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

Toxin-Based Therapeutic Approaches

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin

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

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

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

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

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

Impact of Reactive Sulfur Species on <i>Entamoeba histolytica</i>: 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

SNO trapping-based analysis of S-nitrosylation in A549 cells.

<p><b>(</b>A) Schematic of the proteomic approach. Digitonin cell lysates, obtained from A549 treated with NO donor or with cytokines are incubated with a thioredoxin (Trx) trap mutant, Trx(C35S). In the trap mutant the resolving cysteine is replaced by serine (-OH). The protein also contains a streptavidin binding peptide. Trx(C35S) forms mixed disulfide bonds with nitrosylated substrates and the resulting complexes are pulled-down using avidin agarose. Identification of nitrosylation sites is assisted by differential thiol labeling, involving the sequential application of N-ethylmaleimide (NEM) and iodoacetamide (IAM). Proteins captured in the Trx pull-down are analyzed by SDS-PAGE or liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) A549 cells were treated with or without 500 μM S-nitrosocysteine (CysNO) for 10 min and thereafter digitonin lysates were incubated with Trx(C35S). Proteins captured by Trx were released by DTT and then analyzed by SDS-PAGE. Gels were stained with Krypton fluorescent protein stain and visualized using the Odyssey infrared imaging system. (C) A549 cells were treated for 72 h with LPS (0.5 mg/ml) and a cytokine mixture that included TNFα (20 ng/ml), IFN-γ (10 ng/ml) and IL-1β (10 ng/ml). Trx-based trapping of nitrosylated proteins was performed as in B.</p