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During land plant evolution, the number of genes encoding for components of the thiol redox regulatory network and the generator systems of reactive oxygen species (ROS) expanded, tentatively indicating that they have a role in tailored environmental acclimatization. This hypothesis has been validated both experimentally and theoretically during the last few decades. Recent developments of dynamic redox-sensitive GFP (roGFP)-based in vivo sensors for H2O2 and the redox potential of the glutathione pool have paved the way for dissecting the kinetics changes that occur in these crucial parameters in response to environmental stressors. The versatile cellular redox sensory and response regulatory system monitors alterations in redox metabolism and controls the activity of redox target proteins, and thereby affects most, if not all, cellular processes ranging from transcription to translation and metabolism. This review uses examples to describe the role of the redox- and ROS-dependent regulatory network in realising the appropriate responses to diverse environmental stresses. The selected case studies concern different environmental challenges, namely excess excitation energy, the heavy metal cadmium and the metalloid arsenic, nitrogen or phosphate shortages as examples for nutrient deficiency, wounding, and nematode infestation. Each challenge affects the redox-regulatory and ROS network, but our present state of knowledge also points toward pressing questions that remain open in relation to the translation of redox regulation to environmental acclimatization. [ABSTRACT FROM AUTHOR]

Cysteine thiols are susceptible to various oxidative posttranslational modifications (PTMs) due to their high chemical reactivity. Thiol‐based PTMs play a crucial role in regulating protein functions and are key contributors to cellular redox signaling. Although reversible thiol‐based PTMs, such as disulfide bond formation, S‐nitrosylation, and S‐glutathionylation, have been extensively studied for their roles in redox regulation, thiol sulfinic acid (–SO2H) modification is often perceived as irreversible and of marginal significance in redox signaling. Here, we revisit this narrow perspective and shed light on the redox regulatory roles of –SO2H in plant stress signaling. We provide an overview of protein sulfinylation in plants, delving into the roles of hydrogen peroxide‐mediated and plant cysteine oxidase‐catalyzed formation of –SO2H, highlighting the involvement of –SO2H in specific regulatory signaling pathways. Additionally, we compile the existing knowledge of the –SO2H reducing enzyme, sulfiredoxin, offering insights into its molecular mechanisms and biological relevance. We further summarize current proteomic techniques for detecting –SO2H and furnish a list of experimentally validated cysteine –SO2H sites across various species, discussing their functional consequences. This review aims to spark new insights and discussions that lead to further investigations into the functional significance of protein –SO2H‐based redox signaling in plants. Summary statement: This review expands upon the conventional understanding of cysteine thiol sulfinic acid modification and provides an updated overview of its redox‐dependent regulatory functions in the context of plant stress signaling pathways. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism. Using redox phenylarsine oxide (PAO)-affinity chromatography, we report that PAO-binding vicinal thiols on creatine kinase B and alpha-enolase from healthy rat brains were preferentially oxidized compared to other selected proteins, including neuron-specific (gamma) enolase, under conditions designed to trap in vivo protein thiol redox states. Moreover, measures of the extents of oxidations of vicinal thiols on total protein, and on creatine kinase B and alpha-enolase, showed that vicinal thiol-linked redox states were stable over the lifespan of rats and revealed a transient reductive shift in these redox couples following decapitation-induced global ischemia. Finally, formation of disulfide-linked complexes between peroxiredoxin-2 and brain proteins was demonstrated on redox blots, supporting a link between protein vicinal thiol redox states and the peroxidase activities of peroxiredoxins. The implications of these findings with respect to underappreciated aspects of brain redox metabolism in health, aging, and ischemia are discussed. [ABSTRACT FROM AUTHOR]

Osteoporosis is the most common metabolic bone disease worldwide. The decrease in bone mass is primarily accompanied by a decrease in the number and activity of osteoblasts. Peroxiredoxins (PRDXs) are proteins that detect extremely low peroxide levels and act as sensors that regulate oxidation signals, thereby regulating various cellular functions. This study aimed to evaluate the effects of PRDX1 and estrogen on the biological behavior of osteoblasts, including their proliferation and differentiation. Ovariectomized (OVX) mice were used to establish a model of osteoporosis and perform morphological and immunohistochemical analyses. Prdx1 gene knockout and overexpression were performed in mouse MC3T3-E1 pre-osteoblasts to assess proliferation and osteogenic differentiation using the cell counting kit-8, quantitative reverse transcription polymerase chain reaction, western blotting (WB), Alizarin Red S staining, etc. The OVX mice exhibited osteoporosis and PRDX1 expression increased. In vitro experiments showed that during the osteogenic differentiation of osteoblasts, PRDX1 expression decreased, while the expression of COL1 and RUNX2 increased. After Prdx1 knockout, the proliferation of osteoblasts decreased; expression of Runx2, ALP, and COL1 increased; and mineralization increased. However, after Prdx1 overexpression, osteoblast proliferation was enhanced, whereas osteogenic differentiation and mineralization were inhibited. Estrogen inhibits the H 2 O 2 -induced decrease in osteoblastic differentiation and increase in PRDX1 expression. WB revealed that when LY294002 inhibited the AKT signaling pathway, the levels of p -AKT1, p-P65, and PRDX1 protein in MC3T3-E1 cells decreased. However, when pyrrolidine dithiocarbamate (PDTC) inhibited the NF-κB signaling pathway, the expression of p -AKT1 and PRDX1 did not change except for a significant reduction of p-P65 expression. Furthermore, PDTC reversed the decreased expression of RUNX2, ALP, and COL1 caused by PRDX1 overexpression. PRDX1 promotes the proliferation of osteoblasts and inhibits osteogenic differentiation. Estrogen regulated osteoblastic differentiation by affecting the expression of PRDX1 in osteoblasts, and the effect is related to the AKT1/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Climate change, which causes periods with relatively high temperatures in winter in Poland, can lead to a shortening or interruption of the cold hardening of crops. Previous research indicates that cold acclimation is of key importance in the process of acquiring cereal tolerance to stress factors. The objective of this work was to verify the hypothesis that both natural temperature fluctuations and the plant genotype influence the content of metabolites as well as proteins, including antioxidant enzymes and photosystem proteins. The research material involved four winter triticale genotypes, differing in their tolerance to stress under controlled conditions. The values of chlorophyll a fluorescence parameters and antioxidant activity were measured in their seedlings. Subsequently, the contribution of selected proteins was verified using specific antibodies. In parallel, the profiling of the contents of chlorophylls, carotenoids, phenolic compounds, and proteins was carried out by Raman spectroscopy. The obtained results indicate that a better PSII performance along with a higher photosystem II proteins content and thioredoxin reductase abundance were accompanied by a higher antioxidant activity in the field-grown triticale seedlings. The Raman studies showed that the cold hardening led to a variation in photosynthetic dyes and an increase in the phenolic to carotenoids ratio in all DH lines. [ABSTRACT FROM AUTHOR]

Abstract Peroxiredoxin 3 (PRDX3), a mitochondrial hydrogen peroxide scavenger, is known to be upregulated during tumorigenesis and cancer progression. In this study, we provide evidence for the first time that PRDX3 could regulate cellular signaling pathways associated with Matrix Metalloproteinase-1 (MMP-1) expression and activity in breast cancer progression. We show that shRNA-mediated gene silencing of PRDX3 inhibits cell migration and invasion in two triple-negative breast cancer cell lines. Reciprocal experiments show that PRDX3 overexpression promotes invasion and migration of the cancer cells, processes which are important in the metastatic cascade. Notably, this phenomenon may be attributed to the activation of MMP-1, which is observed to be upregulated by PRDX3 in the breast cancer cells. Moreover, immunohistochemical staining of breast cancer tissues revealed a positive correlation between PRDX3 and MMP-1 expression in both epithelial and stromal parts of the tissues. Further pathway reporter array and luciferase assay demonstrated that activation of ERK signaling is responsible for the transcriptional activation of MMP-1 in PRDX3-overexpressed cells. These findings suggest that PRDX3 could mediate cancer spread via ERK-mediated activation of MMP-1. Targeted inhibition of ERK signaling may be able to inhibit tumor metastasis in triple-negative breast cancer.

Background/Aim: Cisplatin [cis-diamminedichloroplatinum(II), CDDP] is a widely used and effective antitumor drug in clinical settings, notorious for its nephrotoxic side effects. This study investigated the mechanisms of CDDPinduced damage in African green monkey kidney (Vero) cells, with a focus on the role of Peroxiredoxin I (Prx I) and Peroxiredoxin II (Prx II) of the peroxiredoxin (Prx) family, which scavenge reactive oxygen species (ROS). Materials and Methods: We utilized the Vero cell line derived from African green monkey kidneys and exposed these cells to various concentrations of CDDP. Cell viability, apoptosis, ROS levels, and mitochondrial membrane potential were assessed. Results: CDDP significantly compromised Vero cell viability by elevating both cellular and mitochondrial ROS, which led to increased apoptosis. Pretreatment with the ROS scavenger Nacetyl-L-cysteine (NAC) effectively reduced CDDP-induced ROS accumulation and subsequent cell apoptosis. Furthermore, CDDP reduced Prx I and Prx II levels in a doseand time-dependent manner. The inhibition of Prx I and II exacerbated cell death, implicating their role in CDDPinduced accumulation of cellular ROS. Additionally, CDDP enhanced the phosphorylation of MAPKs (p38, ERK, and JNK) without affecting AKT. The inhibition of these pathways significantly attenuated CDDP-induced apoptosis. Conclusion: The study highlights the involvement of Prx proteins in CDDPinduced nephrotoxicity and emphasizes the central role of ROS in cell death mediation. These insights offer promising avenues for developing clinical interventions to mitigate the nephrotoxic effects of CDDP. [ABSTRACT FROM AUTHOR]

Ferroptosis, a unique type of non-apoptotic cell death resulting from iron-dependent lipid peroxidation, has a potential physiological function in tumor suppression, but its underlying mechanisms have not been fully elucidated. Here, we report that the long non-coding RNA (lncRNA) LncFASA increases the susceptibility of triple-negative breast cancer (TNBC) to ferroptosis. As a tumor suppressor, LncFASA drives the formation of droplets containing peroxiredoxin1 (PRDX1), a member of the peroxidase family, resulting in the accumulation of lipid peroxidation via the SLC7A11-GPX4 axis. Mechanistically, LncFASA directly binds to the Ahpc-TSA domain of PRDX1, inhibiting its peroxidase activity by driving liquid-liquid phase separation, which disrupts intracellular ROS homeostasis. Notably, high LncFASA expression indicates favorable overall survival in individuals with breast cancer, and LncFASA impairs the growth of breast xenograft tumors by modulating ferroptosis. Together, our findings illustrate the crucial role of this lncRNA in ferroptosis-mediated cancer development and provide new insights into therapeutic strategies for breast cancer. [ABSTRACT FROM AUTHOR]

Oxidative stress is implicated in the development and progression of type 2 diabetes (T2D). Peroxiredoxin-4 is an antioxidant protein, which may serve as biomarker of oxidative stress, and has previously been associated with new-onset T2D. In this study, we investigated associations between circulating peroxiredoxin-4 and the risk of developing new-onset microvascular complications in T2D patients. Serum peroxiredoxin-4 was measured in 536 patients with T2D with (n = 257) and without (n = 279) baseline microvascular complications who participated in a primary-care based cohort study (Zwolle Outpatient Diabetes project Integrating Available Care [ZODIAC] study). Over a median follow-up of 3.4 years, 38 (13.6%) developed nephropathy, defined as albuminuria in two consecutive urine samples. In multivariable Cox proportional hazards regression analyses, peroxiredoxin-4 was associated with new-onset nephropathy (hazard ratio [HR] per doubling 1.78 [95% CI: 1.27–2.49], P < 0.001) after adjustment for potential confounding factors, including age, sex, disease duration, HbA1c levels, macrovascular complications, systolic blood pressure, and even high-sensitive C-reactive protein. There was no interaction of peroxiredoxin-4 with hs-CRP impacting on new-onset nephropathy. No significant associations were found with new-onset retinopathy or neuropathy. In conclusion, circulating peroxiredoxin-4 associates positively with an increased risk of developing nephropathy in T2D independent and irrespective of low-grade inflammation. [Display omitted] • Circulating peroxiredoxin-4 levels may represent a systemic biomarker of oxidative stress. • High serum peroxiredoxin-4 levels associate with the development of diabetic nephropathy. • Serum peroxiredoxin-4 levels and new-onset diabetic nephropathy associated independently of low-grade inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Prdx2 is a peroxiredoxin (Prx) family protein that protects cells from attack via reactive oxygen species (ROS), and it has an important role in improving the resistance and scavenging capacity of ROS in fungi. Arthrobotrys oligospora is a widespread nematode-trapping fungus that can produce three-dimensional nets to capture and kill nematodes. In this study, AoPrdx2, a homologous protein of Prx5, was investigated in A. oligospora via gene disruption, phenotypic analysis, and metabolomics. The deletion of Aoprdx2 resulted in an increase in the number of mycelial septa and a reduction in the number of nuclei and spore yield. Meanwhile, the absence of Aoprdx2 increased sensitivity to oxidative stresses, whereas the ∆Aoprdx2 mutant strain resulted in higher ROS levels than that of the wild-type (WT) strain. In particular, the inactivation of Aoprdx2 severely influenced trap formation and pathogenicity; the number of traps produced by the ∆Aoprdx2 mutant strain was remarkably reduced and the number of mycelial rings of traps in the ∆Aoprdx2 mutant strain was less than that of the WT strain. In addition, the abundance of metabolites in the ∆Aoprdx2 mutant strain was significantly downregulated compared with the WT strain. These results indicate that AoPrdx2 plays an indispensable role in the scavenging of ROS, trap morphogenesis, and secondary metabolism. [ABSTRACT FROM AUTHOR]

The immunomodulatory potential of the excretory‐secretory (E/S) proteins of the helminths has been shown in previous investigations. This study evaluated the effects of the recombinants and excretory‐secretory proteins of the Fasciola hepatica on induced colitis in Balb/c mice. The F. hepatica Recombinant proteins, Cathepsin L1 and Peroxiredoxin, and E/S proteins were intraperitoneally injected into the three mice groups as the case groups, while the control groups received PBS. Colitis was induced in mice by intraluminal administration of the 2, 4, 6‐Trinitrobenzenesulfonic acid solution (TNBS). After 8 h, the case groups received the second dosage of the treatments, and it was repeated 24 h later. The immunological, pathological, and macroscopic changes were evaluated 3 days after colitis induction. The macroscopic evaluation revealed significantly lower inflammatory scores in the mice treated with recombinant Peroxiredoxin (rPRX) and recombinant Cathepsin L1 (rCL1). Despite the macroscopic observation, the pathological finding was insignificant between the groups. IFN‐γ secretion was significantly lower in splenocytes of the groups that received rPRX, rCL1, and E/S than the controls. IL‐10 showed significantly higher levels in groups treated with rPRX and rCL1 than controls, whereas the level of IL‐4 was not statistically significant. Excretory‐secretory proteins of the F. hepatica showed immunomodulatory potency and the main effects observed in this study were through the reduction of inflammatory cytokine and inflammation manifestation as well as induction of anti‐inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are abundant and ubiquitous proteins that participate in different cellular functions, such as oxidant detoxification, protein folding, and intracellular signaling. Under different cellular conditions, peroxiredoxins can be secreted by different parasites, promoting the induction of immune responses in hosts. In this work, we demonstrated that the cytosolic tryparedoxin peroxidase of Trypanosoma cruzi (cTXNPx) is secreted by epimastigotes and trypomastigotes associated with extracellular vesicles and also as a vesicle-free protein. By confocal microscopy, we show that cTXNPx can enter host cells by an active mechanism both through vesicles and as a recombinant protein. Transcriptomic analysis revealed that cTXNPx induces endoplasmic reticulum stress and interleukin-8 expression in epithelial cells. This analysis also suggested alterations in cholesterol metabolism in cTXNPx-treated cells, which was confirmed by immunofluorescence showing the accumulation of LDL and the induction of LDL receptors in both epithelial cells and macrophages. BrdU incorporation assays and qPCR showed that cTXNPx has a mitogenic, proliferative, and proinflammatory effect on these cells in a dose–dependent manner. Importantly, we also demonstrated that cTXNPx acts as a paracrine virulence factor, increasing the susceptibility to infection in cTXNPx-pretreated epithelial cells by approximately 40%. Although the results presented in this work are from in vitro studies and likely underestimate the complexity of parasite–host interactions, our work suggests a relevant role for this protein in establishing infection. [ABSTRACT FROM AUTHOR]

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2 in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

Schwann cells and oligodendrocytes secrete proteins that promote neuron survival, but their role in amyotrophic lateral sclerosis (ALS) is unclear. To address this question, we evaluated the effect of molecules secreted by Schwann cells on reactive oxygen species (ROS)-induced motor neuronal cell death. We observed that in motor neuron cell line NSC-34 cultures, the conditioned medium (CM) from Schwann cell line YST-1 (YST-1 CM) cultures had a protective effect against hydrogen peroxide-induced cell death. However, this protective effect of YST-1 CM was abolished by removing peroxiredoxin 1–4 (PRDX1–4) from the CM. We found that the expression of PRDX1 mRNA was markedly downregulated in the lumbar spinal cord of the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We also found that transient transfection of YST-1 cells with G93A SOD1 resulted in reduced PRDX1 mRNA expression. Additionally, in the mutant transfected cells, YST-1 CM showed decreased neuroprotective effect against hydrogen peroxide-induced NSC-34 cell death compared to those transfected with WT SOD1. Our results suggest that Schwann cells protect motor neurons from oxidative stress by secreting PRDX1 and that the reduction of PRDX secreted from Schwann cells contributes to increased ROS and associated motor neuronal death in ALS.

Thiol/disulfide‐based redox regulation in plant chloroplasts is essential for controlling the activity of target proteins in response to light signals. One of the examples of such a role in chloroplasts is the activity of the chloroplast ATP synthase (CFoCF1), which is regulated by the redox state of the CF1γ subunit and involves two cysteines in its central domain. To investigate the mechanism underlying the oxidation of CF1γ and other chloroplast redox‐regulated enzymes in the dark, we characterized the Arabidopsis cbsx2 mutant, which was isolated based on its altered NPQ (non‐photochemical quenching) induction upon illumination. Whereas in dark‐adapted WT plants CF1γ was completely oxidized, a small amount of CF1γ remained in the reduced state in cbsx2 under the same conditions. In this mutant, reduction of CF1γ was not affected in the light, but its oxidation was less efficient during a transition from light to darkness. The redox states of the Calvin cycle enzymes FBPase and SBPase in cbsx2 were similar to those of CF1γ during light/dark transitions. Affinity purification and subsequent analysis by mass spectrometry showed that the components of the ferredoxin‐thioredoxin reductase/thioredoxin (FTR‐Trx) and NADPH‐dependent thioredoxin reductase (NTRC) systems as well as several 2‐Cys peroxiredoxins (Prxs) can be co‐purified with CBSX2. In addition to the thioredoxins, yeast two‐hybrid analysis showed that CBSX2 also interacts with NTRC. Taken together, our results suggest that CBSX2 participates in the oxidation of the chloroplast redox‐regulated enzymes in darkness, probably through regulation of the activity of chloroplast redox systems in vivo. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) cause damage to various cellular processes in almost all organisms, in particular photosynthetic organisms that depend on the electron transfer chain for CO2 fixation. However, the detoxifying process to mitigate ROS damage has not been studied intensively in microalgae. Here, we characterized the ROS detoxifying role of a bZIP transcription factor, BLZ8, in Chlamydomonas reinhardtii.To identify downstream targets of BLZ8, we carried out comparative genome‐wide transcriptomic profiling of BLZ8 OX and its parental CC‐4533 under oxidative stress conditions. Luciferase reporter activity assays and RT‐qPCR were performed to test whether BLZ8 regulates downstream genes. We performed an in silico functional gene network analysis and an in vivo immunoprecipitation assay to identify the interaction between downstream targets of BLZ8.Comparative transcriptomic analysis and RT‐qPCR revealed that overexpression of BLZ8 increased the expression levels of plastid peroxiredoxin1 (PRX1) and ferredoxin‐5 (FDX5) under oxidative stress conditions. BLZ8 alone could activate the transcriptional activity of FDX5 and required bZIP2 to activate transcriptional activity of PRX1. Functional gene network analysis using FDX5 and PRX1 orthologs in A. thaliana suggested that these two genes were functionally associated. Indeed, our immunoprecipitation assay revealed the physical interaction between PRX1 and FDX5. Furthermore, the complemented strain, fdx5 (FDX5), recovered growth retardation of the fdx5 mutant under oxidative stress conditions, indicating that FDX5 contributes to oxidative stress tolerance.These results suggest that BLZ8 activates PRX1 and FDX5 expression, resulting in the detoxification of ROS to confer oxidative stress tolerance in microalgae. [ABSTRACT FROM AUTHOR]

The transporter Str3 promotes heme import in Schizosaccharomyces pombe cells that lack the heme receptor Shu1 and are deficient in heme biosynthesis. Under microaerobic conditions, the peroxiredoxin Tpx1 acts as a heme scavenger within the Str3‐dependent pathway. Here, we show that Srx1, a sulfiredoxin known to interact with Tpx1, is essential for optimal growth in the presence of hemin. The expression of Srx1 is induced in response to low iron and repressed under iron repletion. Coimmunoprecipitation and bimolecular fluorescence complementation experiments show that Srx1 interacts with Str3. Although the interaction between Srx1 and Str3 is weakened, it is still observed in tpx1Δ mutant cells or when Str3 is coexpressed with a mutant form of Srx1 (mutD) that cannot bind Tpx1. Further analysis by absorbance spectroscopy and hemin‐agarose pull‐down assays confirms the binding of Srx1 to hemin, with an equilibrium constant value of 2.56 μM. To validate the Srx1‐hemin association, we utilize a Srx1 mutant (mutH) that fails to interact with hemin. Notably, when Srx1 binds to hemin, it partially shields hemin from degradation caused by hydrogen peroxide. Collectively, these findings elucidate an additional function of the sulfiredoxin Srx1, beyond its conventional role in oxidative stress defense. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) widely exist in organisms and can prevent oxidative damage. Here, the characterization and biological function of NdPrx3 from Neocaridina denticulata sinensis were analyzed. The coding sequence of NdPrx3 consists of 684 bp open reading frame (ORF), encoding 227 amino acids with a predicted molecular weight of 24.7 kDa and theoretical pI 6.49. Multiple sequence alignments showed that the conserved domains of NdPrx3, including catalytic triad, dimer interface, decamer interface, peroxidatic, and resolving cysteines, were similar to those of other organisms. The phylogenetic relationship demonstrated that NdPrx3 clustered in the Prx3 class. The highest relative expression of NdPrx3 mRNA was confirmed in gill among the nine tissues from healthy shrimp. The transcript level of NdPrx3 was significantly upregulated from 0 h to 48 h and decreased in 72 h under copper challenge, indicating that NdPrx3 may play an important role in the copper challenge of N. denticulata sinensis. In addition, NdPrx3 was recombinantly expressed in E. coli and purified to one band on SDS-PAGE. The DNA protection of rNdPrx3 was verified. The enzymatic assay of the recombinant NdPrx3 indicated that it had the oxidoreductase function and was stable at a low temperature (10–30 °C).

Abstract Thiol/disulfide‐based redox regulation in plant chloroplasts is essential for controlling the activity of target proteins in response to light signals. One of the examples of such a role in chloroplasts is the activity of the chloroplast ATP synthase (CFoCF1), which is regulated by the redox state of the CF1γ subunit and involves two cysteines in its central domain. To investigate the mechanism underlying the oxidation of CF1γ and other chloroplast redox‐regulated enzymes in the dark, we characterized the Arabidopsis cbsx2 mutant, which was isolated based on its altered NPQ (non‐photochemical quenching) induction upon illumination. Whereas in dark‐adapted WT plants CF1γ was completely oxidized, a small amount of CF1γ remained in the reduced state in cbsx2 under the same conditions. In this mutant, reduction of CF1γ was not affected in the light, but its oxidation was less efficient during a transition from light to darkness. The redox states of the Calvin cycle enzymes FBPase and SBPase in cbsx2 were similar to those of CF1γ during light/dark transitions. Affinity purification and subsequent analysis by mass spectrometry showed that the components of the ferredoxin‐thioredoxin reductase/thioredoxin (FTR‐Trx) and NADPH‐dependent thioredoxin reductase (NTRC) systems as well as several 2‐Cys peroxiredoxins (Prxs) can be co‐purified with CBSX2. In addition to the thioredoxins, yeast two‐hybrid analysis showed that CBSX2 also interacts with NTRC. Taken together, our results suggest that CBSX2 participates in the oxidation of the chloroplast redox‐regulated enzymes in darkness, probably through regulation of the activity of chloroplast redox systems in vivo.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Cellular oxidation–reduction (redox) systems, which encompass pro‐ and antioxidant molecules, are integral components of a plethora of essential cellular processes. Any dysregulation of these systems can cause molecular imbalances between the pro‐ and antioxidant moieties, leading to a state of oxidative stress. Long‐lasting oxidative stress can manifest clinically as a variety of chronic illnesses including cancers, neurodegenerative disorders, cardiovascular disease, and metabolic diseases like diabetes. As such, this review investigates the impact of oxidative stress on the human body with emphasis on the underlying oxidants, mechanisms, and pathways. It also discusses the available antioxidant defense mechanisms. The cellular monitoring and regulatory systems that ensure a balanced oxidative cellular environment are detailed. We critically discuss the notion of oxidants as a double‐edged sword, being signaling messengers at low physiological concentrations but causative agents of oxidative stress when overproduced. In this regard, the review also presents strategies employed by oxidants including redox signaling and activation of transcriptional programs such as those mediated by the Nrf2/Keap1 and NFk signaling. Likewise, redox molecular switches of peroxiredoxin and DJ‐1 and the proteins they regulate are presented. The review concludes that a thorough comprehension of cellular redox systems is essential to develop the evolving field of redox medicine. [ABSTRACT FROM AUTHOR]

Peroxiredoxins play central roles in the detoxification of reactive oxygen species and have been modelled across multiple organisms using a variety of kinetic methods. However, the peroxiredoxin dimer-to-decamer transition has been underappreciated in these studies despite the 100-fold difference in activity between these forms. This is due to the lack of available kinetics and a theoretical framework for modelling this process. Using published isothermal titration calorimetry data, we obtained association and dissociation rate constants of 0.050 µM−4·s−1 and 0.055 s−1, respectively, for the dimer–decamer transition of human PRDX1. We developed an approach that greatly reduces the number of reactions and species needed to model the peroxiredoxin decamer oxidation cycle. Using these data, we simulated horse radish peroxidase competition and NADPH-oxidation linked assays and found that the dimer–decamer transition had an inhibition-like effect on peroxidase activity. Further, we incorporated this dimer–decamer topology and kinetics into a published and validated in vivo model of PRDX2 in the erythrocyte and found that it almost perfectly reconciled experimental and simulated responses of PRDX2 oxidation state to hydrogen peroxide insult. By accounting for the dimer–decamer transition of peroxiredoxins, we were able to resolve several discrepancies between experimental data and available kinetic models. [ABSTRACT FROM AUTHOR]

Compounds that cause oxidative stress have recently gained considerable interest as potential anticancer treatment modalities. Nevertheless, their efficiency may be diminished by the antioxidant systems often upregulated in cancer cells. Peroxiredoxins (PRDXs) are antioxidant enzymes that scavenge peroxides and contribute to redox homeostasis. They play a role in carcinogenesis and are upregulated in several cancer types. Here, we assessed the expression pattern of PRDX1 and PRDX2 in glioblastoma (GBM) and examined the efficacy of their inhibitors in GBM cell lines and patient-derived GBM cells. Both PRDX1 and PRDX2 were upregulated in GBM compared to non-tumor brain tissues and their considerable amounts were observed in GBM cells. Adenanthin, a compound inhibiting PRDX1 activity, slightly decreased GBM cell viability, while conoidin A (CONA), a covalent PRDX2 inhibitor, displayed high toxicity in GBM cells. CONA elevated the intracellular reactive oxygen species (ROS) level. Pre-treatment with an ROS scavenger protected cells from CONA-induced death, indicating that ROS accumulation plays a crucial role in this phenomenon. Menadione or celecoxib, both of which are ROS-inducing agents, potentiated the anticancer activity of CONA. Collectively, our results unveil PRDX1 and PRDX2 as potential targets for GBM therapy, and substantiate the further exploration of their inhibitors. [ABSTRACT FROM AUTHOR]

Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction–oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Our recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. We identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. Here, we found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, we demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) are autoimmune disorders of the gut. It is increasingly clear that voluntary exercise (VE) may exert protection against IBDs, but the exact background mechanism needs to be elucidated. In the present study, we aimed to investigate the possible role of NETosis and the antioxidant peroxiredoxin (Prdx) enzyme family in VE-induced protection. Wistar Han rats were randomly divided into two groups: sedentary (SED) and VE. After the 6-week voluntary wheel running, animals were treated with 2,4,6-trinitrobenzene sulphonic acid (TNBS) as a model of colitis. Here, we found that VE significantly decreased inflammation and ulceration of the colon in the VE TNBS group compared with SED TNBS. We also found that VE significantly decreased the expression of protein arginine deiminase 4 (PAD4) and myeloperoxidase (MPO), and markedly reduced citrullinated histone H3 (citH3) compared with SED TNBS. Furthermore, VE caused a significant increase in the levels of Prdx6 in the control and TNBS groups. Taken together, we found that a prior 6-week VE effectively reduces inflammation in TNBS-induced colitis, and we suggest that the protective effect of VE may be mediated via the inhibition of NETosis and upregulation of Prdx6 antioxidant. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Both catalase and peroxiredoxin show high activities of H2O2 decomposition and coexist in the same organism; however, their division of labor in defense against H2O2 is unclear. We focused on the major peroxiredoxin (PrxA) and catalase (CatB) in Aspergillus nidulans at different growth stages to discriminate their antioxidant roles. The dormant conidia lacking PrxA showed sensitivity to high concentrations of H2O2 (>100 mM), revealing that PrxA is one of the important antioxidants in dormant conidia. Once the conidia began to swell and germinate, or further develop to young hyphae (9 h to old age), PrxA-deficient cells (ΔprxA) did not survive on plates containing H2O2 concentrations higher than 1 mM, indicating that PrxA is an indispensable antioxidant in the early growth stage. During these early growth stages, absence of CatB did not affect fungal resistance to either high (>1 mM) or low (<1 mM) concentrations of H2O2. In the mature hyphae stage (24 h to old age), however, CatB fulfills the major antioxidant function, especially against high doses of H2O2. PrxA is constitutively expressed throughout the lifespan, whereas CatB levels are low in the early growth stage of the cells developing from swelling conidia to early growth hyphae, providing a molecular basis for their different contributions to H2O2 resistance in different growth stages. Further enzyme activity and cellular localization analysis indicated that CatB needs to be secreted to be functionalized, and this process is confined to the growth stage of mature hyphae. Our results revealed differences in effectiveness and timelines of two primary anti-H2O2 enzymes in fungus. [ABSTRACT FROM AUTHOR]

Objective: To determine whether the prokaryotic expression product of the red rice OsPrx3 gene, which encodes a peroxiredoxin, can enhance oxidative stress resistance in animals. Methods: OsPrx3 was cloned and expressed in Escherichia coli. After ingestion of the target protein, the animal model Caenorhabditis elega was evaluated for its motility, body length, oxidative stress resistance, reactive oxygen species (ROS) and fat contents, and oxidation-related gene expression levels. Results: This gene was cloned and expressed in E. coli successfully, and the purified expressed protein had high antioxidant activity, which at 1 μg/mL scavenged more than 40% of H2O2 in five minutes. Ingestion of the target protein significantly increased the resistance to oxidative stress within 15 minutes after oxidative treatment and reduced ROS and cellular lipid content in C. elegans compared to the control group, although there were no significant changes in motility or body length. Additionally, the expression levels of several critical antioxidant genes were evidently increased in the treated C. elegans. Conclusion: The OsPrx3 protein had high in vitro antioxidant potency, and when ingested by C. elegans, significantly enhanced the resistance to oxidative stress.

Celiac disease (CD) is an autoimmune enteropathy. Peroxiredoxins (PRDXs) are powerful antioxidant enzymes having an important role in significant cellular pathways including cell survival, apoptosis, and inflammation. This study aimed at investigating the expression levels of all PRDX isoforms (1–6) and their possible relationships with a transcription factor, HIF-1α, in the small intestinal tissue samples of pediatric CD patients. The study groups consisted of first-diagnosed CD patients (n = 7) and non-CD patients with functional gastrointestinal tract disorders as the controls (n = 7). The PRDXs and HIF-1α expression levels were determined by using real-time PCR and Western blotting in duodenal biopsy samples. It was observed that the mRNA and protein expression levels of PRDX 5 were significantly higher in the CD patients, whereas the PRDX 1, -2, and -4 expressions were decreased in each case compared to the control group. No significant differences were detected in the PRDX 3 and PRDX 6 expressions. The expression of HIF-1α was also significantly elevated in CD patients. These findings indicate, for the first time, that PRDXs, particularly PRDX 5, may play a significant role in the pathogenesis of CD. Furthermore, our results suggest that HIF-1α may upregulate PRDX-5 transcription in the duodenal tissue of CD.

Prdx2 is a peroxiredoxin (Prx) family protein that protects cells from attack via reactive oxygen species (ROS), and it has an important role in improving the resistance and scavenging capacity of ROS in fungi. Arthrobotrys oligospora is a widespread nematode-trapping fungus that can produce three-dimensional nets to capture and kill nematodes. In this study, AoPrdx2, a homologous protein of Prx5, was investigated in A. oligospora via gene disruption, phenotypic analysis, and metabolomics. The deletion of Aoprdx2 resulted in an increase in the number of mycelial septa and a reduction in the number of nuclei and spore yield. Meanwhile, the absence of Aoprdx2 increased sensitivity to oxidative stresses, whereas the ∆Aoprdx2 mutant strain resulted in higher ROS levels than that of the wild-type (WT) strain. In particular, the inactivation of Aoprdx2 severely influenced trap formation and pathogenicity; the number of traps produced by the ∆Aoprdx2 mutant strain was remarkably reduced and the number of mycelial rings of traps in the ∆Aoprdx2 mutant strain was less than that of the WT strain. In addition, the abundance of metabolites in the ∆Aoprdx2 mutant strain was significantly downregulated compared with the WT strain. These results indicate that AoPrdx2 plays an indispensable role in the scavenging of ROS, trap morphogenesis, and secondary metabolism.

Peroxiredoxins are abundant and ubiquitous proteins that participate in different cellular functions, such as oxidant detoxification, protein folding, and intracellular signaling. Under different cellular conditions, peroxiredoxins can be secreted by different parasites, promoting the induction of immune responses in hosts. In this work, we demonstrated that the cytosolic tryparedoxin peroxidase of Trypanosoma cruzi (cTXNPx) is secreted by epimastigotes and trypomastigotes associated with extracellular vesicles and also as a vesicle-free protein. By confocal microscopy, we show that cTXNPx can enter host cells by an active mechanism both through vesicles and as a recombinant protein. Transcriptomic analysis revealed that cTXNPx induces endoplasmic reticulum stress and interleukin-8 expression in epithelial cells. This analysis also suggested alterations in cholesterol metabolism in cTXNPx-treated cells, which was confirmed by immunofluorescence showing the accumulation of LDL and the induction of LDL receptors in both epithelial cells and macrophages. BrdU incorporation assays and qPCR showed that cTXNPx has a mitogenic, proliferative, and proinflammatory effect on these cells in a dose–dependent manner. Importantly, we also demonstrated that cTXNPx acts as a paracrine virulence factor, increasing the susceptibility to infection in cTXNPx-pretreated epithelial cells by approximately 40%. Although the results presented in this work are from in vitro studies and likely underestimate the complexity of parasite–host interactions, our work suggests a relevant role for this protein in establishing infection.

Asthenospermia is a common cause of male infertility refers to semen samples with spermatozoa that move slowly or immotile. Recent research has implicated oxidative stress-induced ferroptosis in asthenospermia's pathophysiology. Peroxiredoxins are important players in the antioxidant defense to protect cells against oxidative stress. However, some aspects of the antioxidant response necessary for male fertility are not well understood. This case-control study aimed to elucidate the role of peroxiredoxins in regulating oxidative stress in male fertility via their correlation with ferroptosis. It included 90 fertile and 90 asthenospermic subfertile males from Hilla City, Iraq. Total peroxiredoxin activity, peroxiredoxin-6 level, and peroxiredoxin-4 level were measured alongside the ferroptosis biomarkers glutathione peroxidase-4, malondialdehyde, and the reduced/oxidized protein thiol ratio. Infertile males had significantly higher oxidized thiol and malondialdehyde levels than fertile males (p

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy.

Hydrogen peroxide is the main metabolite effective in redox regulation and it is considered an insulinomimetic agent, with insulin signalling being essential for normal mitochondrial function in cardiomyocytes. Therefore, the aim of this work was to deeply analyse the heart mitochondrial H 2 O 2 metabolism, in the early stage of type 1 diabetes. Diabetes was induced by Streptozotocin (STZ, single dose, 60 mg × kg−1, ip.) in male Wistar rats and the animals were sacrificed 10 days after injection. Mitochondrial membrane potential and ATP production, using malate-glutamate as substrates, in the heart of diabetic animals were like the ones observed in control group. Mn-SOD activity was lower (15%) in the heart of diabetic rats even though its expression was increased (29%). The increment in heart mitochondrial H 2 O 2 production (117%) in diabetic animals was accompanied by an enhancement in the activities and expressions of glutathione peroxidase (26% and 42%) and of catalase (200% and 133%), with no changes in the peroxiredoxin activity, leading to [H 2 O 2 ] ss ∼40 nM. Heart mitochondrial lipid peroxidation and protein nitration were higher in STZ-injected animals (45% and 42%) than in control group. The mitochondrial membrane potential and ATP production preservation suggest the absence of irreversible damage at this early stage of diabetes 1. The increase in mitochondrial [H 2 O 2 ] ss above the physiological range, but still below supraphysiological concentration (∼100 nM) seems to be part of the adaptive response triggered in cardiomyocytes due to the absence of insulin. The signs of mitochondrial dysfunction observed in this very early stage of diabetes are consistent with the mitochondrial entity called ″complex I syndrome″. [Display omitted] • Cardiac mitochondria exhibit "complex I syndrome" after 7 days of hyperglycemia. • An adaptive response is triggered in heart mitochondria due to lack of insulin. • Regulation of heart mitochondrial H 2 O 2 metabolism is a central event in early DM 1. • [H 2 O 2 ] ss in the heart mitochondrial matrix is controlled by GPx and Prx systems. • Heart mitochondrial ΔΨ and ATP generation are preserved 10 days post-STZ injection. [ABSTRACT FROM AUTHOR]

Simple Summary: Reactive oxygen species (ROS) are unstable derivatives of oxygen that are generated naturally as products or biproducts of biological reactions. ROS, such as hydrogen peroxide, can be utilized for defense against infectious agents and as messengers in the biological system. However, excess ROS can damage DNA, lipids, and proteins, possibly inactivating or altering their functions. The molecules utilized by the body to stop production of ROS, to neutralize excess ROS, or to repair damage caused by ROS are known as antioxidants. Peroxiredoxins (Prxs) are one of the antioxidant enzymes expressed in species from bacteria to humans that protect cells against ROS. In recent years, great progress has been made in understanding the role of Prxs in normal physiology and in different diseases. In this review, we have briefly summarized the recent findings regarding Prxs in cancer. Peroxiredoxins (Prxs) are antioxidant enzymes with ubiquitous expression in human tissues. Prxs are expressed in archaea, bacteria, and eukaryota, often in multiple isoforms. Because of their abundant expression in different cellular organelles and extraordinary sensitivity to H2O2, Prxs are among the first defenses against oxidative stress. Prxs undergo reversible oxidation to disulfides, and some family members perform chaperone or phospholipase functions upon further oxidation. Prxs are upregulated in cancer cells. Research has suggested that Prxs can function as tumor promoters in various cancers. The major objective of this review is to summarize novel findings regarding the roles of Prxs in common cancer types. Prxs have been shown to influence differentiation of inflammatory cells and fibroblasts, remodeling of extracellular matrix, and regulation of stemness. Since aggressive cancer cells have higher intracellular levels of ROS that they can utilize to proliferate and metastasize compared to normal cells, it is critical that we understand the regulation and functions of primary antioxidants such as Prxs. These small but mighty proteins could prove to be key for improving cancer therapeutics and patient survival. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 2 (Prdx2) is the third most abundant erythrocyte protein. It was known previously as calpromotin since its binding to the membrane stimulates the calcium-dependent potassium channel. Prdx2 is present mostly in cytosol in the form of non-covalent dimers but may associate into doughnut-like decamers and other oligomers. Prdx2 reacts rapidly with hydrogen peroxide (k > 107 M−1 s−1). It is the main erythrocyte antioxidant that removes hydrogen peroxide formed endogenously by hemoglobin autoxidation. Prdx2 also reduces other peroxides including lipid, urate, amino acid, and protein hydroperoxides and peroxynitrite. Oxidized Prdx2 can be reduced at the expense of thioredoxin but also of other thiols, especially glutathione. Further reactions of Prdx2 with oxidants lead to hyperoxidation (formation of sulfinyl or sulfonyl derivatives of the peroxidative cysteine). The sulfinyl derivative can be reduced by sulfiredoxin. Circadian oscillations in the level of hyperoxidation of erythrocyte Prdx2 were reported. The protein can be subject to post-translational modifications; some of them, such as phosphorylation, nitration, and acetylation, increase its activity. Prdx2 can also act as a chaperone for hemoglobin and erythrocyte membrane proteins, especially during the maturation of erythrocyte precursors. The extent of Prdx2 oxidation is increased in various diseases and can be an index of oxidative stress. [ABSTRACT FROM AUTHOR]

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This protocol describes a detailed fluorometric method for measuring peroxiredoxin (Prx) enzyme activity in vitro. Peroxide dissociation is the rate-limiting step in the Prx-controlled enzymatic reaction. To prevent interference by the catalase enzyme, we developed a peroxiredoxin assay that measures Prx activity using the substrate tert-Butyl hydroperoxide (t-BOOH). Prx enzyme activity is measured by incubating the enzymatic substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in the presence of the desired volume of Prx enzyme. Next, the reagent N-(9-Acridinyl)maleimide (NAM) is used to stop the enzymatic reaction and form a fluorescent end product. Finally, Prx activity is measured by thiol fluorometry using a Box–Behnken design to optimize reaction conditions. This novel protocol was validated by evaluating Prx activity in matched samples against a reference assay. The correlation coefficient between our protocol and the reference assay was 0.9933, demonstrating its precision compared with existing methods. The NAM-Prx protocol instead uses t-BOOH as a substrate to measure Prx activity. Because catalase does not participate in the dissociation of t-BOOH, this approach does not require sodium azide. Furthermore, the method eliminates the need for concentrated acids to terminate the Prx enzymatic reaction since the NAM reagent can inhibit the enzymatic reaction regulated by the Prx enzyme. [ABSTRACT FROM AUTHOR]

Peroxiredoxin IV (Prx4), a typical two-cysteine-containing member of the peroxidase family, functions as an antioxidant to maintain cellular redox homeostasis through the reduction of reactive oxygen species (ROS) via cycles of oxidation–reduction reactions. Under oxidative stress, all Prxs including Prx4 are inactivated as their catalytic cysteines undergo hyperoxidation, and hyperoxidized two-cysteine Prxs can be exclusively repaired and revitalized through the reduction cycle catalyzed by sulfiredoxin (Srx). Previously, we showed that Prx4 is a preferred substrate of Srx, and knockout of Srx in mice leads to resistance to azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis. To further understand the significance of the Srx/Prx4 axis in colorectal cancer development, Prx4−/− mice were established and subjected to standard AOM/DSS protocol. Compared with wildtype littermates, mice with Prx4−/− genotype had significantly fewer and smaller tumors. Histopathological analysis revealed that loss of Prx4 leads to increased cell death through lipid peroxidation and lower infiltration of inflammatory cells in the knockout tumors compared to wildtype. Treatment with DSS alone also showed decreased infiltration of macrophages and lymphocytes in the colon of knockout mice, suggesting a role for Prx4 in inflammatory response. In addition, loss of Prx4 caused alterations in plasma cytokines and chemokines after DSS and AOM/DSS treatments. These findings suggest that loss of Prx4 protects mice from AOM/DSS-induced colon tumorigenesis. Thus, targeting Prx4 may provide novel strategies for colon cancer prevention and treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • OGD/R-induced astrocytes damaged and released PRDX6. • The release of PRDX6 aggravated the neuroapoptosis suffering OGD/R. • The mechanism underlying PRDX6 release could be linked to aiPLA2 activities. • PRDX6's pro-apoptotic role in neurons may be associated with RAGE and JNK signaling pathways. Peroxiredoxin-6 (PRDX6), a member of the peroxiredoxin family, has progressively emerged as a possible therapeutic target for a variety of brain diseases, particularly Alzheimer's disease and ischemic stroke. However, the role of PRDX6 in neurons under ischemic conditions has remained elusive. Here, we found that astrocytes could release PRDX6 extracellularly after OGD/R, and that PRDX6 release actually worsened neuroapoptosis under OGD/R. We discovered a unique PRDX6/RAGE/JNK signaling pathway that contributes to the effect of neuroapoptosis. We applied a specific inhibitor of the RAGE signaling pathway in a mouse MCAO model and observed significant alterations in animal behavior. Considered together, our findings show the crucial role of the astrocyte-released PRDX6 in the process of neuroapoptosis caused by OGD/R, and could provide novel insights for investigating the molecular mechanism of protecting brain function from ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Human peroxiredoxin 3 (HsPrx3) is a thiol-based peroxidase responsible for the reduction of most hydrogen peroxide and peroxynitrite formed in mitochondria. Mitochondrial disfunction can lead to membrane lipoperoxidation, resulting in the formation of lipid-bound fatty acid hydroperoxides (LFA-OOHs) which can be released to become free fatty acid hydroperoxides (fFA-OOHs). Herein, we report that HsPrx3 is oxidized and hyperoxidized by fFA-OOHs including those derived from arachidonic acid and eicosapentaenoic acid peroxidation at position 15 with remarkably high rate constants of oxidation (>3.5 × 107 M−1s−1) and hyperoxidation (~2 × 107 M−1s−1). The endoperoxide-hydroperoxide PGG2, an intermediate in prostanoid synthesis, oxidized HsPrx3 with a similar rate constant, but was less effective in causing hyperoxidation. Biophysical methodologies suggest that HsPrx3 can bind hydrophobic structures. Indeed, molecular dynamic simulations allowed the identification of a hydrophobic patch near the enzyme active site that can allocate the hydroperoxide group of fFA-OOHs in close proximity to the thiolate in the peroxidatic cysteine. Simulations performed using available and herein reported kinetic data indicate that HsPrx3 should be considered a main target for mitochondrial fFA-OOHs. Finally, kinetic simulation analysis support that mitochondrial fFA-OOHs formation fluxes in the range of nM/s are expected to contribute to HsPrx3 hyperoxidation, a modification that has been detected in vivo under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Cyanobacteria can perform both anoxygenic and oxygenic photosynthesis, a characteristic which ensured that these organisms were crucial in the evolution of the early Earth and the biosphere. Reactive oxygen species (ROS) produced in oxygenic photosynthesis and reactive sulfur species (RSS) produced in anoxygenic photosynthesis are closely related to intracellular redox equilibrium. ROS comprise superoxide anion (O2●−), hydrogen peroxide (H2O2), and hydroxyl radicals (●OH). RSS comprise H2S and sulfane sulfur (persulfide, polysulfide, and S8). Although the sensing mechanism for ROS in cyanobacteria has been explored, that of RSS has not been elucidated. Here, we studied the function of the transcriptional repressor PerR in RSS sensing in Synechococcus sp. PCC7002 (PCC7002). PerR was previously reported to sense ROS; however, our results revealed that it also participated in RSS sensing. PerR repressed the expression of prxI and downregulated the tolerance of PCC7002 to polysulfide (H2Sn). The reporter system indicated that PerR sensed H2Sn. Cys121 of the Cys4:Zn2+ site, which contains four cysteines (Cys121, Cys124, Cys160, and Cys163) bound to one zinc atom, could be modified by H2Sn to Cys121-SSH, as a result of which the zinc atom was released from the site. Moreover, Cys19 could also be modified by polysulfide to Cys19-SSH. Thus, our results reveal that PerR, a representative of the Cys4 zinc finger proteins, senses H2Sn. Our findings provide a new perspective to explore the adaptation strategy of cyanobacteria in Proterozoic and contemporary sulfurization oceans. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC), the most common type of lung cancer, etiologically associates with tobacco smoking which mechanistically contributes to oxidative stress to facilitate the occurrence of mutations, oncogenic transformation and aberrantly activated signaling pathways. Our previous reports suggested an essential role of Sulfiredoxin (Srx) in promoting the development of lung cancer in humans, and was causally related to Peroxiredoxin IV (Prx4), the major downstream substrate and mediator of Srx-enhanced signaling. To further explore the role of the Srx-Prx4 axis in de novo lung tumorigenesis, we established Prx4−/− and Srx−/−/Prx4−/− mice in pure FVB/N background. Together with wild-type litter mates, these mice were exposed to carcinogenic urethane and the development of lung tumorigenesis was evaluated. We found that disruption of the Srx-Prx4 axis, either through knockout of Srx/Prx4 alone or together, led to a reduced number and size of lung tumors in mice. Immunohistological studies found that loss of Srx/Prx4 led to reduced rate of cell proliferation and less intratumoral macrophage infiltration. Mechanistically, we found that exposure to urethane increased the levels of reactive oxygen species, activated the expression of and Prx4 in normal lung epithelial cells, while knockout of Prx4 inhibited urethane-induced cell transformation. Moreover, bioinformatics analysis found that the Srx-Prx4 axis is activated in many human cancers, and their increased expression is tightly correlated with poor prognosis in NSCLC patients. [ABSTRACT FROM AUTHOR]

The antioxidant activity and chemical stability of 6-amino-6-deoxy-L-ascorbic acid (D1) and N-methyl-6-amino-6-deoxy-L-ascorbic acid (D2) were examined with ABTS and DPPH assays and compared with the reference L-ascorbic acid (AA). In addition, the optimal storing conditions, as well as the pH at which the amino derivatives maintain stability, were determined using mass spectrometry. Comparable antioxidant activities were observed for NH-bioisosteres and AA. Moreover, D1 showed higher stability in an acidic medium than the parent AA. In addition, AA, D1, and D2 share the same docking profile, with wild-type human peroxiredoxin as a model system. Their docking scores are similar to those of dithiothreitol (DTT). This suggests a similar binding affinity to the human peroxiredoxin binding site. [ABSTRACT FROM AUTHOR]

The redox couple formed by NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (Prxs) allows fine-tuning chloroplast performance in response to light intensity changes. Accordingly, the Arabidopsis 2cpab mutant lacking 2-Cys Prxs shows growth inhibition and sensitivity to light stress. However, this mutant also shows defective post-germinative growth, suggesting a relevant role of plastid redox systems in seed development, which is so far unknown. To address this issue, we first analyzed the pattern of expression of NTRC and 2-Cys Prxs in developing seeds. Transgenic lines expressing GFP fusions of these proteins showed their expression in developing embryos, which was low at the globular stage and increased at heart and torpedo stages, coincident with embryo chloroplast differentiation, and confirmed the plastid localization of these enzymes. The 2cpab mutant produced white and abortive seeds, which contained lower and altered composition of fatty acids, thus showing the relevance of 2-Cys Prxs in embryogenesis. Most embryos of white and abortive seeds of the 2cpab mutant were arrested at heart and torpedo stages of embryogenesis suggesting an essential function of 2-Cys Prxs in embryo chloroplast differentiation. This phenotype was not recovered by a mutant version of 2-Cys Prx A replacing the peroxidatic Cys by Ser. Neither the lack nor the overexpression of NTRC had any effect on seed development indicating that the function of 2-Cys Prxs at these early stages of development is independent of NTRC, in clear contrast with the operation of these regulatory redox systems in leaves chloroplasts.