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Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

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According to the World Health Organization (WHO), breast cancer (BC) is the deadliest and the most common type of cancer worldwide in women. Several factors associated with BC exert their effects by modulating the state of stress. They can induce genetic mutations or alterations in cell growth, encouraging neoplastic development and the production of reactive oxygen species (ROS). ROS are able to activate many signal transduction pathways, producing an inflammatory environment that leads to the suppression of programmed cell death and the promotion of tumor proliferation, angiogenesis, and metastasis; these effects promote the development and progression of malignant neoplasms. However, cells have both non-enzymatic and enzymatic antioxidant systems that protect them by neutralizing the harmful effects of ROS. In this sense, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), thioredoxin reductase (TrxR), and peroxiredoxin (Prx) protect the body from diseases caused by oxidative damage. In this review, we will discuss mechanisms through which some enzymatic antioxidants inhibit or promote carcinogenesis, as well as the new therapeutic proposals developed to complement traditional treatments.

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Peroxiredoxins (Prxs) and glutathione peroxidases (GPxs) are the main enzymes of the thiol-dependent antioxidant systems responsible for reducing the H2O2 produced via aerobic metabolism or parasitic organisms by the host organism. These antioxidant systems maintain a proper redox state in cells. The cysticerci of Taenia crassiceps tolerate millimolar concentrations of this oxidant. To understand the role played by Prxs in this cestode, two genes for Prxs, identified in the genome of Taenia solium (TsPrx1 and TsPrx3), were cloned. The sequence of the proteins suggests that both isoforms belong to the class of typical Prxs 2-Cys. In addition, TsPrx3 harbors a mitochondrial localization signal peptide and two motifs (-GGLG- and -YP-) associated with overoxidation. Our kinetic characterization assigns them as thioredoxin peroxidases (TPxs). While TsPrx1 and TsPrx3 exhibit the same catalytic efficiency, thioredoxin-glutathione reductase from T. crassiceps (TcTGR) was five and eight times higher. Additionally, the latter demonstrated a lower affinity (>30-fold) for H2O2 in comparison with TsPrx1 and TsPrx3. The TcTGR contains a Sec residue in its C-terminal, which confers additional peroxidase activity. The aforementioned aspect implies that TsPrx1 and TsPrx3 are catalytically active at low H2O2 concentrations, and the TcTGR acts at high H2O2 concentrations. These results may explain why the T. crassiceps cysticerci can tolerate high H2O2 concentrations.

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We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105 -106 m-1 s-1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

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[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

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TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

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Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 µM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO.

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Marfan syndrome (MFS) is an autosomal dominant disorder caused by a heterozygous mutation of the FBN1 gene. MFS patients present oxidative stress that disturbs redox homeostasis. Redox homeostasis depends in part on the enzymatic antioxidant system, which includes thioredoxin reductase (TrxR) and glutathione peroxidases (GPx), both of which require an adequate concentration of selenium (Se). Therefore, the aim of this study was to determine if Se levels are decreased in the TAA of patients with MFS since this could contribute to the formation of an aneurysm in these patients. The results show that interleukins IL-1ß, IL-6 TGF-ß1, and TNF-α (p ≤ 0.03), and carbonylation (p ≤ 0.03) were increased in the TAA of patients with MFS in comparison with control subjects, while Se, thiols (p = 0.02), TrxR, and GPx (p ≤ 0.001) were decreased. TLR4 and NOX1 (p ≤ 0.03), MMP9 and MMP2 (p = 0.04) and NOS2 (p < 0.001) were also increased. Therefore, Se concentrations are decreased in the TAA of MFS, which can contribute to a decrease in the activities of TrxR and GPx, and thiol groups. A decrease in the activities of these enzymes can lead to the loss of redox homeostasis, which can, in turn, lead to an increase in the pro-inflammatory interleukins associated with the overexpression of MMP9 and MMP2.

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C. glabrata, an opportunistic fungal pathogen, can adapt and resist to different stress conditions. It is highly resistant to oxidant stress compared to other Candida spp and to the phylogenetically related but non-pathogen Saccharomyces cerevisiae. In this work, we describe the Trx/Trr system of C. glabrata composed of Trr1 and Trr2 (thioredoxin reductases) and Trx2 (thioredoxin) that are localized in the cytoplasm and Trx3 present in the mitochondrion. The transcriptional induction of TRR2 and TRX2 by oxidants depends on Yap1 and Skn7 and TRR1 and TRX3 have a low expression level. Both TRR2 and TRX2 play an important role in the oxidative stress response. The absence of TRX2 causes auxotrophy of methionine and cysteine. Trr1 and Trr2 are necessary for survival at high temperatures and for the chronological life span of C. glabrata. Furthermore, the Trx/Trr system is needed for survival in the presence of neutrophils. The role of TRR1 and TRX3 is not clear, but in the presence of neutrophils, they have non-overlapping functions with their TRR2 and TRX2 paralogues.

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Introduction: Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. Methods: To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. Results: MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Conclusion: Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.