Endogenous histidine peptides are physiological antioxidants that prevent oligodendrocyte cell death and myelin loss in vivo.

Histidine dipeptides (HDs) are synthesized in brain oligodendrocytes by carnosine synthase (carns1), but their role is unknown. Using metabolomics and in vivo experiments with both constitutive and oligodendrocyte-selective carns1-KO mouse models, we found that HDs are critical for oligodendrocyte survival and protect against oxidative stress. Carns1-KO mouse models had lower numbers of mature oligodendrocytes, increased lipid peroxidation, and behavioral changes. Cuprizone administration, which increases reactive oxygen species in vivo, resulted in higher oligodendrocyte death, demyelination, axonal alterations, and oxidative damage in the corpus callosum of carns1-KO mice. Gliosis and oxidative damage by cuprizone were prevented by pretreatment with the antioxidant N-acetylcysteine. NADPH levels were increased threefold in the brains of carns1-KO mice as an antioxidant response to oxidative stress through acceleration of the pentose phosphate pathway (PPP). This was due to overexpression of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Likewise, expression of NAD kinase, the biosynthetic enzyme for NADP+, and NAMPT, which replenishes the NAD+ pool, was higher in carns1-KO mice brains than in controls. Our observations suggest that HDs cell-autonomously protect oligodendrocytes from oxidative stress, with implications for demyelinating diseases.

Widespread S-persulfidation in activated macrophages as a protective mechanism against oxidative-inflammatory stress.

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.

Impact of Reactive Sulfur Species on Entamoeba histolytica: Modulating Viability, Motility, and Biofilm Degradation Capacity.

Reactive sulfur species (RSS) like hydrogen sulfide (H2S) and cysteine persulfide (Cys-SSH) emerged as key signaling molecules with diverse physiological roles in the body, depending on their concentration and the cellular environment. While it is known that H2S and Cys-SSH are produced by both colonocytes and by the gut microbiota through sulfur metabolism, it remains unknown how these RSS affect amebiasis caused by Entamoeba histolytica, a parasitic protozoan that can be present in the human gastrointestinal tract. This study investigates H2S and Cys-SSH's impact on E. histolytica physiology and explores potential therapeutic implications. Exposing trophozoites to the H2S donor, sodium sulfide (Na2S), or to Cys-SSH led to rapid cytotoxicity. A proteomic analysis of Cys-SSH-challenged trophozoites resulted in the identification of >500 S-sulfurated proteins, which are involved in diverse cellular processes. Functional assessments revealed inhibited protein synthesis, altered cytoskeletal dynamics, and reduced motility in trophozoites treated with Cys-SSH. Notably, cysteine proteases (CPs) were significantly inhibited by S-sulfuration, affecting their bacterial biofilm degradation capacity. Immunofluorescence microscopy confirmed alterations in actin dynamics, corroborating the proteomic findings. Thus, our study reveals how RSS perturbs critical cellular functions in E. histolytica, potentially influencing its pathogenicity and interactions within the gut microbiota. Understanding these molecular mechanisms offers novel insights into amebiasis pathogenesis and unveils potential therapeutic avenues targeting RSS-mediated modifications in parasitic infections.

Global Thiol Proteome Analysis Provides Novel Insights into the Macrophage Inflammatory Response and Its Regulation by the Thioredoxin System.

Aims: Oxidative modifications of cysteine (Cys) thiols regulate various physiological processes, including inflammatory responses. The thioredoxin (Trx) system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical proinflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the Trx system. Results: In this study, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of Trx system activity by auranofin (AF) by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, oxidation of nearly 10% was selectively affected by LPS or AF treatments. Notably, the proteomic analysis uncovered a subset of ∼100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and Trx. Compared with the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modification as a mechanism that allows for rapid modulation of macrophage activities in response to a proinflammatory or pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione-synthesizing enzyme, glutamate-cysteine ligase, and the autophagy adaptor, SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges. Antioxid. Redox Signal. 38, 388-402.

S-nitrosocysteine and glutathione depletion synergize to induce cell death in human tumor cells: Insights into the redox and cytotoxic mechanisms.

Nitric oxide (NO)-dependent signaling and cytotoxic effects are mediated in part via protein S-nitrosylation. The magnitude and duration of S-nitrosylation are governed by the two main thiol reducing systems, the glutathione (GSH) and thioredoxin (Trx) antioxidant systems. In recent years, approaches have been developed to harness the cytotoxic potential of NO/nitrosylation to inhibit tumor cell growth. However, progress in this area has been hindered by insufficient understanding of the balance and interplay between cellular nitrosylation, other oxidative processes and the GSH/Trx systems. In addition, the mechanistic relationship between thiol redox imbalance and cancer cell death is not fully understood. Herein, we explored the redox and cellular effects induced by the S-nitrosylating agent, S-nitrosocysteine (CysNO), in GSH-sufficient and -deficient human tumor cells. We used l-buthionine-sulfoximine (BSO) to induce GSH deficiency, and employed redox, biochemical and cellular assays to interrogate molecular mechanisms. We found that, under GSH-sufficient conditions, a CysNO challenge (100-500 µM) results in a marked yet reversible increase in protein S-nitrosylation in the absence of appreciable S-oxidation. In contrast, under GSH-deficient conditions, CysNO induces elevated and sustained levels of both S-nitrosylation and S-oxidation. Experiments in various cancer cell lines showed that administration of CysNO or BSO alone commonly induce minimal cytotoxicity whereas BSO/CysNO combination therapy leads to extensive cell death. Studies in HeLa cancer cells revealed that treatment with BSO/CysNO results in dual inhibition of the GSH and Trx systems, thereby amplifying redox stress and causing cellular dysfunction. In particular, BSO/CysNO induced rapid oxidation and collapse of the actin cytoskeletal network, followed by loss of mitochondrial function, leading to profound and irreversible decrease in ATP levels. Further observations indicated that BSO/CysNO-induced cell death occurs via a caspase-independent mechanism that involves multiple stress-induced pathways. The present findings provide new insights into the relationship between cellular nitrosylation/oxidation, thiol antioxidant defenses and cell death. These results may aid future efforts to develop NO/redox-based anticancer approaches.

Oxidants, Antioxidants and Thiol Redox Switches in the Control of Regulated Cell Death Pathways.

It is well appreciated that biological reactive oxygen and nitrogen species such as hydrogen peroxide, superoxide and nitric oxide, as well as endogenous antioxidant systems, are important modulators of cell survival and death in diverse organisms and cell types. In addition, oxidative stress, nitrosative stress and dysregulated cell death are implicated in a wide variety of pathological conditions, including cancer, cardiovascular and neurological diseases. Therefore, much effort is devoted to elucidate the molecular mechanisms linking oxidant/antioxidant systems and cell death pathways. This review is focused on thiol redox modifications as a major mechanism by which oxidants and antioxidants influence specific regulated cell death pathways in mammalian cells. Growing evidence indicates that redox modifications of cysteine residues in proteins are involved in the regulation of multiple cell death modalities, including apoptosis, necroptosis and pyroptosis. In addition, recent research suggests that thiol redox switches play a role in the crosstalk between apoptotic and necrotic forms of regulated cell death. Thus, thiol-based redox circuits provide an additional layer of control that determines when and how cells die.

Opposing effects of polysulfides and thioredoxin on apoptosis through caspase persulfidation.

Hydrogen sulfide has been implicated in a large number of physiological processes including cell survival and death, encouraging research into its mechanisms of action and therapeutic potential. Results from recent studies suggest that the cellular effects of hydrogen sulfide are mediated in part by sulfane sulfur species, including persulfides and polysulfides. In the present study, we investigated the apoptosis-modulating effects of polysulfides, especially on the caspase cascade, which mediates the intrinsic apoptotic pathway. Biochemical analyses revealed that organic or synthetic polysulfides strongly and rapidly inhibit the enzymatic activity of caspase-3, a major effector protease in apoptosis. We attributed the caspase-3 inhibition to persulfidation of its catalytic cysteine. In apoptotically stimulated HeLa cells, short-term exposure to polysulfides triggered the persulfidation and deactivation of cleaved caspase-3. These effects were antagonized by the thioredoxin/thioredoxin reductase system (Trx/TrxR). Trx/TrxR restored the activity of polysulfide-inactivated caspase-3 in vitro, and TrxR inhibition potentiated polysulfide-mediated suppression of caspase-3 activity in situ We further found that under conditions of low TrxR activity, early cell exposure to polysulfides leads to enhanced persulfidation of initiator caspase-9 and decreases apoptosis. Notably, we show that the proenzymes procaspase-3 and -9 are basally persulfidated in resting (unstimulated) cells and become depersulfidated during their processing and activation. Inhibition of TrxR attenuated the depersulfidation and activation of caspase-9. Taken together, our results reveal that polysulfides target the caspase-9/3 cascade and thereby suppress cancer cell apoptosis, and highlight the role of Trx/TrxR-mediated depersulfidation in enabling caspase activation.

Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.

Life on Earth emerged in a hydrogen sulfide (H2S)-rich environment eons ago and with it protein persulfidation mediated by H2S evolved as a signaling mechanism. Protein persulfidation (S-sulfhydration) is a post-translational modification of reactive cysteine residues, which modulate protein structure and/or function. Persulfides are difficult to label and study due to their reactivity and similarity with cysteine. Here, we report a facile strategy for chemoselective persulfide bioconjugation using dimedone-based probes, to achieve highly selective, rapid, and robust persulfide labeling in biological samples with broad utility. Using this method, we show persulfidation is an evolutionarily conserved modification and waves of persulfidation are employed by cells to resolve sulfenylation and prevent irreversible cysteine overoxidation preserving protein function. We report an age-associated decline in persulfidation that is conserved across evolutionary boundaries. Accordingly, dietary or pharmacological interventions to increase persulfidation associate with increased longevity and improved capacity to cope with stress stimuli.

S-Nitrosylation of α1-Antitrypsin Triggers Macrophages Toward Inflammatory Phenotype and Enhances Intra-Cellular Bacteria Elimination.

Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo, hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored. Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection. Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi. Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells. Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways. Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.

Roles of mammalian glutathione peroxidase and thioredoxin reductase enzymes in the cellular response to nitrosative stress.

Mammalian cells employ elaborate antioxidant systems to effectively handle reactive oxygen and nitrogen species (ROS and RNS). At the heart of these systems operate two selenoprotein families consisting of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) enzymes. Although mostly studied in the context of oxidative stress, considerable evidence has amassed to indicate that these selenoenzymes also play important roles in nitrosative stress responses. GPx and TrxR, together with their redox partners, metabolize nitrosothiols and peroxynitrite, two major RNS. As such, these enzymes play active roles in the cellular defense against nitrosative stress. However, under certain conditions, these enzymes are inactivated by nitrosothiols or peroxynitrite, which may exacerbate oxidative and nitrosative stress in cells. The selenol groups in the active sites of GPx and TrxR enzymes are critically involved in these beneficial and detrimental processes. Further elucidation of the biochemical interactions between distinct RNS and GPx/TrxR will lead to a better understanding of the roles of these selenoenzymes in cellular homeostasis and disease.

Correction: Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells.

[This corrects the article DOI: 10.1371/journal.pone.0169862.].

Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells.

Nitrosylation of cysteines residues (S-nitrosylation) mediates many of the cellular effects of nitric oxide in normal and diseased cells. Recent research indicates that S-nitrosylation of certain proteins could play a role in tumor progression and responsiveness to therapy. However, the protein targets of S-nitrosylation in cancer cells remain largely unidentified. In this study, we used our recently developed nitrosothiol trapping approach to explore the nitrosoproteome of human A549 lung carcinoma cells treated with S-nitrosocysteine or pro-inflammatory cytokines. Using this approach, we identified about 300 putative nitrosylation targets in S-nitrosocysteine-treated A549 cells and approximately 400 targets in cytokine-stimulated cells. Among the more than 500 proteins identified in the two screens, the majority represent novel targets of S-nitrosylation, as revealed by comparison with publicly available nitrosoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitrosylation sites in about 150 proteins. The proteomic results were validated for several proteins by an independent approach. Bioinformatic analysis highlighted important cellular pathways that are targeted by S-nitrosylation, notably, cell cycle and inflammatory signaling. Taken together, our results identify new molecular targets of nitric oxide in lung cancer cells and suggest that S-nitrosylation may regulate signaling pathways that are critically involved in lung cancer progression.

Inhibitory nitrosylation of mammalian thioredoxin reductase 1: Molecular characterization and evidence for its functional role in cellular nitroso-redox imbalance.

Mammalian thioredoxin 1 (Trx1) and the selenoprotein Trx reductase 1 (TrxR1) are key cellular enzymes that function coordinately in thiol-based redox regulation and signaling. Recent studies have revealed that the Trx1/TrxR1 system has an S-nitrosothiol reductase (denitrosylase) activity through which it can regulate nitric oxide-related cellular processes. In this study we revealed that TrxR1 is itself susceptible to nitrosylation, characterized the underlying mechanism, and explored its functional significance. We found that nitrosothiol or nitric oxide donating agents rapidly and effectively inhibited the activity of recombinant or endogenous TrxR1. In particular, the NADPH-reduced TrxR1 was partially and reversibly inhibited upon exposure to low concentrations (<10µM) of S-nitrosocysteine (CysNO) and markedly and continuously inhibited at higher doses. Concurrently, TrxR1 very efficiently reduced low, but not high, levels of CysNO. Biochemical and mass spectrometric analyses indicated that its active site selenocysteine residue renders TrxR1 highly susceptible to nitrosylation-mediated inhibition, and revealed both thiol and selenol modifications at the two redox active centers of the enzyme. Studies in HeLa cancer cells demonstrated that endogenous TrxR1 is sensitive to nitrosylation-dependent inactivation and pointed to an important role for glutathione in reversing or preventing this process. Notably, depletion of cellular glutathione with l-buthionine-sulfoximine synergized with nitrosating agents in promoting sustained nitrosylation and inactivation of TrxR1, events that were accompanied by significant oxidation of Trx1 and extensive cell death. Collectively, these findings expand our knowledge of the role and regulation of the mammalian Trx system in relation to cellular nitroso-redox imbalance. The observations raise the possibility of exploiting the nitrosylation susceptibility of TrxR1 for killing tumor cells.

Emerging Roles of Protein S-Nitrosylation in Macrophages and Cancer Cells.

Despite long and intensive investigation, the mechanisms by which nitric oxide (NO) regulates immune function and carcinogenesis remain incompletely understood. Protein S-nitrosylation, the covalent attachment of a nitroso group to a cysteine thiol, has emerged as a central mechanism of NO-dependent cellular regulation. In particular, recent research has revealed important roles for S-nitrosylation/denitrosylation in modulating the activity of macrophage and tumor cell proteins, implicating Snitrosylation in the regulation of macrophage function as well as in tumor development and response to therapy. This review summarizes recent progress in the identification and characterization of S-nitrosylated proteins in macrophages and cancer cells. The review highlights key findings and insights obtained from functional and proteomic studies about the roles of S-nitrosylation in signaling, transcription, apoptosis and other cellular processes relevant to macrophage function and cancer progression. Some of the implications of recent discoveries for the development of novel anticancer approaches are also discussed.

Dual targeting of the thioredoxin and glutathione systems in cancer and HIV.

Although the use of antioxidants for the treatment of cancer and HIV/AIDS has been proposed for decades, new insights gained from redox research have suggested a very different scenario. These new data show that the major cellular antioxidant systems, the thioredoxin (Trx) and glutathione (GSH) systems, actually promote cancer growth and HIV infection, while suppressing an effective immune response. Mechanistically, these systems control both the redox- and NO-based pathways (nitroso-redox homeostasis), which subserve innate and cellular immune defenses. Dual inhibition of the Trx and GSH systems synergistically kills neoplastic cells in vitro and in mice and decreases resistance to anticancer therapy. Similarly, the population of HIV reservoir cells that constitutes the major barrier to a cure for AIDS is exquisitely redox sensitive and could be selectively targeted by Trx and GSH inhibitors. Trx and GSH inhibition may lead to a reprogramming of the immune response, tilting the balance between the immune system and cancer or HIV in favor of the former, allowing elimination of diseased cells. Thus, therapies based on silencing of the Trx and GSH pathways represent a promising approach for the cure of both cancer and AIDS and warrant further investigation.

Nitric oxide and the thioredoxin system: a complex interplay in redox regulation.

BACKGROUND: The free radical nitric oxide (NO) and the thiol oxidoreductase thioredoxin (Trx) play essential roles in cellular redox regulation. Recent biochemical and cellular studies have revealed a complex thiol-dependent crosstalk between NO and Trx that modulates multiple redox-dependent pathways. SCOPE OF REVIEW: This review aims to discuss recent progress, as well as the remaining questions, regarding the interaction and cross regulation between NO and Trx in cellular function and dysfunction. MAJOR CONCLUSIONS: The importance and ubiquity of NO-mediated S-nitrosylation of protein thiols as a signaling mechanism is increasingly recognized as is the central role of Trx in regulating S-nitrosylation processes. By denitrosylating diverse protein substrates, Trx plays an active role in attenuating NO signaling as well as in ameliorating nitrosative stress. Yet, at the same time, Trx can also support the activity of NO synthases, thus promoting NO production and its downstream effects. Finally, NO can reciprocally modulate the redox activity of Trx and Trx reductase. GENERAL SIGNIFICANCE: Further elucidation of the crosstalk between NO and Trx will be important for an improved understanding of the effects of reactive oxygen and nitrogen species on cellular signaling and function.

Blocking IL1ß Pathway Following Paclitaxel Chemotherapy Slightly Inhibits Primary Tumor Growth but Promotes Spontaneous Metastasis.

Acquired resistance to therapy is a major obstacle in clinical oncology, and little is known about the contributing mechanisms of the host response to therapy. Here, we show that the proinflammatory cytokine IL1ß is overexpressed in response to paclitaxel chemotherapy in macrophages, subsequently promoting the invasive properties of malignant cells. In accordance, blocking IL1ß, or its receptor, using either genetic or pharmacologic approach, results in slight retardation of primary tumor growth; however, it accelerates metastasis spread. Tumors from mice treated with combined therapy of paclitaxel and the IL1 receptor antagonist anakinra exhibit increased number of M2 macrophages and vessel leakiness when compared with paclitaxel monotherapy-treated mice, indicating a prometastatic role of M2 macrophages in the IL1ß-deprived microenvironment. Taken together, these findings demonstrate the dual effects of blocking the IL1 pathway on tumor growth. Accordingly, treatments using "add-on" drugs to conventional therapy should be investigated in appropriate tumor models consisting of primary tumors and their metastases.

Thioredoxin-mimetic peptides as catalysts of S-denitrosylation and anti-nitrosative stress agents.

S-nitrosylation, the coupling of a nitric oxide moiety to a reactive cysteine residue to form an S-nitrosothiol (SNO), is an important posttranslational mechanism for regulating protein activity. Growing evidence indicates that hyper-S-nitrosylation may contribute to cellular dysfunction associated with various human diseases. It is also increasingly appreciated that thioredoxin and thioredoxin reductase play significant roles in the cellular catabolism of SNO and protection from nitrosative stress. Here, we investigated the SNO reductase activity and protective effects of thioredoxin-mimetic peptides (TXMs), Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4), both under cell-free conditions and in nitrosatively stressed cultured cells. In vitro biochemical analyses revealed that the TXM peptides reduced small-molecule SNO compounds, such as S-nitrosoglutathione (GSNO), and acted as general and efficient protein-denitrosylating agents. In particular, CB3 was found to be a highly potent SNO-metabolizing agent. Notably, CB3 mimicked the activity of thioredoxin by coupling with thioredoxin reductase to enhance GSNO reduction. Moreover, in a cell-free lysate system, both CB3 and CB4 synergized with an NADPH-dependent activity to denitrosylate proteins. Further investigation revealed that the TXM peptides protect the peroxiredoxin-thioredoxin system from SNO-dependent inhibition. Indeed, SNO-inhibited Prx1 was efficiently denitrosylated and reactivated by CB3 or CB4. In addition, CB3 protected thioredoxin reductase from SNO-mediated inactivation both in vitro and in intact cells. Finally, CB3 and CB4 partially rescued human neuroblastoma SH-SY5Y cells and rat insulinoma INS-1 832/13 cells from GSNO-induced growth inhibition. Collectively, the present findings indicate the efficient denitrosylation activity and protective effects of TXM peptides and suggest their potential therapeutic value in treating pathological conditions related to nitrosative stress.

Suppression of the pro-inflammatory NLRP3/interleukin-1ß pathway in macrophages by the thioredoxin reductase inhibitor auranofin.

BACKGROUND: The thioredoxin/thioredoxin reductase system, which is best known for its essential role in antioxidant defense and redox homeostasis, is increasingly implicated in the regulation of multiple cellular signaling pathways. In the present study, we asked if the thioredoxin system in macrophages might regulate toll-like receptor 4 (TLR4)-dependent gene expression and consequent responses. METHODS: Using microarray analysis we analyzed the effect of auranofin, a highly potent and specific inhibitor of thioredoxin reductase, on the transcriptional program activated in J774 macrophages by the TLR4 agonist, lipopolysaccharide (LPS). We used quantitative real-time PCR (qPCR), Western blotting, ELISA and cytotoxicity assays to confirm and extend the microarray results. RESULTS: Global transcriptional profiling revealed that macrophage treatment with auranofin exerted a selective effect on LPS-induced gene expression, suppressing the induction of a small number of genes. Interestingly, among these suppressed genes were three members of the interleukin-1 (IL-1) family of genes, among which IL-1ß was most affected. qPCR analyses confirmed the repressive effects of auranofin on IL-1 genes. In addition, qPCR and Western blot analyses showed that auranofin impaired TLR4-dependent induction of the inflammasome receptor NLRP3, which plays a critical role in IL-1ß processing. Consistent with these findings, inflammasome-dependent release of IL-1ß from stimulated macrophages was suppressed by auranofin as was inflammasome-mediated cell death. CONCLUSIONS: Our findings suggest a regulatory role for the thioredoxin system in macrophage inflammatory signaling. Inhibition of the thioredoxin system in macrophages exerts an anti-inflammatory effect by repressing the activation of the NLRP3/IL-1ß pathway.