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Skeletal muscle cellular function relies on mitochondrial oxidative phosphorylation and maintenance of the mitochondrial reticulum through naturally occurring fusion and fission. A key molecule in oxidative phosphorylation is lactate, a product of glycolysis formed under aerobic conditions and a major energy source, gluconeogenic precursor, and a signaling molecule. With age progression, mitochondrial function decreases in part due to elevated oxidative damage and increased mitochondrial fission, resulting in the inability to metabolize different energy substrates or “metabolic inflexibility”. Damage caused by oxidative stress, such as lipid peroxidation, leads to altered mitochondrial dynamics, oxidative cell death, and an increased risk of age-related diseases such as sarcopenia. Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme capable of repairing peroxidized membranes and mice lacking Prdx6 exhibit sarcopenia-like phenotypes suggesting an age-related effect of Prdx6. The purpose of this dissertation was to understand the effects of aging on lactate metabolism and mitochondrial dysfunction while studying the role of oxidative stress as a potential driver for age-related changes to mitochondrial metabolism. I hypothesized that aging impairs lactate metabolism in humans while promoting mitochondrial dysfunction in skeletal muscle-derived cells and that oxidative stress derived from age-related reductions in mitochondrial Prdx6 is a primary driver of this dysfunction. The effect of age on the Postprandial Lactate Shuttle and aging-related metabolic inflexibility and mitochondrial dysfunction was revealed. These findings collectively contribute to our understanding of the mechanisms underlying age-related mitochondrial dysfunction, emphasizing the critical role of Prdx6 in preserving mitochondrial integrity in skeletal muscle. These insights pave the way for future research and interventions aimed at improving mitochondrial health in the aging population. Here, I discuss how aging affects skeletal muscle, lactate metabolism, and mitochondrial function, and how oxidative stress is a primary driver of such effects in skeletal muscle.

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.

Acidophilic archaea thrive in anaerobic and aerobic low pH environments (pH < 5) rich in dissolved heavy metals that exacerbate stress caused by the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (·OH) and superoxide (O2−). ROS react with lipids, proteins and nucleic acids causing oxidative stress and damage that can lead to cell death. Herein, genes and mechanisms potentially involved in ROS mitigation are predicted in over 200 genomes of acidophilic archaea with sequenced genomes. These organisms are often be subjected to simultaneous multiple stresses such as high temperature, high salinity, low pH and high heavy metal loads. Some of the topics addressed include: (1) the phylogenomic distribution of these genes and what this can tell us about the evolution of these mechanisms in acidophilic archaea; (2) key differences in genes and mechanisms used by acidophilic versus non-acidophilic archaea and between acidophilic archaea and acidophilic bacteria and (3) how comparative genomic analysis predicts novel genes or pathways involved in oxidative stress responses in archaea and likely horizontal gene transfer (HGT) events.

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 H2O2, 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 peroxi-dases 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.

Melatonin (MT) has been reported to regulate certain plant physiological processes and promote tolerance to different environmental stresses such as salinity. Green bean (Phaseolus vulgaris L. cv. Royal Nel) seedlings were exposed to 200 mM NaCl with or without pre-treatment with 150 μM MT. Salt stress led to a lower chlorophyll content, a reduced photosynthetic activity, increased reactive oxygen species (ROS) contents, and decreased photosystem II (PSII) activity. The application of exogenous MT to green bean seedlings under salt stress improved photosynthetic activity and alleviated the oxidative damages by enhancing the activity of antioxidant enzymes. The expression of catalase (CAT1), glutathione reductase (GR), superoxide dismutase (CuZnSOD1), ascorbate peroxidase (APX), Peroxiredoxin Q (PrxQ), and 2-cysteine peroxiredoxin (2-Cys-Prx) encoding genes was significantly increased under salt stress in green bean seedling compared with the untreated control. However, plants treated with exogenous MT and NaCl had 28.8, 21.1, 26.1, 20, 26.2, and 22.4% higher CuZnSOD, CAT1, APX, GR, PrxQ, and 2-Cys-Prx transcript levels, respectively, compared to NaCl stress alone. Our study revealed the protective mechanisms mediated by exogenous MT application in NaCl stress alleviation and our findings could be used in the management of green bean cultivation in salinity-prone soils.

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.

Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys152 and Cys157) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys152 and Cys157 are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys152 and Cys157 in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.

Objectives Synthetic biology has emerged from molecular biology and engineering approaches and aims to develop novel, biologically-inspired systems for industrial and basic research applications ranging from biocomputing to drug production. Surprisingly, redoxin (thioredoxin, glutaredoxin, peroxiredoxin) and other thiol-based redox systems have not been widely utilized in many of these synthetic biology applications. Methods We reviewed thiol-based redox systems and the development of synthetic biology applications that have used thiol-dependent parts. Results The development of circuits to facilitate cytoplasmic disulfide bonding, biocomputing and the treatment of intestinal bowel disease are amongst the applications that have used thiol-based parts. We propose that genetically encoded redox sensors, thiol-based biomaterials and intracellular hydrogen peroxide generators may also be valuable components for synthetic biology applications. Discussion Thiol-based systems play multiple roles in cellular redox metabolism, antioxidant defense and signaling and could therefore offer a vast and diverse portfolio of components, parts and devices for synthetic biology applications. However, factors limiting the adoption of redoxin systems for synthetic biology applications include the orthogonality of thiol-based components, limitations in the methods to characterize thiol-based systems and an incomplete understanding of the design principles of these systems.

Molecular modelling and design are valued and vital tools in the pharmaceutical research for defining, developing and analyzing active biological and chemical molecules. It is based on the evolution of computational theories and methods to study molecules behavior enabling scientists to hypothesize potent drugs for a particular disease. Quercetin is one of the flavonoid family that possesses pharmacological properties due to its interface with cellular targets including, anti-inflammatory, anti-oxidant, anti-cytotoxicity and anti-cancer activities. Molecular docking analyses were performed to predicted quercetin possible binding action along with absorption, distribution, metabolism and excretion (ADME) study. The molecular docking results revealed the bind of quercetin to the active site of Serine/threonine mammalian sterile-20 (MST3) and peroxiredoxin 5 pockets. Moreover, ADME results show its vital properties absorption and drug mimic where the ability to enter blood brain barrier (BBB) was not high, low permeable and strongly bound. Pre-metabolism analyze results show that 2D9 liver microsomal enzyme has more effect on quercetin than CYP2D6 enzymes. In conclusion, molecular docking study documented some important mechanisms of quercetin as a promising anticancer and antioxidant compound.

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson’s disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Peroxiredoxins (Prxs) are cysteine-based peroxide-reducing enzymes that have been extensively studied over the past two decades. The reduction of the Prx disulfide form is dependent on electron donating enzymes to shift the Prx back into a catalytically active state. However, this reaction is poorly understood and reductant enzymes for this reaction to reduce the Prx disulfide bond have received little attention. Prxs from the bacterium Xanthomonas campestris (XcPrxQ) were used to assess how reductants affect Prx turnover by identifying potential electron transfer interactions between Prxs and reductants, Glutaredoxins (Grxs) and Thioredoxins (Trxs). To examine potential interactions between PrxQ and reducing proteins, we used PrxQ, Grx, and Trx mutants to determine the effect of specific cysteines in each of the proteins. Mutant and wildtype proteins were expressed in E. coli and purified using anion-exchange and gel filtration chromatography and purity was verified by SDS-PAGE. The results clarified the role of the two cysteines in PrxQ, C48 and C84, which are thought to participate in the reduction reaction of hydrogen peroxide. They also show Trx and Grx proteins from the same bacterium are possible reductants for Prx turnover, as reactions with PrxQ have produced complex formation at a larger molecular weight, suggesting the reductant binding to PrxQ and reducing its disulfide bond. This study is significant because it reveals potential binding interactions between reductants and Prxs that regulate Prx turnover into a catalytically active state, allowing the enzyme to break down peroxide and prevent oxidative damage, as well as highlighting Trxs in bacteria as a potential drug target due to their regulation of hydrogen peroxide levels in pathogenic strains.

Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function. Experimental approaches suitable for testing the hypothesis are also outlined., Graphical abstract Image 1, Highlights • It is hypothesised that denervation leads to failed redox signalling and attenuated muscle exercise responses in aging. • Loss of peripheral motor neurons leads to increased mitochondrial peroxide in denervated and neighbouring muscle fibers. • This peroxide stimulates increased expression of cytoprotective proteins to protect the fiber against oxidative damage. • Increased mitochondrial peroxide does not increase cytosolic peroxide, but adaptations reduce the cytosol redox state. • Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation in the cytosol. • Peroxiredoxin oxidation is suppressed during aging by a chronic change to a more reductive local cytosolic environment.

This research was funded by ERDF grants co-financed by the Spanish Ministry of Economy and Competitiveness (Project PGC2018-096405-B-I00), the Junta de Andalucia (group BIO286), the I+D+I project within the framework Programme of FEDER Andalucia 2014-2020 (Reference 1380901), the grants for I+D+I projects, on a competitive basis, within the scope of the Andalusian plan for research, development and innovation (Junta de Andalucia, PAIDI 2020, Reference: PY20_01002), and the funding for the recruitment of researchers under Action 9 and 10 of the Research Support Plan of the University of Jaen (2019-2020, R.02/10/2020; 2020-2021, R.01/01/2022)., Heat stress is one of the abiotic stresses that leads to oxidative stress. To protect themselves, yeast cells activate the antioxidant response, in which cytosolic peroxiredoxin Tsa1 plays an important role in hydrogen peroxide removal. Concomitantly, the activation of the heat shock response (HSR) is also triggered. Nitro-fatty acids are signaling molecules generated by the interaction of reactive nitrogen species with unsaturated fatty acids. These molecules have been detected in animals and plants. They exert their signaling function mainly through a post-translational modification called nitroalkylation. In addition, these molecules are closely related to the induction of the HSR. In this work, the endogenous presence of nitro-oleic acid (NO2-OA) in Saccharomyces cerevisiae is identified for the first time by LC-MS/MS. Both hydrogen peroxide levels and Tsa1 activity increased after heat stress with no change in protein content. The nitroalkylation of recombinant Tsa1 with NO2-OA was also observed. It is important to point out that cysteine 47 (peroxidatic) and cysteine 171 (resolving) are the main residues responsible for protein activity. Moreover, the in vivo nitroalkylation of Tsa1 peroxidatic cysteine disappeared during heat stress as the hydrogen peroxide generated in this situation caused the rupture of the NO2-OA binding to the protein and, thus, restored Tsa1 activity. Finally, the amino acid targets susceptible to nitroalkylation and the modulatory effect of this PTM on the enzymatic activity of Tsa1 are also shown in vitro and in vivo. This mechanism of response was faster than that involving the induction of genes and the synthesis of new proteins and could be considered as a key element in the fine-tuning regulation of defence mechanisms against oxidative stress in yeast., ERDF - Spanish Ministry of Economy and Competitiveness PGC2018-096405-B-I00, Junta de Andalucia BIO286 PY20_01002, I+D+I project within the framework Programme of FEDER Andalucia 2014-2020 1380901, University of Jaen R.02/10/2020 R.01/01/2022

A new series of 4-(3-(2-amino-3,5-dibromophenyl)-1-(4-substitutedbenzoyl)-4,5-dihydro-1H-pyrazol-5-yl)benzonitrile (4a-h) compounds were synthesized and evaluated for in-vitro anti-inflammatory activities. The spectral (IR, NMR) and elemental analyses data of the product indicated the formation of new pyrazoles 4a-h. Compound 4e exhibited potent anti-inflammatory property with 85.45 % inhibitions. This value was compared with standard diclofenac sodium. This data is explained using molecular docking analysis of receptor-ligand binding. These results demonstrated that pyrazole derivatives are potential inhibitors of Human Peroxiredoxin 5 and Tyrosine kinase receptor in the treatment of inflammation related illness.

Continuous and sustained production of good quality eggs and larvae is required to have economically viable and ecologically sustainable rearing of Seriola rivoliana. Nonetheless, the complete production cycle of these species has been challenging to achieve due to high mortality during embryonic and larval stages. The objective of the project was to study the expression of the proteins involved in the embryonic process of almaco jack. Proteomic characterization of fertilized eggs was performed using two-dimensional electrophoresis and mass spectrometry. Different vitellogenin proteins A, B, C and Ab, β-actin, peroxiredoxin, superoxide dismutase 1, alpha subunit proteasome, and keratin II were identified to their functions related to embryonic development, energy metabolism, protein synthesis, cell structure, cytoskeleton, and antioxidant proteins with defense enzymatic activity.

Skeletal muscle generates superoxide during contractions which is rapidly converted to H2O2. This molecule has been proposed to activate signalling pathways and transcription factors that regulate key adaptive responses to exercise but the concentration of H2O2 required to oxidise and activate key signalling proteins in vitro is much higher than the intracellular concentration in muscle fibers following exercise. We hypothesised that Peroxiredoxins (Prx), which reacts with H2O2 at the low intracellular concentrations found in muscle, would be rapidly oxidised in contracting muscle and hence potentially transmit oxidising equivalents to downstream signalling proteins as a method for their oxidation and activation. The aim of this study was to characterise the effects of muscle contractile activity on the oxidation of Prx1, 2 and 3 and determine if these were affected by aging. Prx1, 2 and 3 were all rapidly and reversibly oxidised following treatment with low micromolar concentrations of H2O2 in C2C12 myotubes and also in isolated mature flexor digitalis brevis fibers from adult mice following a protocol of repeated isometric contractions. Significant oxidation of Prx2 was seen within 1 min (i.e. after 12 contractions), whereas significant oxidation was seen after 2 min for Prx1 and 3. In muscle fibers from old mice, Prx2 oxidation was significantly attenuated following contractile activity. Thus we show for the first time that Prx are rapidly and reversibly oxidised in response to contractile activity in skeletal muscle and hypothesise that these proteins act as effectors of muscle redox signalling pathways which are key to adaptations to exercise that are attenuated during aging., Graphical abstract Image 1, Highlights • Hydrogen peroxide is generated by skeletal muscle during contractions. • Peroxiredoxins (Prx) react with H2O2 at the physiological levels generated during contractions. • Prx 1–3 are all oxidised by H2O2 and contractile activity in muscle fibers. • Prx2 oxidation during contractions is attenuated in muscle fibers from old mice. • Prx may act as effectors in activation of redox-regulated adaptations to contractile activity in muscle.

AhpC is a bacterial representative of 2-Cys peroxiredoxins (Prxs) with broad substrate specificity and functional plasticity. However, details underpinning these two important attributes of AhpC remain unclear. Here, we studied the functions and mechanisms of regulation of AhpC in the facultative Gram-negative anaerobic bacterium Shewanella oneidensis, in which AhpC's physiological roles can be conveniently assessed through its suppression of a plating defect due to the genetic loss of a major catalase. We show that successful suppression can be achieved only when AhpC is produced in a dose- and time-dependent manner through a complex mechanism involving activation of the transcriptional regulator OxyR, transcription attenuation, and translation reduction. By analyzing AhpC truncation variants, we demonstrate that reactivity with organic peroxides (OPs) rather than H2O2 is resilient to mutagenesis, implying that OP reduction is the core catalytic function of AhpC. Intact AhpC could be recycled only by its cognate reductase AhpF, and AhpC variants lacking the Prx domain or the extreme C-terminal five residues became promiscuous electron acceptors from the thioredoxin reductase TrxR and the GSH reductase Gor in addition to AhpF, implicating an additional dimension to functional plasticity of AhpC. Finally, we show that the activity of S. oneidensis AhpC is less affected by mutations than that of its Escherichia coli counterpart. These findings suggest that the physiological roles of bacterial AhpCs are adapted to different oxidative challenges, depending on the organism, and that its functional plasticity is even more extensive than previously reported.

Ebselen is an organoselenium compound exhibiting hydroperoxide- and peroxynitrite-reducing activity, acting as a glutathione peroxidase and peroxiredoxin enzyme mimetic. Ebselen reacts with a multitude of protein thiols, forming a selenosulfide bond, which results in pleiotropic effects of antiviral, antibacterial and anti-inflammatory nature. The main protease (Mpro) of the corona virus SARS-CoV-2 is a potential drug target, and a screen with over 10,000 compounds identified ebselen as a particularly promising inhibitor of Mpro (Jin, Z. et al. (2020) Nature 582, 289–293). We discuss here the reaction of ebselen with cysteine proteases, the role of ebselen in infections with viruses and with other microorganisms. We also discuss effects of ebselen in lung inflammation. In further research on the inhibition of Mpro in SARS-CoV-2, ebselen can serve as a promising lead compound, if the inhibitory effect is confirmed in intact cells in vivo. Independently of this action, potential beneficial effects of ebselen in COVID-19 are ascribed to a number of targets critical to pathogenesis, such as attenuation of inflammatory oxidants and cytokines., Graphical abstract Image 1, Highlights • Ebselen, an organoselenium compound, exhibits anti-inflammatory and antiviral activity. • Ebselen is a glutathione peroxidase and peroxiredoxin mimetic. • Ebselen reacts with a multitude of protein thiols, resulting in pleiotropic effects. • Ebselen is an effective inhibitor of Mpro, the main protease of SARS-CoV-2. • Ebselen may serve as lead compound for drugs targeting COVID-19.

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

Clostridium difficile is a spore-forming bacterium and a leading cause of hospital-acquired antibiotic-associated diarrhoea. Symptoms of disease result from secreted toxins, while disease transmission is mediated via resistant endospores. CotE is a bifunctional spore-coat protein with peroxiredoxin and chitinase domains that are implicated in colonization. Here, the structure of the chitinase domain of CotE has been determined, revealing a GH18 family fold and, unexpectedly, a peptide bound in the active site., CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349–712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.

To characterize the high‐value protein content and to discover new bioactive peptides, present in edible organisms, as silkworm pupae, semiquantitative analytical approach has been applied. The combination of appropriate protein extraction methods, semiquantitative high‐resolution mass spectrometry analyses of peptides, in silico bioactivity and gene ontology analyses, allowed protein profiling of silkworm pupae (778 gene products) and the characterization of bioactive peptides. The semiquantitative analysis, based on the measurement of the emPAI, revealed the presence of high‐abundance class of proteins, such as larval storage protein (LSP) class. This class of proteins, beside its nutrient reservoir activity, is of great pharmaceutical interest for their efficacy in cardiovascular diseases. Potential allergens were also characterized and quantified, such as arginine kinase, thiol peroxiredoxin, and Bom m 9. This powerful bioanalytical approach proved the potential industrial applications of Bombyx mori pupae, as source of high‐value proteins in a green and “circular” economy perspective., Protein content by the semiquantitative analyses of Bombyx mori pupae. Identification of high‐value bioactive peptides by mass spectrometry. Potential industrial applications of B. mori pupae, as nutrient reservoir.

The 1-cyseine peroxiredoxin (Prx6) is an importantly antioxidant enzyme that protects cells from oxidative damage caused by excessive production of reactive oxygen species (ROS). In this study, we described the molecular characteristics of the noble scallop Chlamys nobilis peroxiredoxin 6 (designed as CnPrx6), immune responses and DNA protection activity of the recombinant protein. The complete ORF (696 bp) of CnPrx6 encoded a polypeptide (25.5 kDa) of 231 amino acids, harboring a conserved peroxidase catalytic center (41PVCTTE46) and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. The deduced amino acid sequence of CnPrx6 shared a relatively high amino acid sequence similarity (more than 50%). The qRT-PCR revealed that the CnPrx6 mRNA was constitutively expressed in all examined tissues, with the highest expression observed in adductor. Upon immunological challenge with Vibrio parahaemolyticus, lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly I:C), the expression level of CnPrx6 mRNA was significantly up-regulated (P

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic β-cell damage during the pathogenesis of autoimmune diabetes. While β-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in β-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of β-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that β-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 ( Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 ( Prdx2) had no effect. Together, these results suggest that β-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif “31WCXXC35”. We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the repression of Prx1–mediated H2O2 decomposition leading to increased ROS accumulation in infected host cells. Increased ROS in turn prevented nuclear translocation of NF-κB and inhibition of NF-κB target genes, leading to bacterial survival and enhanced replication inside host cells. Targeting Trxlp-mediated virulence promises to attenuate E. piscicida infection.

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is a transcriptional coactivator described as a master regulator of mitochondrial biogenesis and function, including oxidative phosphorylation and reactive oxygen species detoxification. PGC-1α is highly expressed in tissues with high energy demands, and it is clearly associated with the pathogenesis of metabolic syndrome and its principal complications including obesity, type 2 diabetes mellitus, cardiovascular disease, and hepatic steatosis. We herein review the molecular pathways regulated by PGC-1α, which connect oxidative stress and mitochondrial metabolism with inflammatory response and metabolic syndrome. PGC-1α regulates the expression of mitochondrial antioxidant genes, including manganese superoxide dismutase, catalase, peroxiredoxin 3 and 5, uncoupling protein 2, thioredoxin 2, and thioredoxin reductase and thus prevents oxidative injury and mitochondrial dysfunction. Dysregulation of PGC-1α alters redox homeostasis in cells and exacerbates inflammatory response, which is commonly accompanied by metabolic disturbances. During inflammation, low levels of PGC-1α downregulate mitochondrial antioxidant gene expression, induce oxidative stress, and promote nuclear factor kappa B activation. In metabolic syndrome, which is characterized by a chronic low grade of inflammation, PGC-1α dysregulation modifies the metabolic properties of tissues by altering mitochondrial function and promoting reactive oxygen species accumulation. In conclusion, PGC-1α acts as an essential node connecting metabolic regulation, redox control, and inflammatory pathways, and it is an interesting therapeutic target that may have significant benefits for a number of metabolic diseases.

The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel-filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X-ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring-type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid-associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild-type levels of 16:1t. The FAD4-PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co-production of PRXQ with FAD4 was required to produce Δ3-desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site-directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.

The marked heterogeneity of autism spectrum disorders is a challenge for research into biomarkers of these disorders. The present study aimed to assess the association of the thioredoxin (TRX) system and vasopressin with autism in children. Thirty-five autistic children were recruited with thirty-one healthy controls children matched for age and gender. Total antioxidant capacity (TAC), vasopressin, peroxiredoxin (PRDX1, PRDX3), phox domain-containing protein kinase-like protein, and TRX1 levels were measured in plasma and mercury and lead levels were measured in red blood cells. Children with autism had significantly lower levels of PRDX1 and TRX1 and significantly higher levels of lead and mercury compared with controls. Significantly higher levels of vasopressin were also observed in more severe cases of autism. Receiver operating characteristics analysis demonstrated satisfactory specificity and sensitivity of TAC, PRDX1, PRDX3, and TRX1 for the diagnosis of autism. The optimal cut-off value for plasma TRX1 levels, as an indicator of autism, was 10.6 ng/mL. Significant positive correlations existed between TRX system components, lead, and mercury. A negative correlation was found between vasopressin and TRX1, but positive correlations were found between vasopressin and mercury and lead. In conclusion, TAC, PRDX1, PRDX3, and TRX1 may be useful biomarkers for the diagnosis of autism in Saudi populations. Vasopressin-resistance in autistic patients may be due to an increased oxidative burden.

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 A resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

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.

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.

Typical 2-cysteine peroxiredoxins (2-Cys Prxs) are critical peroxidase sensors and could be deactivated by the hyperoxidation under oxidative stress. In plants, 2-Cys Prxs present at a high level in chloroplasts and are repaired by Sulfiredoxin. Whereas many studies have explored the mechanism of Sulfiredoxin from Homo sapiens (HsSrx), the molecular mechanism of Sulfiredoxin in plants with unique photosynthesis remains unclear. Here we report the crystal structure of Sulfiredoxin from Arabidopsis thaliana (AtSrx), which displayed a typical ParB/Srx fold with an ATP bound at a conservative nucleotide binding motif GCHR. Both the ADP binding pocket and the putative AtSrx-AtPrxA interaction surface of AtSrx are more positively charged comparing to HsSrx, suggesting a robust mechanism of AtSrx. These features illustrate the unique mechanisms of AtSrx, which are vital for figure out the strategies of plants to cope with oxidation stress.

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO2 to produce peroxymonocarbonate (HCO4-). Peroxymonocarbonate is more reactive with thiols than H2O2 but it makes up only a small fraction of the H2O2 in physiological bicarbonate buffers so the increase in rate of oxidation of low molecular weight thiols is modest. However, for some thiol proteins such as protein tyrosine phosphatases, the rate enhancement is very much greater. We have investigated the effect of bicarbonate/CO2 on the oxidation of peroxiredoxins (Prdxs) 2 and 3. Using an assay in which reduced Prdx2 inhibits oxidation of horseradish peroxidase by H2O2, we saw no difference between phosphate and bicarbonate buffers (pH 7.4). However, hyperoxidation of both Prdxs in bicarbonate was considerably enhanced. Hyperoxidation involves the reaction of the sulfenic acid formed at the active site with a second H2O2, and prevents its condensation to a disulfide. Using LC/MS analysis, we determined that the presence of 25 mM bicarbonate/CO2 increased the ratio of hyperoxidation compared with condensation 6-fold for Prdx2 and 11-fold for Prdx3. These results imply that Prdx hyperoxidation will occur more readily under physiological conditions than appreciated from in vitro experiments, which seldom use bicarbonate buffers. They also raise the possibility that variations in bicarbonate concentration could provide a mechanism for regulating the cellular level of active Prdxs.

Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organised microdomains, where not all interactors are covalently bound. However, most studies aimed at identifying peroxiredoxin interactors were based on methods that only detect covalently linked partners. Here, we explore the applicability of two thiol-disulphide independent in-cell trapping methodological approaches in combination with mass spectrometry for the identification of interaction partners of peroxiredoxin 2 (Prdx2). The first is biotin-dependent proximity-labelling (BioID) with a biotin ligase A (BirA*)-fused Prdx2, which has never been applied on redox-active proteins. The second is crosslinker co-immunoprecipitation with an N-terminally His-tagged Prdx2. During the initial characterisation of the tagged Prdx2 constructs, we found that the His-tag, but not BirA*, compromises the peroxidase and signalling activities of Prdx2. Further, the Prdx2 interactors identified with each approach showed little overlap. We therefore concluded that BioID is a more reliable method than crosslinker co-immunoprecipitation. After a stringent mass spec data filtering, BioID identified 13 interactors under elevated H2O2 conditions, including subunit five of the COP9 signalosome complex (CSN5). The Prdx2:CSN5 interaction was further confirmed in a proximity ligation assay. Taken together, our results demonstrate that BioID can be used as a method for the identification of interactors of Prdxs, and that caution should be exercised when interpreting protein-protein interaction results using tagged Prdxs., Graphical abstract Image 1, Highlights • BioID is a thiol-disulphide independent method that can be used to trap Prdx2 interactors. • Prdx2 interacts with subunit five of the COP9 signalosome complex (CSN5). • N-terminally His-tagged Prdx2 displays compromised peroxidase and signalling activity. • The tag attached to Prdx2 influences its interactome.

Entamoeba histolytica is the conductive agent of amoebiasis. Upon the parasite's infection, macrophages and neutrophils are activated by Interferon γ (IFN-γ), IL-13, and Tumour Necrosis Factor (TNF-α). These immune cells then carry out the amoebicidal activity by releasing Nitric Oxide Synthase (NOS) and Reactive Oxygen Species (ROS). This review talks about the protective and destructive role of ROS in Eh. E. histolytica has defense strategies against oxidative stress which is a result of excess ROS production. They possess antioxidants for their defense such as L-Cysteine, Flavodiiron proteins (FDPs), Peroxiredoxin, and Trichostatin A, which contribute to the parasite's virulence. The reactive oxygen species are harmful to the host cells as excess ROS production stimulates cell death by mechanisms like apoptosis and necroptosis. NADPH oxidase (NOX) is a key source of ROS in mammalian cells and causes apoptosis of host cells via the protein kinase transduction pathway. This review provides insights into why NOX inhibitors that could be a potent antiparasitic drug, is not effective for in vivo purposes. This paper also gives an insight into a solution that could be a potent source in generating new treatment and vaccines for amoebiasis by targeting parasite development.

Reactive oxygen species (ROS) are recognized as important signaling molecules in healthy skeletal muscle. Redox sensitive proteins can respond to intracellular changes in ROS by oxidation of reactive thiol groups on cysteine (Cys) residues. Exercise is known to induce the generation of superoxide and nitric oxide, resulting in the activation of several adaptive signaling pathways; however, it has been suggested that aging attenuates these redox-regulated adaptations to acute exercise. In the present study, we used redox proteomics to study the vastus lateralis muscles of Adult (n = 6 male, 6 female; 18–30 yrs) and Old (n = 6 male, 6 female; 64–79 yrs) adults. Participants completed a bout of high intensity cycling exercise consisting of five sets of 2-min intervals performed at 80% maximal aerobic power output (PPO), with 2 min recovery cycling at 40% PPO between sets. Muscle biopsies were collected prior to exercise, and immediately following the first, second, and fifth high intensity interval. Global proteomic analysis indicated differences in abundance of a number of individual proteins between skeletal muscles of Adult and Old subjects at rest with a significant exacerbation of these differences induced by the acute exercise. In particular, we observed an exercise-induced decrease in abundance of mitochondrial proteins in muscles from older subjects only. Redox proteome analysis revealed cysteines from five cytosolic proteins in older subjects with lower oxidation (i.e. greater reduction) than was seen in muscle from the young adults at rest. Redox homeostasis was well maintained in Adult subjects following exercise, but there was significant increase in oxidation of multiple mitochondrial and cytosolic protein cysteines in Old subjects. We also observed that oxidation of peroxiredoxin 3 occurred following exercise in both Adult and Old groups, supporting the possibility that this is a key effector protein for mitochondrial redox signaling. Thus, we show, for the first time that exercise reveals a lack of resilience in muscle of older human participants, that is apparent as a loss of mitochondrial proteins and oxidation of multiple protein cysteines that are not seen in younger subjects. The precise consequences of this redox disruption are unclear, but this likely play a role in the attenuation of multiple adaptations to exercise that are classically seen with aging. Such changes were only seen following the acute stress of exercise., highlighting the need to consider not only basal differences seen during aging but also the difference following physiological challenge.

The peroxiredoxin 5 (PRDX5) is a member of peroxiredoxins with antitumor activity. However, as a recombinant protein, PRDX5 is restricted in clinic due to high cost and keeping high dose in medication. The alternative way is to explore the antitumor active fragments of PRDX5 for potential of peptide drugs. According to the sequence, crystal structure and enzyme function of PRDX5, seven peptides were designed and named as IMB-P1∼7. The peptide IMB-P1 (AFTPGCSKTHLPGFVEQAEAL) containing critical residue C47 exhibited antitumor activity similar to PRDX5 in vivo. Transcriptome analysis showed peptide IMB-P1 could make influence on expression of multiple genes involved in tumorigenesis and deterioration. Besides, an important discovery is the down-regulation of oxidation-related genes. In CT26 cells, IMB-P1 carried similar antitumor activity with increasing ROS level to intact PRDX5. The results demonstrated that peptide IMB-P1 with easier synthesis from PRDX5 may serve as a promising antitumor candidate.

Rhynchosia ferruginea (Udusalim, Afan Oromo) is a medicinal plant traditionally used to treat skin infection, intestinal problems and amoebiasis. Silica gel chromatographic separation of dichloromethane/methanol (1:1) roots extract yielded isoflavan (1), isoflavene (2) and 1, 3-dilinoleoyl-2-stearoylglycerol (3), reported herein for the first time from the genus. Antibacterial activity was examined using disc diffusion method against E. coli, S. aureus, P. aeruginosa and S. pyogenes. AutoDoc vina 4.2 soft ware was used for molecular docking analysis of compounds against human peroxidoxin 5 and DNA gyrase B enzymes. Isoflavan (1) displayed zone of inhibition of 9.67 ± 0.58 mm and 10.67 ± 0.58 mm whereas isoflavene (2) showed 10.33 ± 1.15 mm and 10 ± 1.00 mm against E. coli and S. aureus, respectively, compared to ciprofloxacin (15.67±0.58 mm for both strains). DPPH radical scavenging assay of the dichloromethane/methanol (1:1) roots extract and isoflavan (1) exhibited better radical scavenging activity with IC50 value of 17.7 and 32, respectively. Molecular docking analysis revealed that 1 and 2 exhibited similar binding affinity of -7.4 kcal/mol compared to ciprofloxacin (-7.3 kcal/mol). In silico analysis against human peroxidoxin 5 (PDB ID: 1HD2) revealed minimum binding affinity of - 3.7 and - 2.0 kcal/mol for compounds 1 and 2, respectively, compared to ascorbic acid (-4.9 kcal/mol). The in vitro antibacterial and antioxidant activity of compounds 1 and 2 suggest the potential use of these compounds as drug lead candidates which corroborate with the traditional uses of the roots of R. ferruginea.

The dithiocarbamate fungicide maneb (MB) has garnered increasing concern due to its association with Parkinson's disease (PD) and the overall negative human health effects of pesticides in agriculture. As one of several environmental toxicant models of PD, MB has been shown to induce global dehydrogenase inhibition, mitochondrial dysfunction, proteostasis disruption, and delayed cell death in neuronal in vitro models. Recently, our group has reported distinct alterations in bioenergetics and energy metabolism after an acute exposure of MB to neuroblastoma cells. Additionally, this acute MB exposure in neuroblastoma also caused a disruption in peroxiredoxin 3 (Prdx3) oxidation, uncovering significant protein thiol interactions. In the current study, human recombinant Prdx3 was evaluated to evaluate direct thiol-thiol interactions between MB and Prdx3 protein. We report a direct, concentration dependent modification of Prdx3 after MB exposure in our system. Further investigation revealed that MB directly adducts Prdx3 protein through disulfide chemistry, similar to the hyperoxidation or glutathionylation of critical cysteine residues. Computational simulations confirm the disulfide bonding on both reduced monomers and oxidized dimers of Prdx3 protein by a possible MB polymer. The data presented strengthens the argument that MB can preferentially target thiol regulatory switches present in many proteins, giving further credence to the MB model of PD in which many disease-associated proteins contain these functional groups, e.g. ubiquinol-cytochrome c reductase, aconitase, and aldehyde dehydrogenase (ALDH2). Furthermore, MB's ability to mimic hyperoxidation of Prdx3 protein introduces a novel dynamic of toxicity within the toxicant model that accounts for the oxidative distress that cannot be reversed by therapeutic anti-oxidants in PD.

Background Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes and ROS. Methods Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure by immunoblotting and immunohistochemistry. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and its major intermediator acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and knock down of NOX4 using siRNA. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls. Results Acute ethanol exposure of mice over 24 h significantly induced autophagy as measured by LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in huh7 cells in the presence of overexpressed CYP2E1. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA mediated knock down. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1-and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid. Conclusion We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS. These data suggest that suppression of autophagy by ethanol is most likely due to longer alcohol exposure during chronic alcohol consumption with the accumulation of e.g. misfolded proteins., Graphical abstract Image 1, Highlights • Generation of ROS is the major mechanism of autophagy activation during ethanol exposure in acute alcoholic liver disease (ALD). • Non-toxic H2O2 significantly induces autophagy without the engagement of mTOR suppression. • Important ROS-generating enzymes CYP2E1 and NOX4 are strongly induced in a mice model of acute alcohol exposure.

Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H2O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 μM H2O2/min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5–3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation., Graphical abstract Image 1

The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes.

Enhanced production of reactive oxygen species (ROS) triggered by various stimuli, including viral infections, has attributed much attention in the past years. It has been shown that different viruses that cause acute or chronic diseases induce oxidative stress in infected cells and dysregulate antioxidant its antioxidant capacity. However, most studies focused on catalase and superoxide dismutases, whereas a family of peroxiredoxins (Prdx), the most effective peroxide scavengers, were given little or no attention. In the current review, we demonstrate that peroxiredoxins scavenge hydrogen and organic peroxides at their physiological concentrations at various cell compartments, unlike many other antioxidant enzymes, and discuss their recycling. We also provide data on the regulation of their expression by various transcription factors, as they can be compared with the imprint of viruses on transcriptional machinery. Next, we discuss the involvement of peroxiredoxins in transferring signals from ROS on specific proteins by promoting the oxidation of target cysteine groups, as well as briefly demonstrate evidence of nonenzymatic, chaperone, functions of Prdx. Finally, we give an account of the current state of research of peroxiredoxins for various viruses. These data clearly show that Prdx have not been given proper attention despite all the achievements in general redox biology.

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenoplicuyngodon idella). The full-length cDNA of CiPrx2 was 1163 by containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 by with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 by encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.

Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (C(P), C(R)). The first step in catalysis, the reduction of peroxide substrate, is fast, 10(7) − 10(8) M(−1)s(−1) for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of C(P), which occurs when a second molecule of peroxide reacts with the C(P) in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the C(P) sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on different mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Abiotic stresses in plants are commonly caused by antioxidant system imbalance in their tissues. A peroxiredoxin/thioredoxin (Prx/Trx) system is a ubiquitous antioxidant system involved in sensing and detoxifying hydrogen peroxide (H2O2) and other reactive oxygen species (ROS). This study investigated exogenous H2O2 potential in reducing browning and its interaction with the Prx/Trx system in fresh-cut guava fruit during storage at 25 °C. Fresh-cut guava samples were immersed in 0 (control) and 250 mM H2O2 for 10 min, placed on a foam tray, packed inside a polyethylene bag and kept at 25 ± 1 °C for 48 h. The H2O2 treatment reduced browning and maintained fresh-cut guava fruit quality during storage. The H2O2 treatment reduced oxidative membrane damage and an accumulation of ROS, but activated the Prx/Trx system by stimulating the activities of peroxiredoxin and thioredoxin reductase, and NADPH-generating dehydrogenases including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as an increase in NADP redox state. The altered redox state and the activation of the Prx/Trx system by H2O2 was correlated with delayed browning of fresh-cut guava. Thus, the Prx/Trx system is involved in browning development, and H2O2 treatment could delay browning in fresh-cut guava by reducing oxidative membrane damage via stimulating the Prx/Trx system.

Peroxiredoxin-5 (Prdx5) is a multifunctional protein involved in oxidative stress, apoptosis and inflammatory responses. However, how Prdx5 functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection increased Prdx5 expression to promote its intracellular growth in macrophages. Further study show that B. abortus infection promoted its intracellular growth by decreasing the production of nitric oxide and reactive oxygen species. In addition, the expression of Prdx5 was independent on live Brucella and the type IV secretion system of Brucella. Instead, its expression was regulated by the lipopolysaccharide of Brucella. Moreover, Brucella infection increased Prdx5 expression in primary macrophage and mice. Collectively, these findings demonstrate for the first time that Prdx5 promotes Brucella intracellular growth by decreasing the production of NO and ROS. This finding provides new insights into the evasive strategies of Brucella and will be useful for the development of novel effective therapeutic approaches to treat Brucella infections.