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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]

In the search for new leads capable of interacting with multiple targets involved in NDD pathogenesis, a series of pyrazine-linked isoxazoline scaffolds were designed, synthesized and evaluated for their acetylcholinesterase and tyrosinase inhibitory potency and antioxidant activity. Isoxazolines 4a, 4d and 4h exhibited better molecular interaction with cholinesterases, tyrosinases and peroxiredoxin enzymes. Isoxazolines 4a, 4d and 4h interacted with acetylcholinesterase with the highest docking score of −9.083, −8.68 and −7.87 kcal/mol, respectively. Compound 4h ranked top when interacting with butyrylcholinesterase with a docking score of −7.926 kcal/mol, followed by 4a (−6.327 kcal/mol). 4a exhibited a robust interaction with 1HD2 with a docking score of −3.103 kcal/mol followed by 4d and 4h. 4a, 4d and 4h exhibited better docking scores of −5.47 kcal/mol, −4.63 kcal/mol and −5.157 kcal/mol with the enzyme tyrosinase. Based on the in-silico data, we have proceeded further to synthesis and in-vitro studies. Chalcones were synthesized by the Claisen-Schmidt reaction, which was cyclised to isoxazolines by the cycloaddition of hydroxylamine HCl. FTIR, 1HNMR, 13CNMR, and mass spectral studies further characterized the compounds. The prediction of pharmacokinetic parameters also supports the study, and all the compounds passed the screening. In-vitro studies were performed to evaluate acetylcholinesterase and tyrosinase inhibition. Compound 4h displayed excellent action against acetylcholinesterases and tyrosinase enzymes. Hydrogen peroxide assay determined the antioxidant effect, which found that 4h and 4d compounds exhibited higher strength as peroxide scavengers. Thus, the study shows that pyrazine-based isoxazolines with electron-withdrawing groups can be used as leads to develop a drug of choice for NDD, as it has excellent acetylcholinesterase and tyrosinase inhibitory action and tremendous peroxide scavenging effect. [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]

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]

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]

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]

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]

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]

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]

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]

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]

[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]

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 temporal and spatial patterns of reactive oxygen species (ROS) in cells and tissues decisively determine the plant acclimation response to diverse abiotic and biotic stresses. Recent progress in developing dynamic cell imaging probes provides kinetic information on changes in parameters like H 2 O 2 , glutathione (GSH/GSSG) and NAD(P)H/NAD(P)+, that play a crucial role in tuning the cellular redox state. Central to redox-based regulation is the thiol-redox regulatory network of the cell that integrates reductive information from metabolism and oxidative ROS signals. Sensitive proteomics allow for monitoring changes in redox-related posttranslational modifications. Thiol peroxidases act as sensitive peroxide and redox sensors and play a central role in this signal transduction process. Peroxiredoxins (PRX) and glutathione peroxidases (GPX) are the two main thiol peroxidases and their function in ROS sensing and redox signaling in plants is emerging at present and summarized in this review. Depending on their redox state, PRXs and GPXs act as redox-dependent binding partners, direct oxidants of target proteins and oxidants of thiol redox transmitters that in turn oxidize target proteins. With their versatile functions, the multiple isoforms of plant thiol peroxidases play a central role in plant stress acclimation, e.g. to high light or osmotic stress, but also in ROS-mediated immunity and development. [Display omitted] • Thiol peroxidases play a crucial role in H 2 O 2 - and redox-dependent signaling. • Thiol peroxidases have five modes of action. Modes: tuning peroxide concentration, transmitter-dependent thiol oxidase, chaperone, covalent binder and contact thiol oxidase functions. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme, a unique member of the peroxiredoxin family, with an important role in antioxidant defense. Moreover, it has also been linked with the biosynthesis of anti-inflammatory and anti-diabetic lipids called fatty acid esters of hydroxy fatty acids (FAHFAs) and many diseases, including cancer, inflammation, and metabolic disorders. Here, we performed metabolomic and lipidomic profiling of subcutaneous adipose tissue from mouse models with genetically modified Prdx6. Deletion of Prdx6 resulted in reduced levels of FAHFAs containing 13-hydroxylinoleic acid (13-HLA). Mutation of Prdx6 C47S impaired the glutathione peroxidase activity and reduced FAHFA levels, while D140A mutation, responsible for phospholipase A2 activity, showed only minor effects. Targeted analysis of oxidized phospholipids and triacylglycerols in adipocytes highlighted a correlation between FAHFA and hydroxy fatty acid production by Prdx6 or glutathione peroxidase 4. FAHFA regioisomer abundance was negatively affected by the Prdx6 deletion, and this effect was more pronounced in longer and more unsaturated FAHFAs. The predicted protein model of Prdx6 suggested that the monomer-dimer transition mechanism might be involved in the repair of longer-chain peroxidized phospholipids bound over two monomers and that the role of Prdx6 in FAHFA synthesis might be restricted to branching positions further from carbon 9. In conclusion, our work linked the peroxidase activity of Prdx6 with the levels of FAHFAs in adipose tissue. [Display omitted] • Loss or mutation of Prdx6 alters subcutaneous adipose tissue lipidome in mice. • Peroxiredoxin 6 Cys47 is important for the unsaturated FAHFA synthesis pathway. • Prdx6 deletion affects position-specific FAHFA regioisomer abundance. • Prdx6 and Gpx4 are possibly involved in hydroxy-fatty acid synthesis for FAHFAs. • Prdx6 protein model supports phospholipid binding over two monomers. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) signaling via the accumulation of unfolded and misfolded proteins. ER stress leads to the production of reactive oxygen species (ROS), which are necessary to maintain redox homeostasis in the ER. Although peroxiredoxin 1 (Prx1) is an antioxidant enzyme that regulates intracellular ROS levels, the link between Prx1 and ER stress remains unclear. In this study, we investigated the role of Prx1 in X-box binding protein 1 (XBP-1) activation, the C/EBP homologous protein (CHOP) pathway, and apoptosis in response to ER stress. We observed that Prx1 overexpression inhibited the nuclear localization of XBP-1 and the expression of XBP-1 target genes and CHOP after thapsigargin (Tg) treatment to induce ER stress. In addition, Prx1 inhibited apoptosis and ROS production during ER stress. The ROS scavenger inhibited ER stress-induced apoptosis but did not affect XBP-1 activation and CHOP expression. Therefore, the biological role of Prx1 in ER stress may have important implications for ER stress-related diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanomas and the melanocytes from which they arise are subject to the damaging effects of reactive oxygen species (ROS) from exogenous and endogenous sources. Many attempts have been made to counteract these effects with antioxidant drugs and natural products. Here, we demonstrate that the antioxidant enzyme thioredoxin reductase-1 controls the stability and function of MITF, the master regulator of melanocytes and melanoma. We believe that understanding these phenomena will generate new melanoma treatment and prevention strategies that are far more effective than brute-force approaches that attempt to simply eliminate ROS from vulnerable tissues and tumors. TR1 and other selenoproteins have paradoxical effects in melanocytes and melanomas. Increasing selenoprotein activity with supplemental selenium in a mouse model of UV-induced melanoma prevents oxidative damage to melanocytes and delays melanoma tumor formation. However, TR1 itself is positively associated with progression in human melanomas and facilitates metastasis in melanoma xenografts. Here, we report that melanocytes expressing a microRNA directed against TR1 (TR1low) grow more slowly than control cell lines and contain significantly less melanin. This phenotype is associated with lower tyrosinase (TYR) activity and reduced transcription of tyrosinase-like protein-1 (TYRP1). Melanoma cells in which the TR1 gene (TXNRD1) was disrupted using Crispr/Cas9 showed more dramatic effects including the complete loss of the melanocyte-specific isoform of MITF; other MITF isoforms were unaffected. We provide evidence that TR1 depletion results in oxidation of MITF itself. This newly discovered mechanism for redox modification of MITF has profound implications for controlling both pigmentation and tumorigenesis in cells of the melanocyte lineage. [ABSTRACT FROM AUTHOR]

Peroxiredoxin IV (Prx4) is a 2-Cysteine peroxidase with ubiquitous expression in human tissues. Prx4 scavenges hydrogen peroxide and participates in oxidative protein folding in the endoplasmic reticulum. In addition, Prx4 is secreted outside the cell. Prx4 is upregulated in several cancers and is a potential therapeutic target. We have summarized historical and recent advances in the structure, function and biological roles of Prx4, focusing on inflammatory diseases and cancer. Oxidative stress is known to activate pro-inflammatory pathways. Chronic inflammation is a risk factor for cancer development. Hence, redox enzymes such as Prx4 are important players in the crosstalk between inflammation and cancer. Understanding molecular mechanisms of regulation of Prx4 expression and associated signaling pathways in normal physiological and disease conditions should reveal new therapeutic strategies. Thus, although Prx4 is a promising therapeutic target for inflammatory diseases and cancer, further research needs to be conducted to bridge the gap to clinical application. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) is an antioxidant protein that widely exists in various organisms. To further investigate the role of Prx in the antioxidant and immune responses of Neocaridina denticulata sinensis , the full-length cDNA sequence of a Prx gene (Nd-Prx) from N. denticulata sinensis was obtained. The open reading frame (ORF) of Nd-Prx is 597 bp and encodes 198 amino acids. Amino acid similarity alignment showed that Nd-Prx contained a conserved sequence region "FYPLDFTFVCPTEI". qRT-PCR assay showed that Nd-Prx was expressed in all tested tissues and its expression was highest in the ovary. Nd-Prx was most highly expressed at 36 h after copper stimulation. Nd-Prx expression levels in hepatopancreas were significantly upregulated after Vibrio parahaemolyticus challenge (P < 0.05). In addition, the recombinant Nd-Prx was prepared and its enzyme activity was most stable at 70 °C with pH of 6.0. The antioxidant activity and DNA protection of recombinant Nd-Prx was also demonstrated. In summary, this study investigated the role of Prx in antioxidant and immune responses of N. denticulata sinensis , which might provide a foundation for further exploring Prx in immune system of crustaceans and for the application in disease control. [Display omitted] • Nd-Prx gene was cloned and its recombinant protein was prepared to analyze its enzymatic activity. • Nd-Prx was expressed in all tested tissues. • Expression of Nd-Prx was elevated under copper exposure or V. parahaemolyticus challenge. • The recombinant Nd-Prx exhibited high reducibility. [ABSTRACT FROM AUTHOR]

Transmission of reductive and oxidative cues from the photosynthetic electron transport chain to redox regulatory protein networks plays a crucial role in coordinating photosynthetic activities. The tight balance between these two signals dictates the cellular response to changing light conditions. While the role of reductive signals in activating chloroplast metabolism is well established, the role of their counterbalanced oxidative signals is still unclear, mainly due to monitoring difficulties. Here, we introduced chlroGFP2-PrxΔCR, a 2-Cys peroxiredoxin-based biosensor, into Arabidopsis thaliana chloroplasts to monitor the dynamic changes in photosynthetically derived oxidative signaling. We showed that chl-roGFP2-PrxΔCR oxidation states reflected oxidation patterns similar to those of endogenous 2-Cys peroxiredoxin under varying light conditions. By employing a set of genetically encoded biosensors, we showed the induction of 2-Cys peroxiredoxin-dependent oxidative signals, throughout the day, under varying light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals. Furthermore, we demonstrated the induction of 2-Cys peroxiredoxin-derived oxidative signals during a dark--to--low-light transition and uncovered a faster increase in carbon assimilation rates during the photosynthesis induction phase in plants deficient in 2-Cys peroxiredoxins compared with wild type, suggesting the involvement of oxidative signals in attenuating photosynthesis. The presented data highlight the role of oxidative signals under nonstress conditions and suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system. [ABSTRACT FROM AUTHOR]

Oxidative stress has been implied in cellular injury even in the early phases of multiple sclerosis (MS). In this study, we quantified levels of biomarkers of oxidative stress and antioxidant capacity in cerebrospinal fluid (CSF) in newly diagnosed MS patients and their associations with brain atrophy and iron deposits in the brain tissue. Consecutive treatment-naive adult MS patients (n = 103) underwent brain MRI and CSF sampling. Healthy controls (HC, n = 99) had brain MRI. CSF controls (n = 45) consisted of patients with non-neuroinflammatory conditions. 3T MR included isotropic T1 weighted (MPRAGE) and gradient echo (GRE) images that were processed to quantitative susceptibility maps. The volume and magnetic susceptibility of deep gray matter (DGM) structures were calculated. The levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), 8-iso prostaglandin F2α (8-isoPG), neutrophil gelatinase-associated lipocalin (NGAL), peroxiredoxin-2 (PRDX2), and malondialdehyde and hydroxyalkenals (MDA + HAE) were measured in CSF. Compared to controls, MS patients had lower volumes of thalamus, pulvinar, and putamen, higher susceptibility in caudate nucleus and globus pallidus, and higher levels of 8-OHdG, PRDX2, and MDA + HAE. In MS patients, the level of NGAL correlated negatively with volume and susceptibility in the dentate nucleus. The level of 8-OHdG correlated negatively with susceptibility in the caudate, putamen, and the red nucleus. The level of PRDX2 correlated negatively with the volume of the thalamus and both with volume and susceptibility of the dentate nucleus. From MRI parameters with significant differences between MS and HC groups, only caudate susceptibility and thalamic volume were significantly associated with CSF parameters. Our study shows that increased oxidative stress in CSF detected in newly diagnosed MS patients suggests its role in the pathogenesis of MS. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are multifunctional enzymes that play a key role in protecting cells from stresses and maintaining the homeostasis of many cellular processes. Peroxiredoxins were firstly identified as antioxidant enzymes that can be found in all living organisms. Later studies demonstrated that peroxiredoxins also act as redox signaling regulators, chaperones, and proinflammatory factors and play important roles in oxidative defense, redox signaling, protein folding, cycle cell progression, DNA integrity, inflammation, and carcinogenesis. The versatility of peroxiredoxins is mainly based on their unique active center cysteine with a wide range of redox states and the ability to switch between low- and high-molecular-weight species for regulating their peroxidase and chaperone activities. Understanding the molecular mechanisms of peroxiredoxin in these processes will allow the development of new approaches to enhance longevity and to treat various cancers. In this article, we briefly review the history of peroxiredoxins, summarize recent advances in our understanding of peroxiredoxins in aging- and cancer-related biological processes, and discuss the future perspectives of using peroxiredoxins in disease diagnostics and treatments. [ABSTRACT FROM AUTHOR]

Neospora caninum, an apicomplexan parasite, is the etiological agent of neosporosis, a disease that leads to neurological symptoms in dogs and abortion in cattle. Vaccine or drug treatments for neosporosis remain to be determined. Therefore, it is of undeniable relevance to investigate new molecules involved in the parasite's successful survival within the host cell. The aim of this study was to characterize the N. caninum peroxiredoxin (NcPrx), an enzyme involved in the redox system of the parasite. The NcPrx amino acid sequence showed high identity and similarity compared to homologues representatives of Apicomplexa phylum. The recombinant NcPrx (rNcPrx) was cloned and expressed in Escherichia coli (BL21) with the predicted molecular weight (22 kDa), and the identity of monomer and dimer forms of rNcPrx was confirmed by mass spectrometry. Native and recombinant NcPrx were detected by ELISA and western blot, using the polyclonal anti-rNcPrx serum. Multiphoton analysis showed that NcPrx is localized in tachyzoite cytosol. H2O2 treatment increased the rNcPrx dimerization in vitro, and associated with the in silico data, we suggest that NcPrx belongs to typical 2-Cys Prx group (AhpC/Prx1 family). rNcPrx also increased the H2O2 clearance and protected plasmidial DNA under oxidative conditions. Finally, H2O2 increased the NcPrx dimerization in intracellular and extracellular tachyzoites suggesting that it is enrolled in H2O2 clearance and sensing in N. caninum. [ABSTRACT FROM AUTHOR]

Rotator cuff degeneration is one of the several factors that lead to rotator cuff tears. Oxidative stress and superoxide dismutase have been reported to be related to rotator cuff degeneration; however, the precise mechanism still remains unclear. In this study, we investigated the relationship of oxidative stress and superoxide dismutase to the degeneration of the rotator cuff using rat models. Eighty‐four rats were used to create a collagenase‐induced rotator cuff injury model (injury model) and a rotator cuff tear model (tear model). The controls were administered saline and had only a deltoid incision, respectively. We evaluated degeneration morphology of the rotator cuff using a degeneration score; dihydroethidium fluorescence intensity, which detects oxidative stress; gene expression; and superoxide dismutase activity. The rotator cuffs in the injury and tear models significantly increased degeneration scores and dihydroethidium fluorescence intensity. On the other hand, gene expression of superoxide dismutase isoform, superoxide dismutase 1, and superoxide dismutase activity were significantly decreased in the injury model but showed no significant difference in the tear model. These findings suggested that superoxide dismutase might not be associated with rotator cuff degeneration after tear but may be involved in degenerative rotator cuff without tear. However, we found that rotator cuff degeneration involves oxidative stress both with and without tear. Based on these findings, it is presumed that different treatments may be appropriate, depending on the state of rotator cuff degeneration, because the mechanisms of the degeneration may be different. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high‐affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria‐released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2‐mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions. Synopsis: Mitochondria are main producers of H2O2 in cells and its release is an important indicator of their functional state. Here, nutrients affecting mitochondrial metabolism and cytosolic antioxidative systems are found to modulate H2O2 gradients and signaling in intact human cells. Mitochondrial H2O2 can be detected in the cytosol in intact human cells, but not the nucleus or at the plasma membrane.Mitochondrial H2O2 gradients are steep and controlled by peroxiredoxins 1 and 2.Peroxiredoxins 1 and 2 complement each other and are present with a substantial reserve capacity.Metabolism‐induced changes of reducing processes control peroxiredoxin activity. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PHE), are common pollutants found in coastal areas where shrimp farming is developed. Even though PAHs can have adverse effects on physiology, shrimp can detoxify and metabolize toxic compounds and neutralize the reactive oxygen species (ROS) produced during this process. This requires the activation of multiple antioxidant enzymes, including peroxiredoxin 6 (Prx6). Prx6 uses glutathione (GSH) to reduce phospholipid hydroperoxides, a function shared with GSH peroxidase 4 (GPx4). Prx6 has been scarcely studied in crustaceans exposed to pollutants. Herein, we report a novel Prx6 from the shrimp Penaeus vannamei that is abundantly expressed in gills and hepatopancreas. To elucidate the involvement of Prx6 in response to PAHs, we analyzed its expression in the hepatopancreas of shrimp sub-lethally exposed to PHE (3.3 μg/L) and acetone (control) for 24, 48, 72, and 96 h, along with GPx4 expression, GSH-dependent peroxidase activity, and lipid peroxidation (indicated by TBARS). We found that GPx4 expression is not affected by PHE, but Prx6 expression and peroxidase activity decreased during the trial. This might contribute to the rise of TBARS found at 48 h of exposure. However, maintaining GPx4 expression could aid to minimize lipid damage during longer periods of exposure to PHE. [Display omitted] • A novel Prx6 from P. vannamei was cloned and characterized in silico. • Prx6 expression is higher in gills and hepatopancreas than in muscle. • Phenanthrene down-regulated Prx6 in hepatopancreas but did not affect GPx4. • GSH-dependent activity decreased in shrimp exposed to phenanthrene. • TBARS increased in hepatopancreas after 48 h of exposure to phenanthrene. [ABSTRACT FROM AUTHOR]

Neospora caninum, an obligate intracellular parasitic protozoan discovered by Dubey in 1988, is the pathogen of neosporosis, which causes neurological symptoms in dogs and abortions in cows. Since there is no effective drug or vaccine against N. caninum , a deeper understanding of the molecules critical to parasite survival inside host cells is necessary. This study aimed to determine the role of N. caninum peroxiredoxin 1 (NcPrx1) in maintaining redox homeostasis and virulence of N. caninum. By determining the localization of NcPrx1 protein and establishing NcPrx1 gene knockout strain (ΔNcPrx1), the roles of NcPrx1 in N. caninum for invasion, replication, growth, oxidative stress, as well as pathogenicity were investigated. Our results showed that a predicted Alkyl Hydroperoxide1 (AHP1) domain was found in the amino acid sequence of NcPrx1, which displayed a high degree of similarity to homologs of several protozoa. Immunofluorescence assay (IFA) indicated that NcPrx1 was a cytoplasmic protein in N. caninum tachyzoites. Compared to wild type (WT) strain, ΔNcPrx1 strain showed reduced plaque area, invasion and egress rates. Reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated, and total antioxidant capacity (T-AOC) was attenuated in ΔNcPrx1 tachyzoites, which indicated that ΔNcPrx1 strain was more sensitive to oxidative stress. Furthermore, ΔNcPrx1 strain-infected C57BL/6 mice showed improved survival rate, reduced parasite burden, alleviated pathological changes in tissues, and decreased secretions of IL-6, IL-12, TNF-α, and IFN-γ in serum compared to the WT strain group. These findings suggested that NcPrx1 was a virulence factor of N. caninum which played an important role in maintaining the redox homeostasis of the parasite. • NcPrx1 protein localized in the cytoplasm of Neospora caninum with Prx activity. • NcPrx1 deficiency impaired the growth, invasion, egression and antioxidant stress capacity of N. caninum. • Deletion of NcPrx1 attenuated the virulence of N. caninum. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanocytes are the pigment-producing cells in the skin that help to protect it against damaging solar ultraviolet radiation. As they engage in this function, they are subject to an inordinate amount of oxidative stress. To protect themselves, they utilize numerous antioxidant systems to reduce the amount of reactive oxygen and nitrogen species present in the cells, and this activity then contributes towards the prevention of cancer formation. However, after the formation of melanomas these same antioxidant systems are often co-opted by the cancer in order to promote its uncontrolled growth and metastasis. The purpose of this review is to highlight how the melanocyte's antioxidant systems, regulated by the transcription factor NRF2 and its targets, are co-opted in melanomas and, therefore, could be targeted for novel therapies to treat melanomas going forward. Melanocytes are dendritic, pigment-producing cells located in the skin and are responsible for its protection against the deleterious effects of solar ultraviolet radiation (UVR), which include DNA damage and elevated reactive oxygen species (ROS). They do so by synthesizing photoprotective melanin pigments and distributing them to adjacent skin cells (e.g., keratinocytes). However, melanocytes encounter a large burden of oxidative stress during this process, due to both exogenous and endogenous sources. Therefore, melanocytes employ numerous antioxidant defenses to protect themselves; these are largely regulated by the master stress response transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2). Key effector transcriptional targets of NRF2 include the components of the glutathione and thioredoxin antioxidant systems. Despite these defenses, melanocyte DNA often is subject to mutations that result in the dysregulation of the proliferative mitogen-activated protein kinase (MAPK) pathway and the cell cycle. Following tumor initiation, endogenous antioxidant systems are co-opted, a consequence of elevated oxidative stress caused by metabolic reprogramming, to establish an altered redox homeostasis. This altered redox homeostasis contributes to tumor progression and metastasis, while also complicating the application of exogenous antioxidant treatments. Further understanding of melanocyte redox homeostasis, in the presence or absence of disease, would contribute to the development of novel therapies to aid in the prevention and treatment of melanomas and other skin diseases [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a worldwide protozoan parasite that endangers human health and causes enormous economic losses to the animal production sector. A safe and effective vaccine or treatment is needed to reduce these hazards. In this study, we revealed the cyto-nuclear and mitochondrial localization of TgPrx1 and TgPrx3 proteins, respectively. We knocked out the T. gondii peroxiredoxin (TgPrxKO) 1 and 3 genes using a parental type II Prugniaud strain lacking KU80 and HXGPRT genes (PruΔku80Δhxgprt) via CRISPR-Cas9 technology. The successful KO was confirmed using PCR, IFAT, and Western blotting in two clones of both target genes, named TgPrx1KO and TgPrx3KO. Regarding in vitro assays, no significant variations between any of the knocked-out clones in TgPrx1KO or TgPrx3KO parasite strains, or even PruΔku80Δhxgprt, were obtained in rates of infection, proliferation, or egress. Nevertheless, mice that were infected with tachyzoites of the TgPrx3KO strain showed a marked decrease in survival rate compared with TgPrx1KO- and PruΔku80Δhxgprt-infected mice. This effect was confirmed using different mouse strains (ICR and C57BL/6J mice), sexes (male and female), and immunological backgrounds (ICR and SCID mice). In addition, TgPrx1KO and TgPrx3KO induced high levels of interferon gamma (IFN-γ) in infected mice at 8 days post infection, and increased IL-6 and IL-12p40 production from murine macrophages cultivated in vitro. The results of the present study suggested that TgPrx3 can induce anti-T. gondii immune responses that protect the mice from fatal consequences of toxoplasmosis. The results of our current and previous studies represent TgPrx3 as an excellent candidate for sub-unit vaccines, suggesting it may contribute to the control of toxoplasmosis for susceptible humans and animals. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) is an antioxidant protein family, which widely exists in organisms and plays an important role in innate immunity. In this study, the full-length cDNA of a Prx gene (NdPrx) was obtained from Neocaridina denticulata sinensis , which contains a 735 bp open reading frame (ORF) and encodes a polypeptide of 244 amino acids. It is inferred that the molecular weight of the encoded amino acid is 27261.20 Da and the theoretical isoelectric point is 6.16. Phylogenetic analysis shows that NdPrx and Prx4 have high homology, so it was named NdPrx4. Multiple alignment analysis showed that the amino acid sequence of NdPrx4 had high homology with Prx4 of other species, and the similarity with Homarus americanus was the highest, 92.86%. Quantitative real-time PCR analysis showed that NdPrx4 was expressed in various tissues of N. denticulata sinensis , and the expression in ovary was the highest. It was speculated that NdPrx4 may be related to maternal immune function. Under the stimulation of Cu2+, the expression of NdPrx4 reached the peak at 36 h, and showed a downward trend until 72 h, indicating that NdPrx4 may play an important role in the stress response of N. denticulata sinensis. Then, NdPrx4 was recombinantly expressed in E. coli , and its enzymatic characteristics of rNdPrx4 were detected. The result showed that the activity of rNdPrx4 was the highest at pH 5.0 and 55 °C. It was found that Mn2+ and Ca2+ can inhibit the activity of rNdPrx4, and Zn2+ increases the activity of rNdPrx4. • The full-length cDNA sequence of NdPrx4 was cloned and analyzed. • NdPrx4 was predominantly expressed in ovary of N. denticulata sinensis. • NdPrx4 was upregulated after copper ion stimulation. • NdPrx4 was recombinantly expressed and the enzymatic characterization was determined. [ABSTRACT FROM AUTHOR]

The slowing-down de novo drug-discovery emphasized the importance of repurposing old drugs. This is particularly true when combating infections caused by therapy-refractory microorganisms, such as Scedosporium species and Lomentospora prolificans. Recent studies on Scedosporium responses to oxidative stress underscored the importance of targeting the underlying mechanisms. Auranofin, ebselen, PX-12, honokiol, and to a lesser extent, conoidin A are known to disturb redox-homeostasis systems in many organisms. Their antifungal activity was assessed against 27 isolates belonging to the major Scedosporium species: S. apiospermum, S. aurantiacum, S. boydii, S. dehoogii, S. minutisporum, and Lomentospora prolificans. Auranofin and honokiol were the most active against all Scedosporium species (mean MIC50 values of 2.875 and 6.143 μg/ml, respectively) and against L. prolificans isolates (mean MIC50 values of 4.0 and 3.563 μg/ml respectively). Combinations of auranofin with voriconazole or honokiol revealed additive effects against 9/27 and 18/27 isolates, respectively. Synergistic interaction between auranofin and honokiol was only found against one isolate of L. prolificans. The effects of auranofin upon exposure to oxidative stress were also investigated. For all species except S. dehoogii, the maximal growth in the presence of auranofin significantly decreased when adding a sublethal dose of menadione. The analysis of the expression of genes encoding oxidoreductase enzymes upon exposure of S. apiospermum to honokiol unveiled the upregulation of many genes, especially those coding peroxiredoxins, thioredoxin reductases, and glutaredoxins. Altogether, these data suggest that auranofin and honokiol act via dampening the redox balance and support their repurposing as antifungals against Scedosporium species and L. prolificans. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prdxs) are known to play a critical role in regulating male fertility as antioxidant enzymes. Although several studies have suggested a close association between Prdxs and male fertility, few studies have explored the efficacy of Prdxs to predict male fertility. Therefore, the current study was designed to discover the most efficient biomarkers among the Prdxs with six isoforms. Our study showed a significant positive correlation between the litter size and the levels of PRDX 4 among all isoforms in spermatozoa. Subsequently, a regression analysis using a combination of markers was conducted to increase efficacy for fertility prediction. Nevertheless, PRDX4 had the highest efficacy compared to other combination models to predict litter size. The prediction accuracy of male fertility was further evaluated through receiver operating characteristic curve analysis, which showed that PRDX 4 could predict the litter size with high overall accuracy of 95%. Moreover, litter size was increased by 1.55 piglets after predicting high litter size using PRDX 4. This is the first study to comprehensively elucidate the role of all isoforms of PRDXs on male fertility to the best of our knowledge. PRDX 4 was tested and evaluated up to a practical level. Data here reported suggesting PRDX 4 marker allowed the highest accuracy for male fertility prediction and diagnosis, leading to a measurable improvement in the male fertility outcome. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Accurate estimation of Prx activity poses many complications and interferences. The present protocol is free of interference and provides an effective alternative for the assessment of peroxide with high sensitivity. The assay can be used in clinical pathology laboratories since it is simple, rapid, and inexpensive. The systematic reagent consisted of AFS/ASA which acted as a sensitive probe for peroxide. Methods: Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA). Results: Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of strong acid to inhibit the Prx reaction. Conclusions: We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method. [ABSTRACT FROM AUTHOR]

Many studies reported that redox enzymes, particularly thioredoxin and peroxiredoxin, can be released by cells and act as soluble mediators in immunity. Recently, it became clear that peroxiredoxins can be secreted via the exosome-release route, yet it remains unclear how this exactly happens and why. This review will first introduce briefly the possible redox states of protein cysteines and the role of redox enzymes in their regulation. We will then discuss the studies on the extracellular forms of some of these enzymes, their association with exosomes/extracellular vesicles and with exosome micro-RNAs (miRNAs)/mRNAs involved in oxidative processes, relevant in infection and inflammation. [Display omitted] • Redox changes are associated with inflammation. • Cysteine oxidation of peroxiredoxins 1 and 2 targets them to secretion via extracellular vesicles. • Secreted peroxiredoxins can act as damage-associated molecular patterns that play a role in sterile inflammation. • Extracellular vesicles also contain microRNAs that can be transferred to target cells regulating redox responses. [ABSTRACT FROM AUTHOR]

Soil salinity affects sugarcane (Saccharum officinale L.) production in arid and semiarid climates, severely reducing productivity. This study aimed to identify differentially regulated proteins in two cultivars that differ markedly in tolerance of saline soil. Plants were grown for 30 days and then subjected to treatments of 0 and 160 mM NaCl for 15 days. The tolerant cultivar showed a 3-fold upregulation of lipid metabolising enzymes, GDSL-motif lipases, which are associated with defence to abiotic stress, and which were not upregulated in the sensitive cultivar. Lipoxygenase was 2-fold upregulated in the tolerant cultivar but not in the sensitive cultivar, as were Type III chlorophyll a/b binding proteins. Other differences were that in the sensitive cultivar, the key enzyme of C4 photosynthesis, phosphoenolpyruvate carboxylase was downregulated, along with other chloroplast enzymes. Na+ concentrations had not reached toxic concentrations in either cultivar by this time of exposure to salt, so these changes would be in response to the osmotic effect of the soil salinity, and likely be in common with plants undergoing drought stress. Sugarcane is an important crop globally for food and biofuel production, and its yield is being affected by the expansion of saline land. This study compared two cultivars of differing salt tolerance and found that proteins involved in defence, control of reactive oxygen species, and photosynthetic energy production were upregulated in the tolerant variety and downregulated in the sensitive variety. [ABSTRACT FROM AUTHOR]

The limitations that hypoxia imparts on mitochondrial oxygen supply are circumvented by the activation of anaerobic metabolism and prosurvival mechanisms in hypoxia-tolerant animals. To deal with the hypoxia that jumbo squid (Dosidicus gigas) experience in the ocean’s depth, they depress their metabolic rate by up to 52% relative to normoxic conditions. This is coupled with molecular reorganization to facilitate their daily descents into the ocean’s oxygen minimum zone, where they face not only low oxygen levels but also higher pressures and colder frigid waters. Our current study explores the tissue-specific hypoxia responses of three central processes: (1) antioxidant enzymes responsible for defending against oxidative stress, (2) early apoptotic machinery that signals the activation of cell death, and (3) mitogen-activated protein kinases (MAPKs) that act as central regulators of numerouscellular processes. LuminexxMAP technology was used to assess protein levels and phosphorylation states under normoxic and hypoxic conditions in brains, branchial hearts, and mantle muscles. Hypoxic brains were found to activate apoptosis via up-regulation of phospho-p38, phospho-p53, activated caspase 8, and activated caspase 9, whereas branchial hearts were the only tissue to show an increase in antioxidant enzyme levels. Hypoxic muscles seemed the least affected by hypoxia. Our results suggest that hypoxic squid do not undergo large dynamic changes in the phosphorylation states of key apoptotic and central MAPK factors, except for brains, suggesting that these mechanisms are involved in squid hypometabolic responses. [ABSTRACT FROM AUTHOR]

Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to post‐translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol‐reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry‐based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH‐thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2‐CysPRXs or type‐II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning. The contribution of thiol reductases to the signalling network governing stomatal functioning remains poorly known. Using proteomics, we show that numerous thiol reductases are abundant in a guard cell‐enriched fraction, and we unveil a stomatal phenotype in Arabidopsis mutants for thiol reductases. [ABSTRACT FROM AUTHOR]

Summary: Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c‐TXNPx) is a 2‐Cys peroxiredoxin (Prx) with an important role in detoxifying host cell oxidative molecules during parasite infection. c‐TXNPx is a virulence factor, as its overexpression enhances parasite infectivity and resistance to exogenous oxidation. As Prxs from other organisms possess immunomodulatory properties, we studied the effects of c‐TXNPx in the immune response and analysed whether the presence of the peroxidatic cysteine is necessary to mediate these properties. To this end, we used a recombinant c‐TXNPx and a mutant version (c‐TXNPxC52S) lacking the peroxidatic cysteine. We first analysed the oligomerization profile, oxidation state and peroxidase activity of both proteins by gel filtration, Western blot and enzymatic assay, respectively. To investigate their immunological properties, we analysed the phenotype and functional activity of macrophage and dendritic cells and the T‐cell response by flow cytometry after injection into mice. Our results show that c‐TXNPx, but not c‐TXNPxC52S, induces the recruitment of IL‐12/23p40‐producing innate antigen‐presenting cells and promotes a strong specific Th1 immune response. Finally, we studied the cellular and humoral immune response developed in the context of parasite natural infection and found that only wild‐type c‐TXNPx induces proliferation and high levels of IFN‐γ secretion in PBMC from chronic patients without demonstrable cardiac manifestations. In conclusion, we demonstrate that c‐TXNPx possesses pro‐inflammatory properties that depend on the presence of peroxidatic cysteine that is essential for peroxidase activity and quaternary structure of the protein and could contribute to rational design of immune‐based strategies against Chagas disease. [ABSTRACT FROM AUTHOR]

Str3 is a transmembrane protein that mediates low‐affinity heme uptake in Schizosaccharomyces pombe. Under iron‐limiting conditions, Str3 remains at the cell surface in the presence of increasing hemin concentrations. Using a proximity‐dependent biotinylation approach coupled to mass spectrometry and coimmunoprecipitation assays, we report that the peroxiredoxin Tpx1 is a binding partner of Str3. Under microaerobic conditions, cells deficient in heme biosynthesis and lacking the heme receptor Shu1 exhibit poor hemin‐dependent growth in the absence of Tpx1. Analysis of membrane protein preparations from iron‐starved hem1Δ shu1Δ str3Δ tpx1Δ cells coexpressing Str3‐GFP and TAP‐Tpx1 showed that TAP‐Tpx1 is enriched in membrane protein fractions in response to hemin. Bimolecular fluorescence complementation assays brought additional evidence that an interaction between Tpx1 and Str3 occurs at the plasma membrane. Results showed that Tpx1 exhibits an equilibrium constant value of 0.26 μM for hemin. The association of Tpx1 with hemin protects hemin from degradation by H2O2. The peroxidase activity of hemin is lowered when it is bound to Tpx1. Taken together, these results revealed that Tpx1 is a novel interacting partner of Str3. Our data are the first example of an interaction between a cytoplasmic heme‐binding protein and a cell‐surface heme transporter. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOClsensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. [ABSTRACT FROM AUTHOR]

2-Cys peroxiredoxins are abundant thiol proteins that react efficiently with a wide range of peroxides. Unlike other enzymes, their exceptionally high reactivity does not rely on cofactors. The mechanism of oxidation and reduction of peroxiredoxins places them in a good position to act as antioxidants as well as key players in redox signaling. Understanding of the intimate details of peroxiredoxin functioning is important for translational research. [ABSTRACT FROM AUTHOR]

Ischemia and reperfusion events, such as myocardial infarction (MI), are reported to induce remote organ damage severely compromising patient outcomes. Tissue survival and functional restoration relies on the activation of endogenous redox regulatory systems such as the oxidoreductases of the thioredoxin (Trx) family. Trxs and peroxiredoxins (Prxs) are essential for the redox regulation of protein thiol groups and for the reduction of hydrogen peroxide, respectively. Here, we determined whether experimental MI induces changes in Trxs and Prxs in the heart as well as in secondary organs. Levels and localization of Trx1, TrxR1, Trx2, Prx1, and Prx2 were analyzed in the femur, vertebrae, and kidneys of rats following MI or sham surgery. Trx1 levels were significantly increased in the heart (P = 0.0017) and femur (P < 0.0001) of MI animals. In the femur and lumbar vertebrae, Trx1 upregulation was detected in bone-lining cells, osteoblasts, megakaryocytes, and other hematopoietic cells. Serum levels of Trx1 increased significantly 2 days after MI compared to sham animals (P = 0.0085). Differential regulation of Trx1 in the bone was also detected by immunohistochemistry 1 month after MI. N-Acetyl-cysteine treatment over a period of 1 month induced a significant reduction of Trx1 levels in the bone of MI rats compared to sham and to MI vehicle. This study provides first evidence that MI induces remote organ upregulation of the redox protein Trx1 in the bone, as a response to ischemia–reperfusion injury in the heart. [ABSTRACT FROM AUTHOR]

The slowing-down de novo drug-discovery emphasized the importance of repurposing old drugs. This is particularly true when combating infections caused by therapy-refractory microorganisms, such as Scedosporium species and Lomentospora prolificans. Recent studies on Scedosporium responses to oxidative stress underscored the importance of targeting the underlying mechanisms. Auranofin, ebselen, PX-12, honokiol, and to a lesser extent, conoidin A are known to disturb redox-homeostasis systems in many organisms. Their antifungal activity was assessed against 27 isolates belonging to the major Scedosporium species: S. apiospermum, S. aurantiacum, S. boydii, S. dehoogii, S. minutisporum, and Lomentospora prolificans. Auranofin and honokiol were the most active against all Scedosporium species (mean MIC50 values of 2.875 and 6.143 µg/ml, respectively) and against L. prolificans isolates (mean MIC50 values of 4.0 and 3.563 µg/ml respectively). Combinations of auranofin with voriconazole or honokiol revealed additive effects against 9/27 and 18/27 isolates, respectively. Synergistic interaction between auranofin and honokiol was only found against one isolate of L. prolificans. The effects of auranofin upon exposure to oxidative stress were also investigated. For all species except S. dehoogii, the maximal growth in the presence of auranofin significantly decreased when adding a sublethal dose of menadione. The analysis of the expression of genes encoding oxidoreductase enzymes upon exposure of S. apiospermum to honokiol unveiled the upregulation of many genes, especially those coding peroxiredoxins, thioredoxin reductases, and glutaredoxins. Altogether, these data suggest that auranofin and honokiol act via dampening the redox balance and support their repurposing as antifungals against Scedosporium species and L. prolificans. [ABSTRACT FROM AUTHOR]