NAME, a non-selective NOS inhibitor), 5 mg/kg of l-N6-(1-iminoethyl)-lysine (l-NIL, an inducible NOS inhibitor), 25 mg/kg of 7-Nitroindazole (7-NI, a neuronal NOS inhibitor) and 10 mg/Kg of l-N-(1-iminoethyl)ornithine (l-NIO, a selective eNOS inhibitor) 15 min before the induction of tMCAo. Cortical IkB-alpha expression was evaluated by western blotting and its decrease was considered as an indicator of NF-κB activation. IkB-alpha expression decreased in ischemic cortices when compared with the cortices of the sham group, thus confirming the activation of NF-κB in ischemic conditions. Pre-treatment with l-NAME, l-NIL and 7-NI significantly reduced the infarct volume and prevented ischemia-induced reduction in IkB-alpha expression. Conversely, pretreatment with l-NIO was associated with a significant increase in infarct volume and a reduction in IkB-alpha expression. These findings suggest that NO of neuronal and inducible origin promotes NF-κB activation via IkB-alpha modulation and mediates ischemic-related damage in the brain following ischemia.

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^☆: F. M. AusubelR. BrentR. E. KingstonD. D. MooreJ. G. SeidmanJ. A. SmithK. Struhl, Eds.

¹: Address all correspondence to Paul C. Kuo, 29 S. Greene St., #200, Baltimore, MD 21201. Fax: (410) 329-3837. E-mail: pkuo@surg ery1.ab.umd.edu.

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Regular ArticleAlteration of NF-κB p50 DNA Binding Kinetics by S-Nitrosylation☆

Abstract

Journal of Molecular Biology

Journal of Biological Chemistry

Proc. Natl. Acad. Sci.

Nature

FEBS Letters

Regular Article
Alteration of NF-κB p50 DNA Binding Kinetics by S-Nitrosylation☆