No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Tyrosine Nitration of IκBα: A Novel Mechanism for NF-κB Activation † , ‡
Abstract
The NF-kappaB family of transcription factors is an important component of stress-activated cytoprotective signal transduction pathways. Previous studies demonstrated that some activation mechanisms require phosphorylation, ubiquitination, and degradation of the inhibitor protein, IkappaBalpha. Herein, it is demonstrated that ionizing radiation in the therapeutic dose range stimulates NF-kappaB activity by a mechanism in which IkappaBalpha tyrosine 181 is nitrated as a consequence of constitutive NO* synthase activation, leading to dissociation of intact IkappaBalpha from NF-kappaB. This mechanism does not appear to require IkappaBalpha kinase-dependent phosphorylation or proteolytic degradation of IkappaBalpha. Tyrosine 181 is involved in several noncovalent interactions with the p50 subunit of NF-kappaB stabilizing the IkappaBalpha-NF-kappaB complex. Evaluation of hydropathic interactions of the IkappaBalpha-p50 complex on the basis of the crystal structure of the complex is consistent with nitration disrupting these interactions and dissociating the IkappaBalpha-NF-kappaB complex. Tyrosine nitration is not commonly studied in the context of signal transduction. However, these results indicate that tyrosine nitration is an important post-translational regulatory modification for NF-kappaB activation and possibly for other signaling molecules modulated by mild and transient oxidative and nitrosative stresses.
... Although previous reports show that IκB is one of the candidates to be nitrated on its tyrosine [33,35,36], further investigations using immunoprecipitation and/or immunohistochemistry analyses in calvaria would enable us to identify the protein to which the tyrosine is nitrated by ONOO − . Taken together, these findings raise the possibility that once calvaria is stimulated with LPS, neutrophils generate a great amount of O 2 − , which can produce cytotoxic ONOO − , by reacting with the NO produced by osteoblasts and/or activated macrophages. ...
... The hypothesis is based on the experimental findings in the present study and published reports. We hypothesized that persistent inflammation leading to tissue damage might be caused by the signaling pathway as follows: (a) the LPS-stimulated production of O2 − by neutrophils; (b) peroxynitrite, which was produced from superoxide anions and NO, freely passes through the cell membrane; and (c) nitration of the tyrosine residue of IκB, which was reported to be a candidate for nitration target [33,35,36]; (d) translocation of activated NF-κB into the nucleus and thereby, (e) upregulation of COX-2 and iNOS mRNA expression, which, in turn, increases the production of (f) PGE2 and (g) NO. This pathway from (a) to (g) is a vicious cycle that leads to persistent inflammation. ...
... The hypothesis is based on the experimental findings in the present study and published reports. We hypothesized that persistent inflammation leading to tissue damage might be caused by the signaling pathway as follows: (a) the LPS-stimulated production of O 2 − by neutrophils; (b) peroxynitrite, which was produced from superoxide anions and NO, freely passes through the cell membrane; and (c) nitration of the tyrosine residue of IκB, which was reported to be a candidate for nitration target [33,35,36]; (d) translocation of activated NF-κB into the nucleus and thereby, (e) upregulation of COX-2 and iNOS mRNA expression, which, in turn, increases the production of (f) PGE 2 and (g) NO. This pathway from (a) to (g) is a vicious cycle that leads to persistent inflammation. ...
Bisphosphonates (BPs) are classified into two groups, according to their side chain structures, as nitrogen-containing BPs (NBPs) and non-nitrogen-containing BPs (non-NBPs). In this study, we examined the effects of NBPs and non-NBPs on inflammatory responses, by quantifying the inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), in cultured neonatal mouse calvaria. All examined NBPs (pamidronate, alendronate, incadronate, risedronate, zoledronate) stimulated lipopolysaccharide (LPS)-induced PGE2 and NO production by upregulating COX-2 and iNOS mRNA expression, whereas non-NBPs (etidronate, clodronate, tiludronate) suppressed PGE2 and NO production, by downregulating gene expression. Additionally, [4-(methylthio) phenylthio] methane bisphosphonate (MPMBP), a novel non-NBP with an antioxidant methylthio phenylthio group in its side chain, exhibited the most potent anti-inflammatory activity among non-NBPs. Furthermore, results of immunohistochemistry showed that the nuclear translocation of NF-κB/p65 and tyrosine nitration of cytoplasmic protein were stimulated by zoledronate, while MPMBP inhibited these phenomena, by acting as a superoxide anion (O2−) scavenger. These findings indicate that MPMBP can act as an efficacious agent that causes fewer adverse effects in patients with inflammatory bone diseases, including periodontitis and rheumatoid arthritis.
... Studies have shown that iNOS is expressed by T cells, macrophages, and mature dendritic cells (mDCs), and regulates the differentiation and function of immune cells via nitration of key molecules involved in transcriptional or signaling pathways [6][7][8]. This article reviews and discusses recent experimental research. ...
... By inactivating and destroying infectious agents, NO is an important pro-inflammatory cytotoxic agent that protects the host against various pathogens [6][7][8][9]. Interestingly, NO also plays a key inhibitory role in immunity [10][11][12]. ...
... However, under these conditions, we did not observe significant differences in AHR protein expression between CD4 + T cells from WT and iNOS −/− mice, suggesting that AHR protein expression cannot explain the effect of iNOS produced by T cells on Th17 cell differentiation. A previous study showed that a tyrosine in IKBα is nitrated following activation of NOS, resulting in dissociation of IKBα from NF-κβ [6]. Other studies have stated that nitration of specific tyrosines in proteins may be structurally and functionally important [23], and we have reported a novel mechanism for modulating Th17 cell development through nitration of RORγt tyrosine residues. ...
In recent years, there have been many studies on the function of nitric oxide synthase (NOS) in experimental animals and humans. This review analyzes and explores the relationship between inducible nitric oxide synthase (iNOS) and T cells, macrophages, and dendritic cell et al. differentiation using data based on laboratory research, highlighting recent NOS laboratory research. Our insights into research prospects and directions are also presented.
... Mild oxidative stress such as radiation doses <10 Gy activates NF-kB in diverse cell types including breast cancer cells by a mechanism requiring constitutive eNOS or neuronal NOS (nNOS) and nitration of Tyr181 of the inhibitor protein IkBα. Tyr181 nitration results in dissociation of IkBα from the active transcription factor complex, p50/p65 [ 51 ]. Low basal levels of Tyr-nitrated IkBα are also observed, probably as a consequence of the elevated levels of ROS/RNS generated by tumor cell metabolism and the infl ammatory microenvironment. ...
... When coupled, the primary product of all NOS isoforms is •NO and downstream signaling is dominated by •NO-dependent pathways (eg, sGC/PKG). Uncoupled NOS, on the other hand, produces potent oxidants, e.g., ONOO − and O 2 •− initiating different downstream signaling that is pro-proliferative and antiapoptotic, e.g., NF-kB [ 51 ]. Recoupling NOS by increasing BH4:BH2 with an orally provided BH4 metabolic precursor inhibits the growth of both a spontaneous mammary carcinoma and a colorectal tumor. ...
... In addition, many of the changes that occur during pneumonitis, such as increased TGFβ expression do not return to basal levels but remain elevated until the occurrence of the late phase which is often characterized by the appearance of fi brosis [ 100 , 116 , 117 , 119 ]. Our proteomic study of the rat lung after a 28 Gy hemithoracic radiation exposure [ 120 ] demonstrated changes in protein expression that correlated with these functional and structural events including transient increases (days 1-3) in expression of an anti-infl ammatory and anti-fi brotic enzyme HO-1, and an increase in IkBα Tyr nitration (an indicator of RNS-dependent NF-kB activation [ 51 ], followed at 6-8 weeks with irreversible decreases in a number of structural proteins (e.g. talin and fi lamin), anti-oxidant proteins (biliverdin reductase and peroxidoxin 1). ...
High levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are hallmarks of solid tumors, promoting genomic instability as well as uncontrolled proliferation. In inflammatory diseases such as diabetes, hypertension, atherosclerosis and cancer, loss of nitric oxide (NO) production is a common feature. Recent experiments demonstrated that under these conditions the relative levels of the nitric oxide synthase (NOS) cofactor, tetrahydrobiopterin (BH4), are relatively low resulting in an “uncoupled NOS” and reduced NO bioavailability and increased ROS/RNS. Similar evidence suggest that NOS uncoupling is also a critical “switching mechanism” essential for tumor progression. Furthermore, uncoupling can be exploited therapeutically as both in vitro and in vivo treatment with the BH4 precursor, Sepiapterin (SP), restores NOS coupling and shifts the balance of signaling from ROS/RNS and pro-proliferative to NO dependent and anti-proliferative pathways.
... Hence, this continuous and long-term activation of NF-κB may exacerbate the inflammatory responses mediated by this transcription factor. This was shown for various cell types including muscle myoblasts and epithelial cells [302,321]. ...
... By site-directed mutagenesis, tyrosine 181 was identified as the main nitrosylated residue in IκBα. IκBα tyrosine 181 nitration leads to dissociation of IκBα from NF-κB [321]. This mechanism does not require IκBα kinase (IKK) dependent phosphorylation or proteolytic degradation. ...
... This mechanism does not require IκBα kinase (IKK) dependent phosphorylation or proteolytic degradation. The importance of tyrosine 181 is its ability to stabilise the IκBα-NF-κB complex through non-covalent interactions with the p50 subunit as shown by the crystal structure [321]. Thus, tyrosine nitration becomes an important post-translational modification involved, in this case, in an aberrant NF-κB activation. ...
... PTEN also has an active site Cys sensitive to oxidation, and its oxidation by RNS inhibits PTEN-activating antiapoptotic Akt signaling (9). RNS activate cytoprotective NF-kB transcriptional activity by nitrating Tyr 181 of IkBa, causing its dissociation from NF-kB (10). Simulating a chronic inflammatory environment by treating MCF10A cells with low levels of RNS donors or coculturing with activated macrophages stimulates Tyr nitration and activity of PP2A, leading to the downregulation of BRCA1 expression and reduced homologous recombination DNA repair. ...
... Three hours later, the medium was changed with fresh sepiapterin, and luciferase activity was measured 24 hours later. A luciferase reporter plasmid (Clontech) was used to measure NF-kB promoter activity (10). ...
... This is clearly seen in the experiments demonstrating that increasing BH4:BH2 results in decreased tyrosine nitration of IkBa but increased Snitrosylation of the p65 subunit of NF-kB partially inhibiting the latter's transcriptional activity (Fig. 3). Other regulatory proteins potentially involved in tumor growth are also posttranslationally modified by either Tyr nitration, e.g., PP2A, p53 (10,11,35), or Cys S-nitrosylation or oxidation, e.g., protein Tyr phosphatases, ras (6,7). ...
Here evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast to normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on HPLC analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin: dihydrobiopterin ratio (BH4:BH2)found in breast, colorectal, epidermoid and head and neck tumors compared to normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling including increased cGMP-dependent protein kinase (PKG) activity, decreased beta-catenin expression and TCF4 promoter activity, and reduced NF-kappaB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by clonogenic assay, Ki67 staining and 18F-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and as a consequence nitric oxide synthase activity generates more peroxynitrite and superoxide anion than nitric oxide resulting in important tumor growth promoting and anti-apoptotic signaling properties. Implications: The synthetic BH4, Kuvan®, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.
Copyright © 2015, American Association for Cancer Research.
... Peroxynitrite can also alter the structure of DNA, resulting in over-activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP-1) that consumes nicotinamide adenine dinucleotide (NAD +) and adenosine triphosphate (ATP) for (ADP-ribose) (PAR) formation, leading to apoptosis and necrosis [6]. Peroxynitrite can enhance nuclear factor kappa B (NF-KB) mediated proinflammatory pathways, therefore increasing inflammatory cytokines production [7]. Previous studies have shown that peroxynitrite is implicated in the pathogenesis of AKI, and the use of peroxynitrite scavengers may protect against renal I/R injury [8,9]. ...
... Peroxynitrite-induced nitrosative stress causes lipid peroxidation, DNA destruction, and protein nitration [35][36][37]. This damage eventually results in cell apoptosis and tissue inflammation [4,7]. In the current study, we reported an increase in TNFα expression and caspase 3 activation in rats with renal I/R injury, indicating the presence of inflammation and apoptosis along with impaired kidney function. ...
Background
Acute kidney injury (AKI), secondary to renal ischemia/reperfusion (I/R), is a serious problem associated with high mortality. The pathophysiology of AKI after renal I/R involves peroxynitrite production; hence, scavenging this metabolite may rescue AKI. Entacapone is a catechol-O-methyl transferase (COMT) inhibitor which elicits antioxidant activity by scavenging peroxynitrite. Therefore, we hypothesized that the peroxynitrite scavenging activity of entacopone protects against AKI after renal I/R injury in rats.
Methods
Male Wistar rats were given either entacapone or a well-known peroxynitrite scavenger (FeTPPS) daily for 10 days before I/R procedures. I/R was induced by occluding both renal pedicles for 45 min followed by reperfusion for 24 h.
Results
Pre-treatment with either entacapone or FeTPPS improved renal function as indicated by a significant reduction in serum creatinine and urea when compared to I/R group (P < 0.05). I/R injury increased renal levels of NO (4-folds, P < 0.05), iNOS (4-folds, P < 0.05), and 3-nitrotyrosine (5-folds, P < 0.05) compared to sham control. These effects were abrogated in animals pre-treated with entacapone or FeTPPS before being subjected to I/R (P < 0.05). In addition, entacapone or FeTPPS significantly inhibited I/R-induced elevation in renal TNF-α levels (78% and 58%, respectively) and caspase-3 activity (72% and 56%, respectively) indicating the reduction of both inflammation and apoptosis in the kidney (P < 0.05). The two drugs also improved kidney and liver functions in rats with renal I/R injury.
Conclusion
Our study showed that entacapone and FeTPPS protected against AKI and remote liver damage associated with renal I/R and this effect might be due to scavenging peroxynitrite and reducing nitrosative stress.
... Notably, HDAC3 is a critical regulator of clock function [50]. NO in turn exerts feed-forward amplification of NF-κB signaling [51]. As multiple clock genes contain NF-κB binding motifs in their promoters, cytokine regulated β-cell clock gene expression could be NF-κB signaling dependent as in other cells, raising the possibility that KDACs and nitroxidative stress mediate cytokine-induced β-cell clock gene expression. ...
... This observation further contributes to the possibility that the observed effect on clock genes during inflammatory stress is NF-κB dependent, as iNOS is a prototypic NF-κB driven gene producing NO harmful for the β-cell [49]. NO exerts a feed-forward effect on NF-κB activity [51,64], and thus by inhibiting NO synthesis, NF-κB signaling is blunted. Alternatively, iNOS inhibition de-represses the transcriptional activity of REV-ERBα as has been shown in other cells [65], which might explain the reduction in cytokine-induced E-box controlled gene expression observed in INS-1 cells. ...
Pancreatic β-cell-specific clock knockout mice develop β-cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine interleukin-1β (IL-1β) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1β and interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/β agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast, low (<5,0 μM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1β mediated β-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3, and immunoproteasome activity.
... Notably, HDAC3 is a critical regulator of clock function [47]. NO in turn exerts feed-forward amplification of NF-κB signaling [48]. As multiple clock genes contain NF-κB binding motifs in their promoters, cytokine regulated β-cell clock gene expression could be NF-κB signaling dependent as in other cells, raising the possibility that KDACs and nitroxidative stress mediate cytokine-induced β-cell clock gene expression. ...
... This observation further contributes to the possibility that the observed effect on clock genes during inflammatory stress is NF-κB dependent, as iNOS is a prototypic NF-κB driven gene producing NO harmful for the β-cell [46]. NO exerts a feed-forward effect on NF-κB activity [48,61], and thus by inhibiting NO synthesis, NF-κB signaling is blunted. Alternatively, iNOS inhibition de-represses the transcriptional activity of REV-ERBα as has been shown in other cells [62], which might explain the reduction in cytokine-induced E-box controlled gene expression observed in INS-1 cells. ...
Pancreatic β-cell-specific clock knock-out mice develop β cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine Interleukin-1β (IL-1β) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1β and Interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of Reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/β agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced Brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell-viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast low (<5,0 μM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1β mediated β-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3 and immunoproteasome activity.
... Redox PTMs can also regulate the activity of several transcription factors (Brigelius-Flohé and Flohé, 2011). For instance, oxidation, S-nitrosation or nitration of p53, activating protein 1, myocyte enhancer factor 2 or nuclear factor kappalight-chain-enhancer of activated B cells (NF-kB) modulate the DNA binding and activity of these important transcription factors (Abate et al., 1990;Schreck et al., 1991;Rainwater et al., 1995;Klatt et al., 1999;Kabe et al., 2005;Yakovlev et al., 2007;Ljubuncic et al., 2008;Okamoto et al., 2014;Caviedes et al., 2020). Redox PTMs can also affect the activity of transcription factors through indirect mechanisms. ...
... As a possible underlying molecular mechanism, it has been suggested that redox PTM of NF-kB, a pivotal transcription factor regulating inflammation, modulates NF-kB activity and subsequently the inflammatory response during aging (Ljubuncic et al., 2010;Zhang et al., 2013). Indeed, under sustained O/N stresses (as seen in aging), tyrosine residues of IκBα (one of NF-kB subunits) are nitrated, which leads to a prolonged and excessive NF-kB activation and signaling (Adler et al., 2007;Yakovlev et al., 2007). More integrated studies of the complex process of aging would be required to understand the interconnection and crosstalk between redox PTMs of protein and the various factors contributing to brain aging. ...
Reactive oxygen species and reactive nitrogen species (RONS) are by-products of aerobic metabolism. RONS trigger a signaling cascade that can be transduced through oxidation-reduction (redox)-based post-translational modifications (redox PTMs) of protein thiols. This redox signaling is essential for normal cellular physiology and coordinately regulates the function of redox-sensitive proteins. It plays a particularly important role in the brain, which is a major producer of RONS. Aberrant redox PTMs of protein thiols can impair protein function and are associated with several diseases. This mini review article aims to evaluate the role of redox PTMs of protein thiols, in particular S-nitrosation, in brain aging, and in neurodegenerative diseases. It also discusses the potential of using redox-based therapeutic approaches for neurodegenerative conditions.
... However, it was observed that, following exposure of certain cell types to low doses of ionizing radiation, the inhibitory protein IκBα results nitrated at tyrosine 108, leading this modification to the dissociation of the complex and the release of active NF-κB without the need of phosphorylation of the serine residue of IκBα. 739 Computational studies showed that nitration of this residue disrupts certain noncovalent interactions of the native tyrosine with the p50 subunit of NF-κB, promoting this way the release of the active transcription factor. 739 Similarly, it was shown that tyrosine nitration could activate the transcription factor p53 by promoting its oligomerization and nuclear translocation. ...
... 739 Computational studies showed that nitration of this residue disrupts certain noncovalent interactions of the native tyrosine with the p50 subunit of NF-κB, promoting this way the release of the active transcription factor. 739 Similarly, it was shown that tyrosine nitration could activate the transcription factor p53 by promoting its oligomerization and nuclear translocation. ...
Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•–) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.
... In Human Lung Microvascular Endothelial Cells (HLMVC) exposed to lipopolysaccharide (LPS) Activation and endothelial barrier destabilization (78) IB Y181 CHO cells exposed to ionizing radiation NFB activation (79) p53 Y327 ...
... 316,317 NO is a well-known substance which can increase tissue oxygenation by dilating the tumor blood vessels by several routes. [318][319][320] In contrast, the use of nitric oxide synthase inhibitors, such as NG-nitro-L-arginine (L-NNA), can inhibit anti-apoptotic pathways 321 and decrease tumor blood flow leading to chronic hypoxia, cancer cell death, as well as protection of normal cells under radiation. 322,323 Additionally, NO is an important player in the bystander effect, owing to its rapid diffusion across cell membranes. ...
Radiosensitizers are compounds or nanostructures, which can improve the efficiency of ionizing radiation to kill cells. Radiosensitization increases the susceptibility of cancer cells to radiation‐induced killing, while simultaneously reducing the potentially damaging effect on the cellular structure and function of the surrounding healthy tissues. Therefore, radiosensitizers are therapeutic agents used to boost the effectiveness of radiation treatment. The complexity and heterogeneity of cancer, and the multifactorial nature of its pathophysiology has led to many approaches to treatment. The effectiveness of each approach has been proven to some extent, but no definitive treatment to eradicate cancer has been discovered. The current review discusses a broad range of nano‐radiosensitizers, summarizing possible combinations of radiosensitizing NPs with several other types of cancer therapy options, focusing on the benefits and drawbacks, challenges, and future prospects.
... Signaling is stopped by the action of phosphatase A2 (PP2A) that dephosphorylates IκBa. It was shown that after cell exposure to ionizing radiation IκBa becomes nitrated at tyrosine 108, leading to the dissociation of the complex and to NF-κB activation (174). More recently, another mechanism by which protein nitration activates NF-κB was described, namely nitration of a selective tyrosine (Y289) in the B56d, the regulatory subunit of protein PP2A, that renders the enzyme inactive. ...
The free radical nitric oxide (·NO) is a key mediator in different physiological processes such as vasodilation, neurotransmission, inflammation, and cellular immune responses, and thus preserving its bioavailability is essential. In several disease conditions, superoxide radical (O2·-) production increases and leads to the rapid "inactivation" of ·NO by a diffusion-controlled radical termination reaction that yields a potent and short-lived oxidant, peroxynitrite. This reaction not only limits ·NO bioavailability for physiological signal transduction but also can divert and switch the biochemistry of ·NO toward nitrooxidative processes. Indeed, since the early 1990s peroxynitrite (and its secondary derived species) has been linked to the establishment and progression of different acute and chronic human diseases and also to the normal aging process. Here, we revisit an earlier and classical review on the role of peroxynitrite in human physiology and pathology (Pacher P, Beckman J, Liaudet L. Physiol Rev 87: 315-424, 2007) and further integrate, update, and interpret the accumulated evidence over 30 years of research. Innovative tools and approaches for the detection, quantitation, and sub- or extracellular mapping of peroxynitrite and its secondary products (e.g., protein 3-nitrotyrosine) have allowed us to unambiguously connect the complex biochemistry of peroxynitrite with numerous biological outcomes at the physiological and pathological levels. Furthermore, our current knowledge of the ·NO/O2·- and peroxynitrite interplay at the cell, tissue, and organ levels is assisting in the discovery of therapeutic interventions for a variety of human diseases.
... NF-kB is a family of five master transcription factors, influencing the expression of various genes, which is deregulated in many cancers with various effects depending on the cellular context. Radiation stimulates the pathway by enhancing the DNA binding affinity of NF-kB, its expression, the dissociation of the IKB complex and consequently its activation (182,183). Thalidomide is a for multiple myeloma U.S. Food and Drug Administration (FDA) approved NF-kB modulator that interferes with the NF-kB activation and is currently investigated to reduce urinary complications, a normal tissue complication, upon irradiation of the pelvic region (184). As radiation activates NF-kB-and PI3K/Aktsignaling and thereby affects the radiation response modulation of those signaling pathways, thalidomide can contribute to enhance the therapeutic window (185,186). ...
To meet the anabolic demands of the proliferative potential of tumor cells, malignant cells tend to rewire their metabolic pathways. Although different types of malignant cells share this phenomenon, there is a large intracellular variability how these metabolic patterns are altered. Fortunately, differences in metabolic patterns between normal tissue and malignant cells can be exploited to increase the therapeutic ratio. Modulation of cellular metabolism to improve treatment outcome is an emerging field proposing a variety of promising strategies in primary tumor and metastatic lesion treatment. These strategies, capable of either sensitizing or protecting tissues, target either tumor or normal tissue and are often focused on modulating of tissue oxygenation, hypoxia-inducible factor (HIF) stabilization, glucose metabolism, mitochondrial function and the redox balance. Several compounds or therapies are still in under (pre-)clinical development, while others are already used in clinical practice. Here, we describe different strategies from bench to bedside to optimize the therapeutic ratio through modulation of the cellular metabolism. This review gives an overview of the current state on development and the mechanism of action of modulators affecting cellular metabolism with the aim to improve the radiotherapy response on tumors or to protect the normal tissue and therefore contribute to an improved therapeutic ratio.
... NO also inhibits NF-κB activation in rat vSMCs via a cGMP-independent inhibition of IκB phosphorylation. NF-κB activity is also stimulated through the nitration of IκB.strα following activation of NO synthase [135]. On the other hand, NO can also have anti-apoptotic effects, through modulation of several proteins of the Bcl-2 family via sGC activation or inhibition by S-nitrosation of a large number of apoptotic proteins (e.g., caspases) [136]. ...
Monocytes/macrophages and vascular smooth muscle cells (vSMCs) are the main cell types implicated in atherosclerosis development, and unlike other mature cell types, both retain a remarkable plasticity. In mature vessels, differentiated vSMCs control the vascular tone and the blood pressure. In response to vascular injury and modifications of the local environment (inflammation, oxidative stress), vSMCs switch from a contractile to a secretory phenotype and also display macrophagic markers expression and a macrophagic behaviour. Endothelial dysfunction promotes adhesion to the endothelium of monocytes, which infiltrate the sub-endothelium and differentiate into macrophages. The latter become polarised into M1 (pro-inflammatory), M2 (anti-inflammatory) or Mox macrophages (oxidative stress phenotype). Both monocyte-derived macrophages and macrophage-like vSMCs are able to internalise and accumulate oxLDL, leading to formation of “foam cells” within atherosclerotic plaques. Variations in the levels of nitric oxide (NO) can affect several of the molecular pathways implicated in the described phenomena. Elucidation of the underlying mechanisms could help to identify novel specific therapeutic targets, but to date much remains to be explored. The present article is an overview of the different factors and signalling pathways implicated in plaque formation and of the effects of NO on the molecular steps of the phenotypic switch of macrophages and vSMCs.
... It has been further shown, that peroxynitrite also affects kinases and phosphatases, that regulate endothelial barrier function, such as Akt, PP2A, PTP1B, and PTEN (Delgado-Esteban, Martin-Zanca, Andres-Martin, Almeida, & Bolanos, 2007;Wu & Wilson, 2009). A further important impact on signaling is nitration of the NF-KB inhibitor IKBα, leading to disruption of the inhibitor binding and an increase in the activity of this transcription factor (Yakovlev et al., 2007). This is further interesting in the light of mRNA upregulation of the NF-KB subunit p50 under long-term exposure to hypoxic/hyperoxic O 2 oscillations. ...
The pulmonary endothelium is an immediate recipient of high oxygen concentrations upon oxygen therapy and mediates downstream responses. Cyclic collapse and reopening of atelectatic lung areas during mechanical ventilation with high fractions of inspired oxygen result in the propagation of oxygen oscillations in the hypoxic/hy-peroxic range. We used primary murine lung endothelial cell cultures to investigate cell responses to constant and oscillating oxygen conditions in the hypoxic to hyper-oxic range. Severe constant hyperoxia had pro-inflammatory and cytotoxic effects including an increase in expression of ICAM1, E-selectin, and RAGE at 24 hr exposure. The coagulative/fibrinolytic system responded by upregulation of uPA, tPA, and vWF and PAI1 under constant severe hyperoxia. Among antioxidant enzymes, the upregulation of SOD2, TXN1, TXNRD3, GPX1, and Gstp1 at 24 hr, but down-regulation of SOD3 at 72 hr constant hyperoxia was evident. Hypoxic/hyperoxic oscillating oxygen conditions induced pro-inflammatory cytokine release to a lesser extent and later than constant hyperoxia. Gene expression analyses showed upregula-tion of NFKB p65 mRNA at 72 hr. More evident was a biphasic response of NOS3 and ACE1 gene expression (downregulation until 24 hr and upregulation at 72 hr). ACE2 mRNA was upregulated until 72 hr, but shedding of the mature protein from the cell surface favored ACE1. Oscillations resulted in severe production of perox-ynitrite, but apart from upregulation of Gstp1 at 24 hr responses of antioxidative proteins were less pronounced than under constant hyperoxia. Oscillating oxygen in the hypoxic/hyperoxic range has a characteristical impact on vasoactive mediators like NOS3 and on the activation of the renin-angiotensin system in the lung endothelium. K E Y W O R D S hyperoxia, intermittent hyperoxia, lung microvascular endothelial cells, oxygen oscillations
... In addition, NO can S-nitrosylate both the p50 and p65 NF-κB subunits at cysteine residues 62 and 38, respectively, reducing their DNA binding and inhibiting target gene transcription [78,79]. Moreover, the tyrosine nitration of IκBα at tyrosine 181 by endogenous NO promotes NF-κB signaling through the dissociation of IκBα from NF-κB [80]. The endogenous production of NO or NO donors such S-nitro-N-acetyl-penicillamine (SNAP) can suppress JNK activation via S-nitrosylation at cysteine 116 [81]. ...
Cellular senescence is a cell state involved in both physiological and pathological processes such as age-related diseases and cancer. While the mechanism of senescence is now well known, its role in tumorigenesis still remains very controversial. The positive and negative effects of senescence on tumorigenesis depend largely on the diversity of the senescent phenotypes and, more precisely, on the senescence-associated secretory phenotype (SASP). In this review, we discuss the modulatory effect of nitric oxide (NO) in SASP and the possible benefits of the use of NO donors or iNOS inducers in combination with senotherapy in cancer treatment.
... An interesting case that could represent a mechanism in which tyrosine nitration could be acting in signaling pathways is the activation of the nuclear factor κB (NF-κB) through the nitration of Tyr108 of its inhibitor protein IκBα. In this case, nitration of Tyr108 would disrupt non-covalent interactions between NF-κB and IκBα which in turn causes the dissociation of the complex and the translocation of the active transcription factor to the nucleus [181]. Many other examples have been reported in the literature regarding the involvement of tyrosine nitration on signaling transduction events, either associated with physiological responses or with programmed cell death [133,[182][183][184][185][186]. ...
Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical–radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO−), the product of the reaction of superoxide radical (O2•−) with •NO, but ONOO−-independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.
... For example, hydrogen peroxide induces p65 phosphorylation and nuclear translocation independent of IκB degradation, activating IκK by inducing tyrosine rather than serine phosphorylation, which does not induce IκB degradation (Sethi et al., 2007;Takada et al., 2003). Further, the IκB protein can undergo both tyrosine nitration and polyubiquitination, leading to destabilization of newly synthesized IκB (Enesa et al., 2008) or the dissociation of intact IκBα from p65 (Yakovlev et al., 2007). ...
Induction of innate immune genes in the brain is thought to be a major factor in the development of addiction to substances of abuse. As the major component of the innate immune system in the brain, aberrant activation of myeloid cells such as macrophages and microglia due to substance use may mediate neuroinflammation and contribute to the development of addiction. All addictive drugs modulate the dopaminergic system and our previous studies have identified dopamine as a pro-inflammatory modulator of macrophage function. However, the mechanism that mediates this effect is currently unknown. Inflammatory activation of macrophages and induction of cytokine production is often mediated by the transcription factor NF-κB, and prior studies have shown that dopamine can modulate NF-κB activity in T-cells and other non-immune cell lines. Here we demonstrated that dopamine can activate NF-κB in primary human macrophages, resulting in the induction of its downstream targets including the NLRP3 inflammasome and the inflammatory cytokine IL-1β. These data also indicate that dopamine primes but does not activate the NLRP3 inflammasome in human macrophages. Activation of NF-κB was required for dopamine-mediated increases in IL-1β, as an inhibitor of NF-κB was able to abrogate the effects of dopamine on production of these cytokines. Connecting an increase in extracellular dopamine to NF-κB activation and inflammation suggests specific intracellular targets that could be used to ameliorate the inflammatory impact of dopamine in neuroinflammatory conditions associated with myeloid cell activation such as addiction.
... Nitric oxide (NO) is an important pro-inflammatory cytotoxic agent that protects the host against various pathogens by inactivating and destroying infectious agents [6][7][8]. Under physiological conditions, the gaseous molecule is an important signaling molecule involved in neurotransmission and in the control of vascular tone [9]. ...
One of the best consolidated paradigms in vascular pharmacology is that an uncontrolled excess of oxidizing chemical species causes tissue damage and loss of function in the endothelial and subendothelial layers. The fact that high-density lipoproteins play an important role in preventing such an imbalance is integrated into that concept, for which the expression and activity of paraoxonases is certainly crucial. The term paraoxonase (aryldialkyl phosphatase, EC 3.1.8.1) encompasses at least three distinct isoforms, with a wide variation in substrate affinity, cell and fluid localization, and biased expression of polymorphism. The purpose of this review is to determine the interactions that paraoxonase 1 has with nitric oxide synthase, its reaction product, nitric oxide (nitrogen monoxide, NO), and its derived reactive species generated in an oxidative medium, with a special focus on its pathological implications.
... 9 iNOS and Arg-1 are expressed by a variety of cells, and the effector activity of immune cells may be regulated by iNOS. [10][11][12] Previous studies have demonstrated that iNOS expression can negatively regulate the differentiation of effector dendritic cells (DCs), 13 and iNOS also acts as a mediator in the suppressive function of myeloid-derived suppressor cells. 14 These findings evidence a regulatory function of iNOS expression on immune cells modulating the immune response against pathogens. ...
Inhalation of Cryptococcus gattii yeasts (causing cryptococcosis) triggers an anti-cryptococcal immune response initiated by macrophages, neutrophils or dendritic cells, and the iNOS expressed by various cells may regulate the function and differentiation of innate and adaptive immune cells. Here, we evaluated the effect of progression of C. gattii infection on the host innate immune response. C. gattii infection in BALB/c mice spreads to several organs by 21 d post infection. The numbers of neutrophils and lymphocytes in the peripheral blood of C. gattii-infected mice were remarkably altered on that day. The frequency of CD11b þ cells and cell concentrations of CD4 þ and CD8 þ T cells was significantly altered in the pulmonary tissue of infected mice. We found a higher frequency of CD11b þ /iNOS þ cells in the lungs of infected mice, accompanied by an increase in frequency of CD11b þ /Arginase-1 þ cells over time. Moreover, the iNOS/Arginase-1 expression ratio in CD11b þ cells reached its lowest value at 21 d post infection. In addition, the cytokine micro-environment in infected lungs did not show a pro-inflammatory profile. Surprisingly, iNOS knockout prolonged the survival of infected mice, while their pulmonary fungal burden was higher than that of infected WT mice. Thus, C. gattii infection alters the immune response in the pulmonary tissue, and iNOS expression may play a key role in infection progression.
... iNOS inhibition reversed cytokine-mediated ER stress as expected, but also upregulation of RNA decay effector mRNAs and downregulation of Ins1/2 mRNA. NO-mediated nitration activates the NF-kB (34,35), p53 (36), AP-1 (37), and other signaling pathways (35). Alternatively, nucleotide nitration leading to 8-nitroguanosine modifications of cellular RNAs may cause ribosomal stalling (7,38) and mediate RDPC expressional upregulation as a protective response. ...
Stress-related changes in β-cell mRNA levels result from a balance between gene transcription and mRNA decay. The regulation of RNA decay pathways has not been investigated in pancreatic β-cells. We found that no-go and nonsense-mediated RNA decay pathway components (RDPC) and exoribonuclease complexes were expressed in INS-1 cells and human islets. Pelo, Dcp2, Dis3L2, Upf2 and Smg1/5/6/7 were up-regulated by inflammatory cytokines in INS-1 cells under conditions where central β-cell mRNAs were down-regulated. These changes in RDPC mRNA or corresponding protein levels were largely confirmed in INS-1 cells and rat/human islets. Cytokine-induced up-regulation of Pelo, Xrn1, Dis3L2, Upf2 and Smg1/6 was reduced by iNOS inhibition, as were ER stress, inhibition of Ins1/2 mRNA and accumulated insulin secretion. ROS inhibition or iron chelation did not affect RDPC expression. Pelo or Xrn1 knock-down aggravated whereas Smg6 knock-down ameliorated cytokine-induced INS-1 cell death without affecting ER stress; both increased insulin biosynthesis and medium accumulation, but not glucose-stimulated insulin-secretion in cytokine-exposed INS-1 cells. In conclusion, RDPC are regulated by inflammatory stress in β-cells. RDPC knock-down improved insulin biosynthesis, likely by preventing Ins1/2 mRNA clearance. Pelo/Xrn1 knock-down aggravated, but Smg6 knock-down ameliorated cytokine-mediated β-cell death, possibly through prevention of pro-apoptotic and anti-apoptotic mRNA degradation, respectively.
... While it has been shown that both exposure to short wavelength UV (UV-C) and gamma radiation induces NF-κB activation through a ubiquitin/proteasome pathway, UV-C-induced NF-κB activation was found to involve the degradation of IκB through phosphorylation at Ser-32 and Ser-36, leading to the activation of IKK, whereas gamma rays-induced NF-κB activation was found to utilize a different pathway; thus, suggesting that both types of radiation may utilize two different mechanisms to activate NF-κB [51]. Constitutive nitric oxide synthase activation following IR in a therapeutic dose range has been shown to cause the nitration and dissociation of IκBα tyrosine 181 from NF-κB in a process that does not involve the phosphorylation or degradation of IκBα [70]. ...
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is responsible for the regulation of a large number of genes that are involved in important physiological processes, including survival, inflammation, and immune responses. At the same time, this transcription factor can control the expression of a plethora of genes that promote tumor cell proliferation, survival, metastasis, inflammation, invasion, and angiogenesis. The aberrant activation of this transcription factor has been observed in several types of cancer and is known to contribute to aggressive tumor growth and resistance to therapeutic treatment. Although NF-κB has been identified to be a major contributor to cancer initiation and development, there is evidence revealing its role in tumor suppression. This review briefly highlights the major mechanisms of NF-κB activation, the role of NF-κB in tumor promotion and suppression, as well as a few important pharmacological strategies that have been developed to modulate NF-κB function.
... This study explores HDAC inhibitors as the novel cancer therapies [55]. In a recent study NF-κB mediated mechanism has been proposed where nitration of tyrosine-181 of IκBα results in activation of nitric oxide synthase constitutively, which causes dissociation of intact IκBα from NF-κB and this mechanism doesn't seem to involve proteolytic degradation of IκBα or IκBα kinase-dependent phosphorylation [56]. This activation of NF-κB leads to inflammation [57]. ...
... Another mechanism of NF-jB activation is IKK-independent and occurs through Tyr-181 nitration in NF-jB, which leads to dissociation of NF-jB from the complex with IjB [385]. This way has been proposed as a mechanism of NF-jB signalling by modulating its transcriptional activity at low NO concentrations [165]. ...
Over the last decade, a dual character of cell response to oxidative stress, eustress vs. distress, has become increasingly recognized. A growing body of evidence indicates that under physiological conditions, low concentrations of reactive oxygen and nitrogen species (RONS) maintained by the activity of endogenous antioxidant system (AOS) allow reversible oxidative/nitrosative modifications of key redox-sensitive residues in regulatory proteins. The reversibility of redox modifications such as Cys S-sulfenylation/S-glutathionylation/S-nitrosylation/S-persulfidation and disulfide bond formation, or Tyr nitration, which occur through electrophilic attack of RONS to nucleophilic groups in amino acid residues provides redox switches in the activities of signaling proteins. Key requirement for the involvement of the redox modifications in RONS signaling including ROS-MAPK, ROS-PI3K/Akt, and RNS-TNF-α/NF-kB signaling is their specificity provided by a residue microenvironment and reaction kinetics. Glutathione, glutathione peroxidases, peroxiredoxins, thioredoxin, glutathione reductases, and glutaredoxins modulate RONS level and cell signaling, while some of the modulators (glutathione, glutathione peroxidases and peroxiredoxins) are themselves targets for redox modifications. Additionally, gene expression, activities of transcription factors and epigenetic pathways are also under redox regulation. The present review focuses on RONS sources (NADPH-oxidases, mitochondrial electron-transportation chain (ETC), nitric oxide synthase (NOS), etc.) and their cross-talks, which influence reversible redox modifications of proteins as physiological phenomenon attained by living cells during the evolution to control cell signaling in the oxygen-enriched environment. We discussed recent advances in investigation of mechanisms of protein redox modifications and adaptive redox switches such as MAPK/PI3K/PTEN, Nrf2/Keap1, and NF-κB/IκB, powerful regulators of numerous physiological processes, also implicated in various diseases.
... Tumor cells, compared to nontransformed cells, generate high levels of reactive oxygen species, e.g. the superoxide anion (O 2 -), likely due to the uncoupling of mitochondrial oxidative phosphorylation [15,16]. NO reacts with O 2 and forms the toxic oxidant peroxy-nitrite (ONOO -) which chemically inactivates DNA, proteins and lipids [17][18][19][20][21]. ...
The present studies focused on the ability of the phosphodiesterase 5 (PDE5) inhibitor sildenafil to enhance the anti-cancer properties of clinically relevant concentrations of the dietary diarylheptanoid curcumin. In gastrointestinal tumor cells, sildenafil and curcumin interacted in a greater than additive fashion to kill. Inhibition of the extrinsic apoptotic pathway suppressed killing by ∼50%, as did blockade of the intrinsic apoptotic pathway. Sildenafil and curcumin reduced mTORC1 and mTORC2 activity and increased Beclin1 levels and the numbers of autophagosomes and autolysosomes in cells in a PERK-eIF2α-dependent fashion. Knock down of Beclin1 or ATG5 partially suppressed killing. In contrast, stable knock out of ATG16-L1 unexpectedly enhanced killing, an effect not altered by Beclin1/ATG5 knock down. Curcumin and sildenafil exposure reduced the expression of MCL-1, BCL-XL, thioredoxin and superoxide dismutase 2 (SOD2) in an eIF2α-dependent fashion. Curcumin and sildenafil interacted in a greater than additive fashion to increase the levels of reactive oxygen species; knock down of thioredoxin or SOD2 enhanced killing and over-expression of thioredoxin or SOD2 suppressed killing. In vivo, curcumin and sildenafil interacted to suppress the growth of colon cancer tumors. Multiplex analyses of plasma taken after drug exposure at animal nadir indicated that the levels of M-CSF, CXCL-9, PDGF and G-CSF were significantly increased by [curcumin + sildenafil] and that expression of CXCL1 and CCL5 were significantly reduced. Cells isolated from in vivo treated [curcumin + sildenafil] tumors were resistant to in vitro [curcumin + sildenafil] exposure, a phenotype that was blocked by the colon cancer therapeutic regorafenib.
... However, depending on the context, NO can either inhibit or enhance NF-kB-dependent transcriptional responses (29). Indeed, increased activation of NF-kB by NO has been observed in multiple inflammatory settings (30,31). Our bioinformatic analyses suggested that in macrophages infected with M. tuberculosis and activated with IFN-g, NO suppresses NF-kB-dependent signaling. ...
IFN-γ is essential for control of Mycobacterium tuberculosis infection in vitro and in vivo. However, the mechanisms by which IFN-γ controls infection remain only partially understood. One of the crucial IFN-γ target genes required for control of M. tuberculosis is inducible NO synthase (iNOS). Although NO produced by iNOS is thought to have direct bactericidal activity against M. tuberculosis, the role of NO as a signaling molecule has been poorly characterized in the context M. tuberculosis infection. In this study, we found that iNOS broadly regulates the macrophage transcriptome during M. tuberculosis infection, activating antimicrobial pathways while also limiting inflammatory cytokine production. The transcription factor hypoxia inducible factor-1α (HIF-1α) was recently shown to be critical for IFN-γ-mediated control of M. tuberculosis infection. We found that HIF-1α function requires NO production, and that HIF-1α and iNOS are linked by a positive feedback loop that amplifies macrophage activation. Furthermore, we found that NO inhibits NF-κB activity to prevent hyperinflammatory responses. Thus, NO activates robust microbicidal programs while also limiting damaging inflammation. IFN-γ signaling must carefully calibrate an effective immune response that does not cause excessive tissue damage, and this study identifies NO as a key player in establishing this balance during M. tuberculosis infection.
... Compared to non-transformed cells, tumor cells generate greater amounts of O 2 -. The reaction of NO with O 2 forms the more potent oxidant peroxynitrite (ONOO -) that causes damaging S-nitrosylation of proteins and lipids [26][27][28][29][30]. ...
Phosphodiesterase 5 (PDE5) inhibitors prevent the breakdown of cGMP that results in prolonged protein kinase G activation and the generation of nitric oxide. PDE5 inhibitors enhanced the anti-NSCLC cell effects of the NSCLC therapeutic pemetrexed. [Pemetrexed + sildenafil] activated an eIF2α - ATF4 - CHOP - Beclin1 pathway causing formation of toxic autophagosomes; activated a protective IRE1 - XBP-1 - chaperone induction pathway; and activated a toxic eIF2α - CHOP - DR4 / DR5 / CD95 induction pathway. [Pemetrexed + sildenafil] reduced the expression of c-FLIP-s, MCL-1 and BCL-XL that was blocked in a cell-type -dependent fashion by either over-expression of HSP90 / GRP78 / HSP70 / HSP27 or by blockade of eIF2α-CHOP signaling. Knock down of PKGI/II abolished the ability of sildenafil to enhance pemetrexed toxicity whereas pan-inhibition of NOS using L-NAME or knock down of [iNOS + eNOS] only partially reduced the lethal drug interaction. Pemetrexed reduced the ATPase activities of HSP90 and HSP70 in an ATM-AMPK-dependent fashion that was enhanced by sildenafil signaling via PKGI/II. The drug combination activated an ATM-AMPK-TSC2 pathway that was associated with reduced mTOR S2448 and ULK-1 S757 phosphorylation and increased ULK-1 S317 and ATG13 S318 phosphorylation. These effects were prevented by chaperone over-expression or by expression of an activated form of mTOR that prevented autophagosome formation and reduced cell killing. In two models of NSCLC, sildenafil enhanced the ability of pemetrexed to suppress tumor growth. Collectively we argue that the combination of [pemetrexed + PDE5 inhibitor] should be explored in a new NSCLC phase I trial.
... Under basal conditions, NF-κB is sequestered in the cytoplasm in a complex with IκBα. In a previous study, we have demonstrated that the nitration of IκBα at Y181 triggers its dissociation from NF-κB leading to its nuclear accumulation [63]. In addition, our prior studies have shown that reducing protein nitration, by over-expressing either DDAH II in the pulmonary endothelium of the mouse [12] or using eNOS −/− mice [16], reduces the inflammatory cytokines normally induced by LPS via NF-κB again implicating eNOS rather than iNOS in the progression of ALI. ...
The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.
... Numerous biologically important proteins have been identified to be impaired by tyrosine nitration, under various pathological conditions and leading to a plethora of consequences. To mention a few examples, tyrosines are site-specifically nitrated in: L-type Ca 2+ channel hCa v 1.2b [42]; microvascular protein phosphatase 2A, an effect that prevents inactivation by phosphorylation and leads to endothelial barrier dysfunction [43]; α subunit of arterial voltage-gated potassium channel K v 1.2, especially observed in diabetic rats [44]; α, β and FXYD subunits of renal Na-K-ATPase, related to renal dysfunction [45]; glyceraldehyde 3-phosphate dehydrogenase, peripheral-type benzodiazepine receptor, MAP kinase Erk-1, and glutamine synthetase in astrocytes [46], and, perhaps, the synaptosomal glutamate transporter [47], presumably effects with consequences for extracellular glutamate concentrations; midbrain dopamine transporter and vesicular monoamine transporter-2 [48], modifications relevant to Parkinson's disease; α subunit of IκB, which leads to activation of the transcription factor NF-κB [49]. ...
Although basal and moderately elevated levels of nitric oxide are physiologically necessary and beneficial, excessive upregulations of this signaling molecule can be a cause of damage and cellular dysfunctions. In the presence of increased amounts of superoxide anions (•O2–) and carbon dioxide, peroxynitrite (ONOO–) and the peroxynitrite-CO2 adduct (ONOOCO2–) generate hydroxyl (•OH), nitrogen dioxide (•NO2) and carbonate (•CO3–) radicals, which damage biomolecules by oxidation/peroxidation, nitration and nitrosation reactions. Nitrosation also occurs with all three NO congeners (NO+, •NO, and HNO = protonated NO–), with •NO especially in combination with electron/hydrogen-abstracting compounds, or with N2O3. 3-Nitrotyrosine, found in low-density lipoprotein particles (LDL), atherosclerotic plaques, ion channels, receptors, transporters, enzymes and respirasomal subunits, is associated with numerous dysfunctions. Damage to the mitochondrial electron transport chain (ETC) is of particular significance and involves nitration, nitrosation and oxidation of proteins, cardiolipin peroxidation, and binding of •NO to ETC irons. Resulting bottlenecks of electron flux cause enhanced electron leakage which leads to elevated •O2–. In combination with high •NO, •O2– initiates a vicious cycle by generating more peroxynitrite that leads to further blockades and electron dissipation. Mitochondrial dysfunction, as induced via the •NO/peroxynitrite pathway, is of utmost importance in inflammatory diseases, especially sepsis, but also relevant to neurodegenerative and various other disorders. It may contribute to processes of aging. Melatonin, hormone of the pineal gland and product of other organs, interacts directly with reactive nitrogen species, but, more importantly, has antiinflammatory properties and downregulates inducible and neuronal NO synthases (iNOS, nNOS). It does not block moderately elevated •NO formation, but rather blunts excessive rises as occurring in sepsis and breaks the vicious cycle of mitochondrial electron leakage. The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) forms stable nitrosation products and efficiently inhibits iNOS and nNOS, in conjunction with other antiinflammatory properties. [J Exp Integr Med 2011; 1(2.000): 67-81]
... Carbonylation and tyrosine (Tyr) nitration (Tyr66 and Tyr152) of RelA inhibits its activation (148,149). In contrast nitration of IκBα (Tyr181) has been shown to activate NF-κB (150). ...
NF-κB is a multifunctional redox sensitive transcription factor. In neurodegenerative disorders such as Alzheimer’s, Huntington’s, Parkinson’s disease and amyotrophic lateral sclerosis (ALS), inducible NF-κB activation is observed in glial cells (astrocytes and microglia) as an inflammatory response that regulates the expression of cytokines, chemokines and adhesion molecules as well as the generation of reactive oxygen (ROS) and nitrogen species (RNS). As such, NF-κB activation is thought to contribute to neuronal loss. However, there is strong evidence demonstrating that in neurons, NF-κB protects against cell death progression by regulation of anti-apoptotic genes. Understanding the molecular pathways involved in NF-κB activation during neurodegeneration and its downstream signaling mediating pro-survival, pro-death and inflammatory responses will provide novel targets for therapeutic intervention. In this chapter we revise the role of NF-κB in neurodegeneration, its role in oxidative stress and antioxidant defense in the brain, and its regulation by redox homeostasis and oxidative stress.
... And we expanded this assay for several other similar studies for tyrosine modifications. Because tyrosines that are post-translationally modified by nitrating oxidants, brominating oxidants, and chloramination oxidants produced from macrophages, neutrophils or eosinophils to form nitrotyrosine, bromotyrosine and chlorotyrosine respectively, have relevance to breast cancers (17)(18)(19)(20)(21)(22)(23). For example, hypochlorous acid/hypochlorite adducts have been reported for breast cancer (24)(25)(26)(27). ...
... Furthermore, we identified Tyr74 and Tyr104 residues of IRF5 are important for induction of IL-12p40 promoter activation. A previous study demonstrated that tyrosines of IkBa are nitrated as a consequence of NO synthase activation, resulting in dissociation of IkBa from NF-kB 31 . Some other studies have presented that nitration of a specific tyrosine in proteins can have structural and functional significance [32][33][34] . ...
Here we show that iNOS-deficient mice display enhanced classically activated M1 macrophage polarization without major effects on alternatively activated M2 macrophages. eNOS and nNOS mutant mice show comparable M1 macrophage polarization compared with wild-type control mice. Addition of N6-(1-iminoethyl)-L-lysine dihydrochloride, an iNOS inhibitor, significantly enhances M1 macrophage polarization while S-nitroso-N-acetylpenicillamine, a NO donor, suppresses M1 macrophage polarization. NO derived from iNOS mediates nitration of tyrosine residues in IRF5 protein, leading to the suppression of IRF5-targeted M1 macrophage signature gene activation. Computational analyses corroborate a circuit that fine-tunes the expression of IL-12 by iNOS in macrophages, potentially enabling versatile responses based on changing microenvironments. Finally, studies of an experimental model of endotoxin shock show that iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage activation phenotype. These results suggest that NO derived from iNOS in activated macrophages suppresses M1 macrophage polarization.
... As both LPS treated DDAH II over-expressing mice and eNOS -/mice have increased DDAH activity, low levels of ADMA, and decreased NOS uncoupling, these 5 cytokines: IL-6, IP-10, MIP-1β, MIP-2, and VEGF, are potentially reduced as a result of decreased NOS uncoupling and peroxynitrite generation. As we have previously shown that the nitration of IκBα at Tyr 181 dissociates IκBα from NF-κB, and subsequently, NF-κB becomes activated [74], the decrease in oxidative and nitrative stress in LPS treated eNOS -/and DDAH II overexpressing mice may prevent the nitration of IκBα, the activation of NF-κB, reducing the expression of these inflammatory cytokines. However it is unclear why the other seven cytokines reduced in the LPS treated eNOS -/mice are not reduced in the LPS exposed DDAH II over-expressing mice and this will need further examination. ...
Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.
... Under basal conditions, NF-κB is sequestered in the cytoplasm in a complex with IκBα. In a previous study, we have demonstrated that the nitration of IκBα at Y181 triggers its dissociation from NF-κB leading to its nuclear accumulation [63]. In addition, our prior studies have shown that reducing protein nitration, by over-expressing either DDAH II in the pulmonary endothelium of the mouse [12] or using eNOS −/− mice [16], reduces the inflammatory cytokines normally induced by LPS via NF-κB again implicating eNOS rather than iNOS in the progression of ALI. ...
... Furthermore, we identified Tyr74 and Tyr104 residues of IRF5 are important for induction of IL-12p40 promoter activation. A previous study demonstrated that tyrosines of IkBa are nitrated as a consequence of NO synthase activation, resulting in dissociation of IkBa from NF-kB 31 . Some other studies have presented that nitration of a specific tyrosine in proteins can have structural and functional significance [32][33][34] . ...
Inducible nitric oxide synthase (iNOS) is a signature molecule for classically activated
macrophages (M1) but the molecular mechanisms for the regulation of M1 macrophages are still
not fully understood. Here, we show that iNOS deficient mice displayed enhanced M1
macrophage differentiation but without major effects on alternatively activated macrophages
(M2). While endothelial NOS (eNOS) or neuronal NOS (nNOS) mutant mice showed
comparable M1 macrophage differentiation compared to wild type control mice. Addition of N6-
(1-iminoethyl)-L-lysine dihydrochloride (L-NIL), the iNOS inhibitor, significantly enhanced M1
macrophage differentiation and S-nitroso-N-acetylpenicillamine (SNAP), the NO donor,
suppressed M1 macrophage differentiation. NO derived from iNOS mediated nitration of
tyrosine residues in IRF5 protein leading to the suppression of IRF5-targetted M1 macrophage
signature gene activation. Such results indicated that NO regulates macrophage differentiation by
modulating IRF5. Computational analyses corroborate a circuit that fine-tunes the expression of
IL-12 by iNOS in macrophages, potentially enabling versatile responses based upon changing
innate environments. Finally, studies of an experimental model of endotoxin shock showed that
iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage
activation phenotype. These results suggest that NO derived from iNOS in activated
macrophages promotes M1 macrophage dedifferentiation and highlight the importance of
intrinsic programs for the control of innate immune responses.
... Moreover, Sonveaux et al. showed that radiation-induced eNOS activation facilitates angiogenesis in vascular endothelial cells after X-irradiation [5], and NO-mediated protein nitration has been shown to have an influence on some signal transduction pathways e.g. IjBa on NFjB pathway [6] or TIMP-1 on Akt pathway [7]. These data indicate that radiation-induced NO plays various roles in determining the characteristics of solid tumors. ...
Tetrahydrobiopterin is a cofactor necessary for the activity of several enzymes, the most studied of which is nitric oxide synthase. The role of this cofactor-enzyme relationship in vascular biology is well established. Recently, tetrahydrobiopterin metabolism has received increasing attention in the field of cancer immunology and immunotherapy due to its involvement in the cytotoxic T cell response. Past research has demonstrated that when the availability of BH4 is low, as it is in chronic inflammatory conditions and tumors, electron transfer in the active site of nitric oxide synthase becomes uncoupled from the oxidation of arginine. This results in the production of radical species that are capable of a direct attack on tetrahydrobiopterin, further depleting its local availability. This feedforward loop may act like a molecular switch, reinforcing low tetrahydrobiopterin levels leading to altered NO signaling, restrained immune effector activity, and perpetual vascular inflammation within the tumor microenvironment. In this review, we discuss the evidence for this underappreciated mechanism in different aspects of tumor progression and therapeutic responses. Furthermore, we discuss the preclinical evidence supporting a clinical role for tetrahydrobiopterin supplementation to enhance immunotherapy and radiotherapy for solid tumors and the potential safety concerns.
Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These post-translational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1,971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.
During the last decades the attempt to enlighten the pathobiological substrate of psychosis, from merely focusing on neurotransmitters, has expanded into new areas like the immune and redox systems. Indeed, the inflammatory hypothesis concerning psychosis etiopathology has exponentially grown with findings reflecting dysfunction/aberration of the immune/redox systems' effector components namely cytokines, chemokines, CRP, complement system, antibodies, pro-/anti-oxidants, oxidative stress byproducts just to name a few. Yet, we still lie far from comprehending the underlying cellular mechanisms, their causality directions, and the moderating/mediating parameters affecting these systems; let alone the inter-systemic (between immune and redox) interactions. Findings from preclinical studies on the stress field have provided evidence indicative of multifaceted interactions among the immune and redox components so tightly intertwined as a Gordian knot. Interestingly the literature concerning the interactions between these same systems in the context of psychosis appears minimal (if not absent) and ambiguous. This review attempts to draw a frame of the immune-redox systems' interactions starting from basic research on the stress field and expanding on clinical studies with cohorts with psychosis, hoping to instigate new avenues of research.
Increased levels of reactive oxygen/nitrogen species are one hallmark of chronic inflammation contributing to the activation of pro-inflammatory/proliferative pathways. In the cancers analyzed, the tetrahydrobiopterin:dihydrobiopterin ratio is lower than that of the corresponding normal tissue, leading to an uncoupled nitric oxide synthase activity and increased generation of reactive oxygen/nitrogen species. Previously, we demonstrated that prophylactic treatment with sepiapterin, a salvage pathway precursor of tetrahydrobiopterin, prevents dextran sodium sulfate–induced colitis in mice and associated azoxymethane-induced colorectal cancer. Herein, we report that increasing the tetrahydrobiopterin:dihydrobiopterin ratio and recoupling nitric oxide synthase with sepiapterin in the colon cancer cell lines, HCT116 and HT29, inhibit their proliferation and enhance cell death, in part, by Akt/GSK-3β–mediated downregulation of β-catenin. Therapeutic oral gavage with sepiapterin of mice bearing azoxymethane/dextran sodium sulfate–induced colorectal cancer decreased metabolic uptake of [18F]-fluorodeoxyglucose and enhanced apoptosis nine-fold in these tumors. Immunohistochemical analysis of both mouse and human tissues indicated downregulated expression of key enzymes in tetrahydrobiopterin biosynthesis in the colorectal cancer tumors. Human stage 1 colon tumors exhibited a significant decrease in the expression of quinoid dihydropteridine reductase, a key enzyme involved in recycling tetrahydrobiopterin suggesting a potential mechanism for the reduced tetrahydrobiopterin:dihydrobiopterin ratio in these tumors. In summary, sepiapterin treatment of colorectal cancer cells increases the tetrahydrobiopterin:dihydrobiopterin ratio, recouples nitric oxide synthase, and reduces tumor growth. We conclude that nitric oxide synthase coupling may provide a useful therapeutic target for treating patients with colorectal cancer.
Glioma cells use intermediate levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for growth and invasion, and suppressing these reactive molecules thus may compromise processes that are vital for glioma survival. Increased oxidative stress has been identified in glioma cells, in particular in glioma stem-like cells. Studies have shown that these cells harbor potent antioxidant defenses, although endogenous protection against nitrosative stress remains understudied. The enhancement of oxidative or nitrosative stress offers a potential target for triggering glioma cell death, but whether oxidative and nitrosative stresses can be combined for therapeutic effects requires further research. The optimal approach of harnessing oxidative stress for anti-glioma therapy should include the induction of free radical-induced oxidative damage and the suppression of antioxidant defense mechanisms selectively in glioma cells. However, selective induction of oxidative/nitrosative stress in glioma cells remains a therapeutic challenge, and research into selective drug delivery systems is ongoing. Because of multifactorial mechanisms of glioma growth, progression, and invasion, prospective oncological therapies may include not only therapeutic oxidative/nitrosative stress but also inhibition of oncogenic kinases, antioxidant molecules, and programmed cell death mediators.
Vascular inflammation plays a decisive role in the formation of foam cells and in the pathophysiology of atherosclerosis. However, the underlying mechanisms of these processes are not clearly understood. Macrophages engulf oxidized low-density lipoproteins (OxLDLs) via a scavenger receptor (SR), an event that mediates the elaboration of proinflammatory cytokines to initiate necrotic core formation in atherogenic plaques. In this study, we demonstrate that Nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) promotes OxLDL uptake and enhances the release of proinflammatory cytokines by macrophages. Conversely, we show that NOS1 inhibition by N(G)-nitro-L-arginine methyl ester (L-NAME) suppresses OxLDL uptake and proinflammatory cytokine expression. Current studies indicate that NOS1 plays a crucial role in vascular inflammation and in the progression of atherosclerosis. Therefore, interference with NOS1 enzymatic activity should serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.
The deregulation of a wide variety of protein kinases is associated with cancer cell initiation and tumor progression. Owing to their indispensable function in signaling pathways driving malignant cell features, protein kinases constitute major therapeutic targets in cancer. Over the past two decades, intense efforts in drug development have been dedicated to this field. The development of protein kinase inhibitors (PKIs) have been a real breakthrough in targeted cancer therapy. Despite obvious successes across patients with different types of cancer, the development of PKI resistance still prevails. Combination therapies are part of a comprehensive approach to address the problem of drug resistance. The therapeutic use of nitric oxide (NO) donors to bypass PKI resistance in cancer has never been tested in clinic yet but several arguments suggest that the combination of PKIs and NO donors may exert a potential anticancer effect. The present review summarized the current state of knowledge on common targets to both PKIs and NO. Herein, we attempt to provide the rationale underlying a potential combination of PKIs and NO donors for future directions and design of new combination therapies in cancer.
Cancer is a complex disorder extremely dependent on its microenvironment and highly regulated by multiple intracellular and extracellular stimuli. Studies show that reactive oxygen and nitrogen species (RONS) play key roles in cancer initiation and progression. Accumulation of RONS caused by imbalance between RONS generation and activity of antioxidant system (AOS) has been observed in many cancer types. This leads to alterations in gene expression levels, signal transduction pathways, and protein quality control machinery, that is, processes that regulate cancer cell proliferation, migration, invasion, and apoptosis. This review focuses on the latest advancements evidencing that RONS-induced modifications of key redox-sensitive residues in regulatory proteins, that is, cysteine oxidation/S-sulfenylation/S-glutathionylation/S-nitrosylation and tyrosine nitration, represent important molecular mechanisms underlying carcinogenesis. The oxidative/nitrosative modifications cause alterations in activities of intracellular effectors of MAPK- and PI3K/Akt-mediated signaling pathways, transcription factors (Nrf2, AP-1, NFκB, STAT3, and p53), components of ubiquitin/proteasomal and autophagy/lysosomal protein degradation systems, molecular chaperones, and cytoskeletal proteins. Redox-sensitive proteins, RONS-generating enzymes, and AOS components can serve as targets for relevant anticancer drugs. Chemotherapeutic agents exert their action via RONS generation and induction of cancer cell apoptosis, while drug resistance associates with RONS-induced cancer cell survival; this is exploited in selective anticancer therapy strategies. Cancer Res; 78(21); 6040-7.
One of the early events in the progression of lipopolysaccharide (LPS)-mediated acute lung injury (ALI) in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY-related High Mobility Group box (Sox) group-F family member, Sox18, is a barrier- protective protein through its ability to increase the expression of the tight junction protein, Claudin-5. Thus, the purpose of this study was to determine if down-regulation of the Sox18-Claudin-5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both Sox18 and Claudin-5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar down-regulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. Sox18 over-expression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced Claudin-5 expression (siRNA) reduced the HLMVEC barrier protective effects of Sox18 over-expression. The mechanism by which LPS decreases Sox18 expression was identified as transcriptional repression through binding of p65 NF-kB to a Sox18 promoter sequence located between -1082 and -1073 bp with peroxynitrite contributing to LPS-mediated NF-kB activation. We conclude that NFkB-dependent decreases in the Sox18-Claudin 5 axis is essentially involved in the disruption of human EC barrier integrity associated with LPS-mediated ALI.
The combination of pemetrexed and sorafenib has significant clinical activity against a wide variety of tumor types in patients and the present studies were performed to determine whether sildenafil enhances the killing potential of [pemetrexed + sorafenib]. In multiple genetically diverse lung cancer cell lines, sildenafil enhanced the lethality of [pemetrexed + sorafenib]. The three-drug combination reduced the activities of AKT, mTOR and STAT transcription factors; increased the activities of eIF2α and ULK-1; lowered the expression of MCL-1, BCL-XL, thioredoxin and SOD2; and increased the expression of Beclin1. Enhanced cell killing by sildenafil was blocked by inhibition of death receptor signaling and autophagosome formation. Enforced activation of STAT3 and AKT or inhibition of JNK significantly reduced cell killing. The enhanced cell killing caused by sildenafil was more reliant on increased PKG signaling than on the generation of nitric oxide. In vivo sildenafil enhanced the anti-tumor properties of [pemetrexed + sorafenib]. Based on our data we argue that additional clinical studies combining pemetrexed, sorafenib and sildenafil are warranted.
Dendritic cells (DCs) play a pivotal role in the development of effective immune defense while avoiding detrimental inflammation and autoimmunity by regulating the balance of adaptive immunity and immune tolerance. However, the mechanisms that govern the effector and regulatory functions of DCs are incompletely understood. Here, we show that DC-derived nitric oxide (NO) controls the balance of effector and regulatory DC differentiation. Mice deficient in the NO-producing enzyme inducible nitric oxide synthase (iNOS) harbored increased effector DCs that produced interleukin-12, tumor necrosis factor (TNF) and IL-6 but normal numbers of regulatory DCs that expressed IL-10 and programmed cell death-1 (PD-1). Furthermore, an iNOSspecific inhibitor selectively enhanced effector DC differentiation, mimicking the effect of iNOS deficiency in mice. Conversely, an NO donor significantly suppressed effector DC development. Furthermore, iNOS-/- DCs supported enhanced T cell activation and proliferation. Finally iNOS-/- mice infected with the enteric pathogen Citrobacter rodentium suffered more severe intestinal inflammation with concomitant expansion of effector DCs in colon and spleen. Collectively, our results demonstrate that DCderived iNOS restrains effector DC development, and offer the basis of therapeutic targeting of iNOS in DCs to treat autoimmune and inflammatory diseases.
The transcription factor nuclear factor (NF)-κB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-α to activate NF-κB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IκB-α did not change after exposure to ROS/RNS, whereas a rapid depletion of IκB-α was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-κB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-κB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-κB by oxidan...
Transient generation of reactive oxygen or nitrogen (ROS/RNS), de- tected with dihydrodichlorofluoroscein by fluorescence microscopy, oc- curs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential (DC) and de- crease in fluorescence of a mitochondria-entrapped dye, calcein, are ob- served coincidentally. Radiation-induced ROS/RNS, DC depolarization, and calcein fluorescence decrease are inhibited by the mitochondrial permeability transition inhibitor, cyclosporin A, but not the structural analogue, cyclosporin H. Radiation-stimulated ROS/RNS is also inhibited by overexpressing the Ca21-binding protein, calbindin 28K, or treating cells with an intracellular Ca21 chelator. Radiation-induced ROS/RNS is observed in several cell types with the exception of ro cells deficient in mitochondrial electron transport. ro cells show neither radiation-induced ROS/RNS production nor DC depolarization. We propose that radiation damage in a few mitochondria is transmitted via a reversible, Ca21- dependent mitochondrial permeability transition to adjacent mitochon- dria with resulting enhanced ROS/RNS generation. Measurements of radiation-induced mitogen-activated protein kinase activity indicate that this sensing/amplification mechanism is necessary for activation of some cytoplasmic signaling pathways by low doses of radiation.
A description of the ab initio quantum chemistry package GAMESS is presented. Chemical systems containing atoms through radon can be treated with wave functions ranging from the simplest closed-shell case up to a general MCSCF case, permitting calculations at the necessary level of sophistication. Emphasis is given to novel features of the program. The parallelization strategy used in the RHF, ROHF, UHF, and GVB sections of the program is described, and detailed speecup results are given. Parallel calculations can be run on ordinary workstations as well as dedicated parallel machines. © John Wiley & Sons, Inc.
Widespread use of MCF-7 human breast carcinoma cells as a model system for breast cancer has led to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, variations in sensitivity to apoptosis have not yet been described. Tumor necrosis factor alpha (TNF-alpha) has been shown to both induce apoptosis and inhibit proliferation in MCF-7 cells. We observed that TNF-alpha inhibited proliferation in MCF-7 cell variants from three different laboratories (designated M, L, and N). MCF-7 M cells were resistant to TNF-alpha-induced apoptosis, whereas MCF-7 L cells were moderately resistant to the effect of TNF-alpha. A third variant, MCF-7 N, underwent apoptosis when exposed to TNF-alpha. Analysis of the p55 TNF-alpha receptor (TNFR) 1 expression revealed the greatest expression in MCF-7 N cells, whereas the MCF-7 L and M cells expressed 89 and 67% of MCF-7 N cell TNFR1 levels, respectively. Ceramide generation occurred in all three variants in response to TNF-alpha treatment, with MCF-7 N cells expressing the greatest increase. Cleavage of the CPP32/caspase 3 substrate poly(ADP-ribose) was observed in MCF-7 N and L cells as early as 3 and 6 h, respectively, but poly(ADP-ribose) cleavage was not observed in MCF-7 M cells. The delayed protease activation in the L variant may represent the mechanism by which these cells display delayed sensitivity to TNF-a-induced apoptosis. Expression of the Bcl-2, Mcl-1, Bcl-X, Bax, and Bak proteins was analyzed to determine whether the differences in MCF-7 cell sensitivity to apoptosis could be correlated to the differential expression of these proteins. Whereas Bak, Bcl-X, and Mcl-1 levels were identical between variants, the levels of Bcl-2 were 3.5-3.8-fold higher and the levels of Bax were 1.5-1.7-fold lower in the resistant variants (M and L) as compared with those of the sensitive variant (N). Taken together, these results suggest that differences in susceptibility to TNF-alpha-induced apoptosis among MCF-7 breast cancer cell variants may be explained by differences in TNFR expression, ceramide generation, differential expression of the Bcl-2 family of proteins, and protease activation.
Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.
The transcription factor NF-κB regulates a wide set of genes involved in the establishment of many cellular processes that
control cell activation, proliferation, and apoptosis. IκB inhibitory subunits integrate NF-κB activation signals through
phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IκB-α N-terminal
domain interactors and therefore potential NF-κB regulators. An interaction of IκB-α with the mitochondrial ATP/ADP translocator
ANT was detected in yeast and confirmed in glutathioneS-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance
to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IκB-α and associated p65 NF-κB
in the mitochondrial intermembrane space. IκB-α·NF-κB appeared to be released from mitochondria upon the induction of apoptosis
by engagement of the Fas receptor. These data suggest that the mitochondrial IκB-α·NF-κB pool participates in the regulation
of apoptosis.
The mechanism of release of proinflammatory cytokines by peripheral blood monocytes is unknown. Peroxynitrite (ONOO(-)) formed by the reaction of nitric oxide (NO) and superoxide is released predominantly by inflammatory cells at the site of injury in several inflammatory pathologies. Here we show that human monocytes treated with ONOO(-) at micromolar concentrations induce a dose-dependent release of proinflammatory cytokines. These effects were not antagonized by up to 100 microm epigallocatechin gallate, an inhibitor of protein nitration. However, the proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leu-CHO and 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride, a cell-permeable scavenger of ONOO(-), almost completely inhibited the release of cytokines and the nuclear translocation of the nuclear factor (NF)-kappaB transcription factor. SDS-PAGE electrophoresis separation with Western blotting of cell extracts also indicated that phosphorylation and nitration of tyrosine residues in IkappaB-alpha molecules correlated with NF-kappaB translocation and cytokine release. In addition, the DNA binding activity of the NF-kappaB from the nuclear extracts also correlated with its nuclear translocation. These findings indicate ONOO(-) plays an essential role in the mechanism of proinflammatory cytokine release by monocytes and that Rel/NF-kappaB activation is the obligatory pathway.
Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.
Using a system that expresses a constitutively kinase-active c-Abl protein [c-Abl(KA)], we identified the protein IkappaBalpha as a novel substrate of c-Abl. This kinase-substrate relationship is not only confirmed at the level of endogenous proteins but is also supported by a physical interaction between the two proteins. Interestingly, the association of c-Abl with IkappaBalpha, which is detectable in the form of nonphosphorylated proteins, is remarkably enhanced by an inducible binding of tyrosine-phosphorylated IkappaBalpha to the c-Abl SH2 domain. In contrast to the serine 32/34 phosphorylation that triggers ubiquitination and degradation of IkappaBalpha, c-Abl-mediated phosphorylation at tyrosine 305 is associated with an increase of the IkappaBalpha protein stability. Significantly, this activity is not shared by the oncogenic Bcr-Abl, because it is unique to the nuclear c-Abl. We also demonstrate that c-Abl targets the nuclear subpopulation of IkappaBalpha for phosphorylation and induces it to accumulate in the nucleus. As a consequence, NF-kappaB transcription activity is abolished, leading to an increased cellular sensitivity to the induction of apoptosis. The functional importance of c-Abl-mediated IkappaBalpha phosphorylation is highlighted by a loss of response of the IkappaBalpha(Y305F) protein to c-Abl-mediated regulation. Using cells expressing the c-Abl(KA) protein under the control of an inducible promoter, we demonstrate inactivation of the NF-kappaB-dependent cell survival pathway as one of the mechanisms for c-Abl-mediated apoptosis.
The transcription factor NF-kappa B has been shown to be predominantly cytoplasmically localized in the absence of an inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor alpha or interleukin-1 induces the degradation of I kappa B, the inhibitor of NF-kappa B, allowing nuclear accumulation of NF-kappa B and regulation of specific gene expression. The degradation of I kappa B is controlled initially by phosphorylation induced by the I kappa B kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-kappa B and I kappa B alpha (but not I kappa B beta) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor alpha leads to the phosphorylation of mitochondrial I kappa B alpha and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochrome c oxidase III and cytochrome b mRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-kappa B by expression of the superrepressor form of I kappa B alpha inhibited the loss of expression of both cytochrome c oxidase III and cytochrome b mRNA. These data indicate that the NF-kappa B regulatory pathway exists in mitochondria and that NF-kappa B can negatively regulate mitochondrial mRNA expression.
Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a "denitrase." In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.
The computational modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field that includes hydrogen bonding, Coulombic, and hydrophobic terms, was used to model the free energy of dimer-tetramer association in a series of deoxy hemoglobin beta 37 double mutants. Five of the analyzed mutants (beta 37W -> Y, beta 37W - A, beta 37W - G, beta 37W - E, and beta 37W - R) have been solved crystallographically and characterized thermodynamically and subsequently made a good test set for the calibration of our method as a tool for free energy prediction. Initial free energy estimates for these mutants were conducted without the inclusion of crystallographically conserved water molecules and systematically underestimated the experimentally calculated loss in free energy observed for each mutant dimer-tetramer association. However, the inclusion of crystallographic waters, interacting at the dimer-dimer interface of each mutant, resulted in HINT free energy estimates that were more accurate with respect to experimental data. To evaluate the ability of our method to predict free energies for de novo protein models, the same beta 37 mutants were computationally generated from native deoxy hemoglobin and similarly analyzed. Our theoretical models were sufficiently robust to accurately predict free energy changes in a localized region around the mutated residue. However, our method did not possess the capacity to generate the long-range secondary structural effects observed in crystallographically solved mutant structures. Final method analysis involved the computational generation of structurally and/or thermodynamically uncharacterized beta 37 deoxy hemoglobin mutants. HINT analysis of these structures revealed that free energy predictions for dimer-tetramer association in these models agreed well with previously observed energy predictions for structurally and thermodynamically characterized beta 37 deoxy hemoglobin mutants.
IκBα regulates the transcription factor NF-κB through the formation of stable IκBα/NF-κB complexes. Prior to induction, IκBα retains NF-κB in the cytoplasm until the NF-κB activation signal is received. After activation, NF-κB is removed from gene promoters through association with nuclear IκBα, restoring the preinduction state. The 2.3 Å crystal structure of IκBα in complex with the NF-κB p50/p65 heterodimer reveals mechanisms of these inhibitory activities. The presence of IκBα allows large en bloc movement of the NF-κB p65 subunit amino-terminal domain. This conformational change induces allosteric inhibition of NF-κB DNA binding. Amino acid residues immediately preceding the nuclear localization signals of both NF-κB p50 and p65 subunits are tethered to the IκBα amino-terminal ankyrin repeats, impeding NF-κB from nuclear import machinery recognition.
The transcription factor NF-κB regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-κB is sequestered in the cytosol by the inhibitor IκB-α and released after serine phosphorylation of IκB-α that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-κB activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-κB activation through tyrosine phosphorylation but not degradation of IκB-α. Pervanadate-induced IκB-α phosphorylation and NF-κB activation required expression of the T cell tyrosine kinase p56lck. Reoxygenation of hypoxic cells appeared as a physiological effector of IκB-α tyrosine phosphorylation. Tyrosine phosphorylation of IκB-α represents a proteolysis-independent mechanism of NF-κB activation that directly couples NF-κB to cellular tyrosine kinase.
The inhibitory protein, IκBα, sequesters the transcription factor, NF-κB, as an inactive complex in the cytoplasm. The structure of the IκBα ankyrin repeat domain, bound to a partially truncated NF-κB heterodimer (p50/p65), has been determined by X-ray crystallography at 2.7 Å resolution. It shows a stack of six IκBα ankyrin repeats facing the C-terminal domains of the NF-κB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an α helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IκBα will occlude the NF-κB DNA-binding cleft. The orientation of IκBα in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions.
The regulation of the transcription factor NF-κB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-κB regulation is the IκB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-κB and IKK.
Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors
of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the
nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well
as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated
in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and
the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed
IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex.
It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-κB (p50−p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-κB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-κB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-κB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFα-induced degradation of IκBα or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFα. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-κB regulation.
A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, G
interaction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, G
interaction–13.6kcal mol–1, is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between –0.4 and –2.3kcalmol–1. Four water molecules bridging between DHFR and MTX contribute an additional –1.7kcalmol–1 stability to the complex. The HINT estimate of the cost of desolvation is +13.9kcalmol–1.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent - solvent, solvent - solute, and solute - solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.
IκBα inhibits transcription factor NF-κB activity by specific binding to NF-κB heterodimers composed of p65 and p50 subunits. It binds with slightly lower affinity to p65 homodimers and with significantly lower affinity to homodimers of p50. We have employed a structure-based mutagenesis approach coupled with protein–protein interaction assays to determine the source of this dimer selectivity exhibited by IκBα. Mutation of amino acid residues in IκBα that contact NF-κB only marginally affects complex binding affinity, indicating a lack of hot spots in NF-κB/IκBα complex formation. Conversion of the weak binding NF-κB p50 homodimer into a high affinity binding partner of IκBα requires transfer of both the NLS polypeptide and amino acid residues Asn202 and Ser203 from the NF-κB p65 subunit. Involvement of Asn202 and Ser203 in complex formation is surprising as these amino acid residues occupy solvent exposed positions at a distance of 20 Å from IκBα in the crystal structures. However, the same amino acid residue positions have been genetically isolated as determinants of binding specificity in a homologous system in Drosophila. X-ray crystallographic and solvent accessibility experiments suggest that these solvent-exposed amino acid residues contribute to NF-κB/IκBα complex formation by modulating the NF-κB p65 subunit NLS polypeptide.
The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the α1β1 and α2β2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the α1α2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the α1β2 and α2β1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the α1β2 and α2β1 interfaces in the T → R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Hb Rothschild (Trp 37β→Arg) with Asp94α1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the α1β2 interface with mutant Hb Ypsilanti (Asp99α→Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the α1β2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures.
Molecular dynamics programs simulate the behavior of biomolecular systems, leading to understanding of their functions. However, the computational complexity of such simulations is enormous. Parallel machines provide the potential to meet this computational challenge. To harness this potential, it is necessary to develop a scalable program. It is also necessary that the program be easily modified by application–domain programmers. The NAMD2 program presented in this paper seeks to provide these desirable features. It uses spatial decomposition combined with force decomposition to enhance scalability. It uses intelligent periodic load balancing, so as to maximally utilize the available compute power. It is modularly organized, and implemented using Charm++, a parallel C++ dialect, so as to enhance its modifiability. It uses a combination of numerical techniques and algorithms to ensure that energy drifts are minimized, ensuring accuracy in long running calculations. NAMD2 uses a portable run-time framework called Converse that also supports interoperability among multiple parallel paradigms. As a result, different components of applications can be written in the most appropriate parallel paradigms. NAMD2 runs on most parallel machines including workstation clusters and has yielded speedups in excess of 180 on 220 processors. This paper also describes the performance obtained on some benchmark applications.
Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pH-dependent binding near physiological pH. Three monoclonal antibodies were selectively modified, each under different conditions, with the resultant loss of binding activity at pH > 8 which is recovered at pH < 6. Recovery and loss of binding are ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of the resulting 3-nitrotyrosine side chain (pKa approximately 7) at the binding site of these antibodies. pH on-off dependency of binding activity, common to all TNM-modified antibodies studied by us so far, may find use in a variety of applications in which controlled modulation under mild conditions is required.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.
We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and gamma radiation, activate transcription factor NF-kappaB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-kappaB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-kappaB inhibitor IkappaBalpha. In both cases, induction of NF-kappaB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and gamma radiation induce degradation of IkappaBs by means of the ubiquitin/proteasome pathway. However, although the induction of IkappaBalpha degradation by gamma rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IkappaBalpha degradation was not dependent on phosphorylation of these residues. Even the "super repressor" IkappaBalpha mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that gamma radiation led to activation of IKK, the protein kinase that phosphorylates IkappaBalpha at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKbeta mutant prevented NF-kappaB activation by gamma radiation, but not by UV-C. These results indicate that gamma radiation and UV-C activate NF-kappaB through two distinct mechanisms.
To investigate the mechanism of NFkappaB activation by X-rays in normal primary rat astrocytes.
Primary cultures of type I astrocytes generated from the cortex of neonatal rats were exposed to X-rays with and without various kinase inhibitors and a protease inhibitor. The nuclear or cytoplasmic protein extracts were collected at specified times after treatment and analysed for NFkappaB-DNA binding activity and IkappaB protein levels.
The NFkappaB-DNA binding activity was induced by X-rays in a dose- and time-dependent manner in the absence of IkappaB protein degradation in astrocytes as well as in the human glioma cell line U-373MG. Whereas a protease inhibitor (calpain inhibitor 1) and a protein kinase C inhibitor (CGP-41251) did not affect X-ray-induced NFkappaB-DNA binding, treatment of astrocytes with the tyrosine kinase inhibitor (erbstatin) completely prevented the increase in NFkappaB activity after irradiation. Erbstatin also reduced the phosphorylation of IkappaBalpha after X-ray exposure.
These results indicate that, in contrast with the more frequently investigated activators of NFkappaB, radiation-induced activation of this transcription factor proceeds in the absence of IkappaBalpha degradation and requires tyrosine phosphorylation.
The inhibitory protein, IkappaBalpha, sequesters the transcription factor, NF-kappaB, as an inactive complex in the cytoplasm. The structure of the IkappaBalpha ankyrin repeat domain, bound to a partially truncated NF-kappaB heterodimer (p50/ p65), has been determined by X-ray crystallography at 2.7 A resolution. It shows a stack of six IkappaBalpha ankyrin repeats facing the C-terminal domains of the NF-kappaB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an alpha helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IkappaBalpha will occlude the NF-kappaB DNA-binding cleft. The orientation of IkappaBalpha in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions.
The transcription factor nuclear factor (NF)-kappaB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-alpha to activate NF-kappaB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IkappaB-alpha did not change after exposure to ROS/RNS, whereas a rapid depletion of IkappaB-alpha was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-kappaB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-kappaB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-kappaB by oxidants. In contrast, TNF activated NF-kappaB in a Ras-independent fashion. Evaluation of members of the mitogen-activated protein kinase (MAPK) family as downstream effectors of Ras revealed the requirement of MAPK/ extracellular-regulated kinase (ERK) kinase kinase (MEKK)1 and c-Jun N-terminal kinases in the induction of NF-kappaB by both oxidants and TNF, whereas the MEK-ERK pathway negatively regulates NF-kappaB. Our findings demonstrate that cytokines and oxidants cooperate in the activation of transcription factors through distinct pathways, and suggest that anti-inflammatory and antioxidant therapies may be required in concert to prevent the activation of NF-kappaB-regulated genes important in the development of inflammatory diseases.
Putative 'protein nitratases,' which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca2+. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO2 + H2O --> Protein-Tyr-H + H+ + NO3-. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.
Because the functional form of neuronal nitric-oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron-shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were significantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems.
The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.
A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.
Transcriptional promoters responsive to low doses of X-irradiation may be useful in developing a new strategy in gene therapy combined with conventional radiotherapy. The retrovirus-mediated gene trap screening identified c-IAP2 as one of genes possessing such promoters. The analysis of the cis-elements responsive to X-irradiation in c-IAP2 promoter revealed that the NF-kappaB binding sites were necessary and sufficient for the X-ray-responsiveness. We constructed the plasmid p4NFB-BAX, which had four tandem repeats of the NF-kappaB binding sites of c-IAP2 promoter (4NFB) and a suicide gene BAX under the control of 4NFB. The human tumor cells transfected with p4NFB-BAX significantly reduced the number of cells that survived 2 Gy irradiation.
The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C-terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3-nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3-derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite-mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.
During the last 30 years, investigation of the transcriptional and translational mechanisms of gene regulation has been a major focus of molecular cancer biology. More recently, it has become evident that cancer-related mutations and cancer-related therapies also can affect post-translational processing of cellular proteins and that control exerted at this level can be critical in defining both the cancer phenotype and the response to therapeutic intervention. One post-translational mechanism that is receiving considerable attention is degradation of intracellular proteins through the multicatalytic 26S proteasome. This follows growing recognition of the fact that protein degradation is a well-regulated and selective process that can differentially control intracellular protein expression levels. The proteasome is responsible for the degradation of all short-lived proteins and 70-90% of all long-lived proteins, thereby regulating signal transduction through pathways involving factors such as AP1 and NFKB, and processes such as cell cycle progression and arrest, DNA transcription, DNA repair/misrepair, angiogenesis, apoptosis/survival, growth and development, and inflammation and immunity, as well as muscle wasting (e.g. in cachexia and sepsis). In this review, we discuss the potential involvement of the proteasome in both cancer biology and cancer treatment.
The regulation of the transcription factor NF-kappaB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-kappaB regulation is the IkappaB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-kappaB and IKK.
The prediction of the binding affinity between a protein and ligands is one of the most challenging issues for computational biochemistry and drug discovery. While the enthalpic contribution to binding is routinely available with molecular mechanics methods, the entropic contribution is more difficult to estimate. We describe and apply a relatively simple and intuitive calculation procedure for estimating the free energy of binding for 53 protein-ligand complexes formed by 17 proteins of known three-dimensional structure and characterized by different active site polarity. HINT, a software model based on experimental LogP(o/w) values for small organic molecules, was used to evaluate and score all atom-atom hydropathic interactions between the protein and the ligands. These total scores (H(TOTAL)), which have been previously shown to correlate with DeltaG(interaction) for protein-protein interactions, correlate with DeltaG(binding) for protein-ligand complexes in the present study with a standard error of +/-2.6 kcal mol(-1) from the equation DeltaG(binding) = -0.001 95 H(TOTAL) - 5.543. A more sophisticated model, utilizing categorized (by interaction class) HINT scores, produces a superior standard error of +/-1.8 kcal mol(-1). It is shown that within families of ligands for the same protein binding site, better models can be obtained with standard errors approaching +/-1.0 kcal mol(-1). Standardized methods for preparing crystallographic models for hydropathic analysis are also described. Particular attention is paid to the relationship between the ionization state of the ligands and the pH conditions under which the binding measurements are made. Sources and potential remedies of experimental and modeling errors affecting prediction of DeltaG(binding) are discussed.
Ionizing radiation (1-5 Gy) activates the epidermal growth factor receptor (EGFR), a major effector of the p42/44 mitogen-activated protein kinase (MAPK) pathway. MAPK and its downstream effector, p90 ribosomal S6 kinase (p90RSK), phosphorylate transcription factors involved in cell proliferation. To establish the role of the EGFR/MAPK pathway in radiation-induced transcription factor activation, MDA-MB-231 human breast carcinoma cells were examined using specific inhibitors of signaling pathways. Gel-shift analysis revealed three different profile groups: 1) transcription factors that responded to both radiation (2 Gy) and epidermal growth factor (EGF) (CREB, Egr, Ets, and Stat3); 2) factors that responded to radiation, but not EGF (C/EBP and Stat1); and 3) those that did not respond significantly to either radiation or EGF (AP-1 and Myc). Within groups 1 and 2, a two- to fivefold maximum stimulation of binding activity was observed at 30-60 min after irradiation. Interestingly, only transcription factors that responded to EGF had radiation responses significantly inhibited by the EGFR tyrosine kinase inhibitor, AG1478; these responses were also abrogated by farnesyltransferase inhibitor (FTI) or PD98059, inhibitors of Ras and MEK1/2, respectively. Moreover, radiation-induced increases in CREB and p90RSK phosphorylation and activation of Stat3 and Egr-1 reporter constructs by radiation were all abolished by AG1478. These data demonstrate a distinct radiation response profile at the transcriptional level that is dependent on enhanced EGFR/Ras/MAPK signaling.
Nuclear localization of the transcriptional activator NF-kappaB (nuclear factor kappaB) is controlled in mammalian cells by three isoforms of NF-kappaB inhibitor protein: IkappaBalpha, -beta, and - epsilon. Based on simplifying reductions of the IkappaB-NF-kappaB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-kappaB activation by the coordinated degradation and synthesis of IkappaB proteins. The model demonstrates that IkappaBalpha is responsible for strong negative feedback that allows for a fast turn-off of the NF-kappaB response, whereas IkappaBbeta and - epsilon function to reduce the system's oscillatory potential and stabilize NF-kappaB responses during longer stimulations. Bimodal signal-processing characteristics with respect to stimulus duration are revealed by the model and are shown to generate specificity in gene expression.
IkappaBalpha inhibits transcription factor NF-kappaB activity by specific binding to NF-kappaB heterodimers composed of p65 and p50 subunits. It binds with slightly lower affinity to p65 homodimers and with significantly lower affinity to homodimers of p50. We have employed a structure-based mutagenesis approach coupled with protein-protein interaction assays to determine the source of this dimer selectivity exhibited by IkappaBalpha. Mutation of amino acid residues in IkappaBalpha that contact NF-kappaB only marginally affects complex binding affinity, indicating a lack of hot spots in NF-kappaB/IkappaBalpha complex formation. Conversion of the weak binding NF-kappaB p50 homodimer into a high affinity binding partner of IkappaBalpha requires transfer of both the NLS polypeptide and amino acid residues Asn202 and Ser203 from the NF-kappaB p65 subunit. Involvement of Asn202 and Ser203 in complex formation is surprising as these amino acid residues occupy solvent exposed positions at a distance of 20A from IkappaBalpha in the crystal structures. However, the same amino acid residue positions have been genetically isolated as determinants of binding specificity in a homologous system in Drosophila. X-ray crystallographic and solvent accessibility experiments suggest that these solvent-exposed amino acid residues contribute to NF-kappaB/IkappaBalpha complex formation by modulating the NF-kappaB p65 subunit NLS polypeptide.
Activation of the I kappa B kinase (IKK) complex by LPS induces phosphorylation and degradation of I kappa B alpha, leading to the nuclear translocation of NF-kappa B. Although it is essential for NF-kappa B activation, emerging evidence has indicated that the nuclear translocation of NF-kappa B is not sufficient to activate NF-kappa B-dependent transcription. Here, we reported that LPS induced the phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages. Using mouse embryonic fibroblasts lacking either IKK alpha or IKK beta, we found that IKK beta played an essential role in LPS-induced p65 phosphorylation on serine 536, while IKK alpha was partially required for the p65 phosphorylation. The LPS-induced p65 phosphorylation on serine 536 was independent of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Furthermore, we found that the phosphorylation on serine 536 increased the p65 transcription activity. In summary, our results demonstrate that IKK beta plays an essential role in the LPS-induced p65 phosphorylation on serine 536, which may represent a mechanism to regulate the NF-kappa B transcription activity by LPS.
The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.