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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 (Hs Srx), the molecular mechanism of Sulfiredoxin in plants with unique photosynthesis remains unclear. Here we report the crystal structure of Sulfiredoxin from Arabidopsis thaliana (At Srx), 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 At Srx- At PrxA interaction surface of At Srx are more positively charged comparing to Hs Srx, suggesting a robust mechanism of At Srx. These features illustrate the unique mechanisms of At Srx, which are vital for figure out the strategies of plants to cope with oxidation stress. • We solved the crystal structure of Sulfiredoxin from Arabidopsis thaliana (At Srx) in complex with ADP. • At Srx has more positive charges compared with human Srx. • The functional charged amino acid is conserved in photosynthetic organisms. • Structural analysis indicated that more positive charges provided a more robust mechanism. [ABSTRACT FROM AUTHOR]

Significance: The lung is a unique organ, as it is constantly exposed to air, and thus it requires a robust antioxidant defense system to prevent the potential damage from exposure to an array of environmental insults, including oxidants. The peroxiredoxin (PRDX) family plays an important role in scavenging peroxides and is critical to the cellular antioxidant defense system. Recent Advances: Exciting discoveries have been made to highlight the key features of PRDXs that regulate the redox tone. PRDXs do not act in isolation as they require the thioredoxin/thioredoxin reductase/NADPH, sulfiredoxin (SRXN1) redox system, and in some cases glutaredoxin/glutathione, for their reduction. Furthermore, the chaperone function of PRDXs, controlled by the oxidation state, demonstrates the versatility in redox regulation and control of cellular biology exerted by this class of proteins. Critical Issues: Despite the long-known observations that redox perturbations accompany a number of pulmonary diseases, surprisingly little is known about the role of PRDXs in the etiology of these diseases. In this perspective, we review the studies that have been conducted thus far to address the roles of PRDXs in lung disease, or experimental models used to study these diseases. Intriguing findings, such as the secretion of PRDXs and the formation of autoantibodies, raise a number of questions about the pathways that regulate secretion, redox status, and immune response to PRDXs. Future Directions: Further understanding of the mechanisms by which individual PRDXs control lung inflammation, injury, repair, chronic remodeling, and cancer, and the importance of PRDX oxidation state, configuration, and client proteins that govern these processes is needed. [ABSTRACT FROM AUTHOR]

When intracellular reactive oxygen species (ROS) increase, cancer cells are more vulnerable to oxidative stress compared to normal cells; thus, the collapse of redox homeostasis can lead to selective death of cancer cells. Indeed, recent studies have shown that inhibition of sulfiredoxin (Srx), which participates in antioxidant mechanisms, induces ROS-mediated cancer cell death. In this paper, we describe how an Srx inhibitor, J14 (4-[[[4-[4-(2-chlor-ophenyl)-1-piperazinyl]-6-phenyl-2-pyrimidinyl]thio]methyl]-benzoic acid), interferes with the antioxidant activity of Srx at the molecular level. We searched for possible binding sites of Srx using a binding site prediction method and uncovered two possible inhibition mechanisms of Srx by J14. Using molecular dynamics simulations and binding free energy calculations, we confirmed that J14 binds to the ATP binding site; therefore, J14 acts as a competitive inhibitor of ATP, settling the question of the two mechanisms. Based on the inhibition mechanism revealed at the atomic level, we designed several derivatives of J14, which led to LMT-328 (4-(((4-(4-(2-Chlorophenyl)piperazin-1-yl)-6-(2,4-dihydroxy-5-isopropylphenyl)pyrimidin-2-yl)thio)methyl)benzoic acid), which is possibly an even more potent inhibitor than J14. Image 1 • Inhibition of sulfiredoxin (Srx) induces ROS-mediated cancer cell death. • Two possible inhibition mechanisms by Srx inhibitor J14 were uncovered. • J14 binds to the ATP binding site and acts as a competitive inhibitor of ATP. • New potent inhibitor LMT-328 was designed based on the revealed inhibition mechanism. [ABSTRACT FROM AUTHOR]

Redox regulation in heterotrophic organisms relies on NADPH, thioredoxins (TRXs), and an NADPH-dependent TRX reductase (NTR). In contrast, chloroplasts harbor two redox systems, one that uses photoreduced ferredoxin (Fd), an Fd-dependent TRX reductase (FTR), and TRXs, which links redox regulation to light, and NTRC, which allows the use of NADPH for redox regulation. It has been shown that NTRC-dependent regulation of 2-Cys peroxiredoxin (PRX) is critical for optimal function of the photosynthetic apparatus. Thus, the objective of the present study was the analysis of the interaction of NTRC and 2-Cys PRX in vivo and the identification of proteins interacting with them with the aim of identifying chloroplast processes regulated by this redox system. To assess this objective, we generated Arabidopsis thaliana plants expressing either an NTRC–tandem affinity purification (TAP)-Tag or a green fluorescent protein (GFP)–TAP-Tag, which served as a negative control. The presence of 2-Cys PRX and NTRC in complexes isolated from NTRC–TAP-Tag-expressing plants confirmed the interaction of these proteins in vivo. The identification of proteins co-purified in these complexes by MS revealed the relevance of the NTRC–2-Cys PRX system in the redox regulation of multiple chloroplast processes. The interaction of NTRC with selected targets was confirmed in vivo by bimolecular fluorescence complementation (BiFC) assays. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor. Four residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined. In this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates. c-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative C P and C R role. This study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prx), thiol-dependent peroxidases, were first identified as H 2 O 2 detoxifiers, and more recently as H 2 O 2 sensors, intermediates in redox-signaling pathways, metabolism modulators, and chaperones. The multifaceted nature of Prx is not only dependent on their peroxidase activity but also strongly associated with specific protein–protein interactions that are being identified, and where the Prx oligomerization dynamics plays a role. Their oxidation by a peroxide substrate forms a sulfenic acid that opens a route to channel the redox signal to diverse protein targets. Recent research underscores the importance of different Prx isoforms in the cellular processes behind disease development with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Sarcopenia, the age-related decline in muscle mass and function, derives from multiple etiological mechanisms. Accumulative research suggests that reactive oxygen species (ROS) generation plays a critical role in the development of this pathophysiological disorder. In this communication, we review the various signaling pathways that control muscle metabolic and functional integrity such as protein turnover, cell death and regeneration, inflammation, organismic damage, and metabolic functions. Although no single pathway can be identified as the most crucial factor that causes sarcopenia, age-associated dysregulation of redox signaling appears to underlie many deteriorations at physiological, subcellular, and molecular levels. Furthermore, discord of mitochondrial homeostasis with aging affects most observed problems and requires our attention. The search for the primary suspect of the fundamental mechanism for sarcopenia will likely take more intense research for the secret of this health hazard to the elderly to be unlocked.

Since Ronald Konopka and Seymour Benzer’s discovery of the gene Period in the 1970s, the circadian rhythm field has diligently investigated regulatory mechanisms and intracellular transcriptional and translation feedback loops involving Period, and these investigations culminated in a 2017 Nobel Prize in Physiology or Medicine for Michael W. Young, Michael Rosbash, and Jeffrey C. Hall. Although research on 24-hour behavior rhythms started with Period, a series of discoveries in the past decade have shown us that post-transcriptional regulation and protein modification, such as phosphorylation and oxidation, are alternatives ways to building a ticking clock.

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

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.

Heat stress (HS) causes molecular dysfunction that adversely affects chicken performance and increases mortality. The responses of chickens to HS are extremely complex. Thus, the aim of this study was to evaluate the influence of acute and chronic exposure to HS on the expression of thioredoxin–peroxiredoxin system genes and DNA methylation in chickens. Chickens at 14 d of age were divided into two groups and reared under either constant normal temperature (25 °C) or high temperature (35 °C) in individual cages for 12 days. Five birds per group at one and 12 days post-HS were euthanized and livers were sampled for gene expression. The liver and Pectoralis major muscle were sampled for cellular analysis. mRNA expression of thioredoxin and peroxiredoxins (Prdx) 1, 3, and 4 in the liver were down-regulated at 12 days post-HS compared to controls. The liver activity of thioredoxin reductase (TXNRD) and levels of peroxiredoxin1 (Prdx1) at 12 days post-HS were significantly decreased. The results reveal that there was a significant decrease in DNA methylation at 12 days post HS in liver tissues. In conclusion, pathway of thioredoxin system under HS may provide clues to nutritional strategies to mitigate the effect of HS in meat-type chicken.

Hydrogen peroxide (H2O2) is a key redox signaling molecule that selectively oxidizes cysteines on proteins. It can accomplish this even in the presence of highly efficient and abundant H2O2 scavengers, peroxiredoxins (Prdxs), as it is the Prdxs themselves that transfer oxidative equivalents to specific protein thiols on target proteins via their redox-relay functionality. The first evidence of a mammalian cytosolic Prdx-mediated redox-relay—Prdx1 with the kinase ASK1—was presented a decade ago based on the outcome of a co-immunoprecipitation experiment. A second such redox-relay—Prdx2:STAT3—soon followed, for which further studies provided insights into its specificity, organization, and mechanism. The Prdx1:ASK1 redox-relay, however, has never undergone such a characterization. Here, we combine cellular and in vitro protein–protein interaction methods to investigate the Prdx1:ASK1 interaction more thoroughly. We show that, contrary to the Prdx2:STAT3 redox-relay, Prdx1 interacts with ASK1 at elevated H2O2 concentrations, and that this interaction can happen independently of a scaffolding protein. We also provide evidence of a Prdx2:ASK1 interaction, and demonstrate that it requires a facilitator that, however, is not annexin A2. Our results reveal that cytosolic Prdx redox-relays can be organized in different ways and yet again highlight the differentiated roles of Prdx1 and Prdx2.

The catalytic activity of peroxiredoxins (Prx) is determined by the conserved peroxidatic cysteine (CysP), which reacts with peroxides to form sulfenic acid (Cys-SOH). Under conditions of oxidative stress, CysP is oxidized to catalytically inactive sulfinic (Cys-SO2) and sulfonic (Cys-SO3) forms. The Cys-SO2 form can be reduced in a reaction catalyzed by sulfiredoxin (Srx). To explore the physiological significance of peroxiredoxin overoxidation, we investigated daily variations in the oxidation state of 2-Cys peroxiredoxins in flies of different ages, or under conditions when the pro-oxidative load is high. We found no statistically significant changes in the 2-Cys Prxs monomer:dimer ratio, which indirectly reflects changes in the Prx catalytic activity. However, we found daily variations in Prx-SO2/3 that were more pronounced in older flies as well as in flies lacking Srx. Unexpectedly, the srx mutant flies did not exhibit a diminished survivorship under normal or oxidative stress conditions. Moreover, the srx mutant was characterized by a higher physiological activity. In conclusion, catalytically inactive forms of Prx-SO2/3 serve not only as a marker of cellular oxidative burden, but may also play a role in an adaptive response, leading to a positive effect on the physiology of Drosophila melanogaster.

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

Objective(s): Ranking as the sixth commonest cancer, esophageal squamous cell carcinoma (ESCC) represents one of the leading causes of cancer death worldwide. One of the main reasons for the low survival of patients with esophageal cancer is its late diagnosis. Materials and Methods: We used proteomics approach to analyze ESCC tissues with the aim of a better understanding of the malignant mechanism and searching candidate protein biomarkers for early diagnosis of esophageal cancer. The differential protein expression between cancerous and normal esophageal tissues was investigated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Then proteins were identified by matrix-assisted laser desorption/ ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and MASCOT web based search engine. Results:We reported 4 differentially expressed proteins involved in the pathological process of esophageal cancer, such as annexinA1 (ANXA1), peroxiredoxin-2 (PRDX2), transgelin (TAGLN) andactin-aortic smooth muscle (ACTA2). Conclusion: In this report we have introduced new potential biomarker (ACTA2). Moreover, our data confirmed some already known markers for EC in our region.

Abstract Background Bioethanol from lignocellulosic materials is of great significance to the production of renewable fuels due to its wide sources. However, multiple inhibitors generated from pretreatments represent great challenges for its industrial-scale fermentation. Despite the complex toxicity mechanisms, lignocellulose-derived inhibitors have been reported to be related to the levels of intracellular reactive oxygen species (ROS), which makes oxidoreductase a potential target for the enhancement of the tolerance of yeasts to these inhibitors. Results A typical 2-Cys peroxiredoxin from Kluyveromyces marxianus Y179 (KmTPX1) was identified, and its overexpression was achieved in Saccharomyces cerevisiae 280. Strain TPX1 with overexpressed KmTPX1 gene showed an enhanced tolerance to oxidative stresses. Serial dilution assay indicated that KmTPX1 gene contributed to a better cellular growth behavior, when the cells were exposed to multiple lignocellulose-derived inhibitors, such as formic acid, acetic acid, furfural, ethanol, and salt. In particular, KmTPX1 expression also possessed enhanced tolerance to a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. The maximum glucose consumption rate and ethanol generation rate in KmTPX1-expressing strain were significantly improved, compared with the control. The mechanism of improved tolerance to FAF depends on the lower level of intracellular ROS for cell survival under stress. Conclusion A new functional gene KmTPX1 from K. marxianus is firstly associated with the enhanced tolerance to multiple lignocellulose-derived inhibitors in S. cerevisiae. We provided a possible detoxification mechanism of the KmTPX1 for further theoretical research; meanwhile, we provided a powerful potential for application of the KmTPX1 overexpressing strain in ethanol production from lignocellulosic materials.

Similar to humans, the normal aged rat population is not homogeneous in terms of cognitive function. Two distinct subpopulations of aged Sprague–Dawley rats can be identified on the basis of spatial memory performance in the hole-board paradigm. It was the aim of the study to reveal protein changes relevant to aging and spatial memory performance. Aged impaired (AI) and unimpaired (AU) male rats, 22–24 months old were selected from a large cohort of 160 animals; young animals served as control. Enriched synaptosomal fractions from dentate gyrus from behaviorally characterized old animals were used for isobaric tags labeling based quantitative proteomic analysis. As differences in peroxiredoxin 6 (PRDX6) levels were a pronounced finding, PRDX6 levels were also quantified by immunoblotting. AI showed impaired spatial memory abilities while AU performed comparably to young animals. Our study demonstrates substantial quantitative alteration of proteins involved in energy metabolism, inflammation and synaptic plasticity during aging. Moreover, we identified protein changes specifically coupled to memory performance of aged rats. PRDX6 levels clearly differentiated AI from AU and levels in AU were comparable to those of young animals. In addition, it was observed that stochasticity in protein levels increased with age and discriminate between AI and AU groups. Moreover, there was a significantly higher variability of protein levels in AI. PRDX6 is a member of the PRDX family and well-defined as a cystein-1 PRDX that reduces and detoxifies hydroxyperoxides. It is well-known and documented that the aging brain shows increased active oxygen species but so far no study proposed a potential target with antioxidant activity that would discriminate between impaired and unimpaired memory performers. Current data, representing so far the largest proteomics data set in aging dentate gyrus (DG), provide the first evidence for a probable role of PRDX6 in memory performance.

As a component of the photosynthetic apparatus in cyanobacteria, the phycobilisome (PBS) plays an important role in harvesting and transferring light energy to the core photosynthetic reaction centers. The size, composition (phycobiliprotein and chromophore), and assembly of PBSs can be dynamic to cope with tuning photosynthesis and associated cellular fitness in variable light environments. Here, we explore the role of PBS-related stress responses by analyzing deletion mutants of cpcF or cpcG1 genes in Synechocystis sp. PCC 6803. The cpcF gene encodes a lyase that links the phycocyanobilin (PCB) chromophore to the alpha subunit of phycocyanin (PC), a central phycobiliprotein (PBP) in PBSs. Deletion of cpcF (i.e., ΔcpcF strain) resulted in slow growth, reduced greening, elevated reactive oxygen species (ROS) levels, together with an elevated accumulation of a stress-related Peroxiredoxin protein (Sll1621). Additionally, ΔcpcF exhibited reduced sensitivity to a photosynthesis-related stress inducer, methyl viologen (MV), which disrupts electron transfer. The cpcG1 gene encodes a linker protein that serves to connect PC to the core PBP allophycocyanin. A deletion mutant of cpcG1 (i.e.,ΔcpcG1) exhibited delayed growth, a defect in pigmentation, reduced accumulation of ROS, and insensitivity to MV treatment. By comparison, ΔcpcF and ΔcpcG1 exhibited similarity in growth, pigmentation, and stress responses; yet, these strains showed distinct phenotypes for ROS accumulation, sensitivity to MV and Sll1621 accumulation. Our data emphasize an importance of the regulation of PBS structure in ROS-mediated stress responses that impact successful growth and development in cyanobacteria.

Light is probably the most important environmental stimulus for plant development. As sessile organisms, plants have developed regulatory mechanisms that allow the rapid adaptation of their metabolism to changes in light availability. Redox regulation based on disulfide-dithiol exchange constitutes a rapid and reversible post-translational modification, which affects protein conformation and activity. This regulatory mechanism was initially discovered in chloroplasts when it was identified that enzymes of the Calvin-Benson cycle (CBC) are reduced and active during the day and become rapidly inactivated by oxidation in the dark. At present, the large number of redox-sensitive proteins identified in chloroplasts extend redox regulation far beyond the CBC. The classic pathway of redox regulation in chloroplasts establishes that ferredoxin (Fdx) reduced by the photosynthetic electron transport chain fuels reducing equivalents to the large set of thioredoxins (Trxs) of this organelle via the activity of a Fdx-dependent Trx reductase (FTR), hence linking redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. The presence in chloroplasts of this NADPH-dependent redox system raises the question of the functional relationship between NTRC and the Fdx-FTR-Trx pathways. Here, we update the current knowledge of these two redox systems focusing on recent evidence showing their functional interrelationship through the action of the thiol-dependent peroxidase, 2-Cys peroxiredoxin (2-Cys Prx). The relevant role of 2-Cys Prxs in chloroplast redox homeostasis suggests that hydrogen peroxide may exert a key function to control the redox state of stromal enzymes. Indeed, recent reports have shown the participation of 2-Cys Prxs in enzyme oxidation in the dark, thus providing an explanation for the long-lasting question of photosynthesis deactivation during the light-dark transition.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m 2 /min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation Akt Ser437 and AS160 Thr642 were inconclusive, but there was good evidence for increased IRS Ser312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1 Thr423 /2 Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake., Competing Interests: Declaration of competing interest We were unable to get the Elsevier online DOI working, so this document covers declarations., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Schwann cells and oligodendrocytes secrete proteins that promote neuron survival, but their role in amyotrophic lateral sclerosis (ALS) is unclear. To address this question, we evaluated the effect of molecules secreted by Schwann cells on reactive oxygen species (ROS)-induced motor neuronal cell death. We observed that in motor neuron cell line NSC-34 cultures, the conditioned medium (CM) from Schwann cell line YST-1 (YST-1 CM) cultures had a protective effect against hydrogen peroxide-induced cell death. However, this protective effect of YST-1 CM was abolished by removing peroxiredoxin 1-4 (PRDX1-4) from the CM. We found that the expression of PRDX1 mRNA was markedly downregulated in the lumbar spinal cord of the superoxide dismutase 1 (SOD1) G93A mouse model of ALS. We also found that transient transfection of YST-1 cells with G93A SOD1 resulted in reduced PRDX1 mRNA expression. Additionally, in the mutant transfected cells, YST-1 CM showed decreased neuroprotective effect against hydrogen peroxide-induced NSC-34 cell death compared to those transfected with WT SOD1. Our results suggest that Schwann cells protect motor neurons from oxidative stress by secreting PRDX1 and that the reduction of PRDX secreted from Schwann cells contributes to increased ROS and associated motor neuronal death in ALS., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background/aim: As a fundamental staple of the Mediterranean diet, olive oil has long been recognized for its health benefits, including its ability to reduce cardiovascular and neurological disease. Oleuropein is the primary phenolic chemical found in all parts of the olive tree, especially in the leaves and fruit. Oleuropein exhibits anti-inflammatory and antioxidant properties, and has been associated with cancer inhibition in various animal and cell models. We investigated the effects of oleuropein on the MCF-7 human breast cancer cell line, and compared it to the non-cancerous MCF-10A breast epithelial line., Materials and Methods: Both cell lines were treated with two different concentrations of oleuropein for 48 and 72 hours. Cytotoxicity, apoptosis, and peroxiredoxin expression were measured., Results: Forty-eight hours of oleuropein treatment induced cytotoxicity in MCF-7 cells, whereas it had no effect on MCF-10A cells. Furthermore, oleuropein-induced cytotoxicity in MCF-7 cells involved a measurable increase in apoptosis. Oleuropein treatment of MCF-7 cells significantly and dramatically increased expression of all six peroxiredoxin mRNAs (Prdx1-Prdx6), whereas oleuropein treatment of MCF-10A cells resulted in only a small increase in Prdx1 and Prdx6 expression, with no change in the expression of the other peroxiredoxins. Together, these data demonstrate differential susceptibility to oleuropein-induced cell death between the two lines, and differential regulation of peroxiredoxins., Conclusion: Oleuropein-induced over-expression of peroxiredoxins in MCF-7 cells may either facilitate its cancer-specific cytotoxicity or, alternatively, is a consequence of an altered response of cancer cells., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Thiol-dependent redox regulation controls central processes in plant cells including photosynthesis. Thioredoxins reductively activate, for example, Calvin-Benson cycle enzymes. However, the mechanism of oxidative inactivation is unknown despite its importance for efficient regulation. Here, the abundant 2-cysteine peroxiredoxin (2-CysPrx), but not its site-directed variants, mediates rapid inactivation of reductively activated fructose-1,6-bisphosphatase and NADPH-dependent malate dehydrogenase (MDH) in the presence of the proper thioredoxins. Deactivation of phosphoribulokinase (PRK) and MDH was compromised in 2cysprxAB mutant plants upon light/dark transition compared to wildtype. The decisive role of 2-CysPrx in regulating photosynthesis was evident from reoxidation kinetics of ferredoxin upon darkening of intact leaves since its half time decreased 3.5-times in 2cysprxAB. The disadvantage of inefficient deactivation turned into an advantage in fluctuating light. Physiological parameters like MDH and PRK inactivation, photosynthetic kinetics and response to fluctuating light fully recovered in 2cysprxAB mutants complemented with 2-CysPrxA underlining the significance of 2-CysPrx. The results show that the 2-CysPrx serves as electron sink in the thiol network important to oxidize reductively activated proteins and represents the missing link in the reversal of thioredoxin-dependent regulation.

α,β-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,β-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin–Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

Oxidative stress originates from an elevated intracellular level of free oxygen radicals that cause lipid peroxidation, protein denaturation, DNA hydroxylation, and apoptosis, ultimately impairing cell viability. Antioxidants scavenge free radicals and reduce oxidative stress, which further helps to prevent cellular damage. Medicinal plants, fruits, and spices are the primary sources of antioxidants from time immemorial. In contrast to plants, microorganisms can be used as a source of antioxidants with the advantage of fast growth under controlled conditions. Further, microbe-based antioxidants are nontoxic, noncarcinogenic, and biodegradable as compared to synthetic antioxidants. The present review aims to summarize the current state of the research on the antioxidant activity of microorganisms including actinomycetes, bacteria, fungi, protozoa, microalgae, and yeast, which produce a variety of antioxidant compounds, i.e., carotenoids, polyphenols, vitamins, and sterol, etc. Special emphasis is given to the mechanisms and signaling pathways followed by antioxidants to scavenge Reactive Oxygen Species (ROS), especially for those antioxidant compounds that have been scarcely investigated so far.

Stefin B (cystatin B) is an inhibitor of endo-lysosomal cysteine cathepsin, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht–Lundborg disease (EPM1), a form of progressive myoclonus epilepsy. Stefin B-deficient mice, a mouse model of the disease, display key features of EPM1, including myoclonic seizures. Although the underlying mechanism is not yet completely clear, it was reported that the impaired redox homeostasis and inflammation in the brain contribute to the progression of the disease. In the present study, we investigated if lipopolysaccharide (LPS)-triggered neuroinflammation affected the protein levels of redox-sensitive proteins: thioredoxin (Trx1), thioredoxin reductase (TrxR), peroxiredoxins (Prxs) in brain and cerebella of stefin B-deficient mice. LPS challenge was found to result in a marked elevation of Trx1 and TrxR in the brain and cerebella of stefin B deficient mice, while Prx1 was upregulated only in cerebella after LPS challenge. Mitochondrial peroxiredoxin 3 (Prx3), was upregulated also in the cerebellar tissue lysates prepared from unchallenged stefin B deficient mice, while after LPS challenge Prx3 was upregulated in stefin B deficient brain and cerebella. Our results imply the role of oxidative stress in the progression of the disease.

Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRX‑IIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell.

A series of genetically encoded sensors have been developed to detect the important signaling molecule H 2 O 2 in living cells. However, more responsive and sensitive biosensors need to be developed. To address these demands, we used E. coli as a platform to develop a novel fluorescent H 2 O 2 sensor, which we refer to as TScGP. This sensor employs a circularly permuted YFP (cpYFP) and is based on a redox relay between peroxiredoxin (Prx) and thioredoxin (Trx). Structurally, cpYFP is sandwiched between a fungal PrxA and a C-terminal cysteine mutated TrxA that can form a stabilized disulfide bond between PrxA and TrxA in response to H 2 O 2. We confirmed that TScGP can be used for detecting exogenous H 2 O 2 in the range of 0.5–5 μM with high selectivity and rapidly detecting H 2 O 2 within 30 s in E. coli. To demonstrate an application, cellular H 2 O 2 production by menadione was detected directly by TScGP. Our results demonstrated that using Prx-Trx combination as a sensing moiety is another strategy in designing H 2 O 2 sensor with high performance. • Prx-Trx redox relay was utilized to construct H 2 O 2 sensor TScGP for the first time. • TScGP exhibited higher responsiveness than the typical H 2 O 2 probes Hyper. • Application test of TScGP verified the menadione-catalyzed H 2 O 2 producing process. [ABSTRACT FROM AUTHOR]

Plant 2-Cys peroxiredoxins (2-CysPRXs) are abundant plastidial thiol-peroxidases involved in key signaling processes such as photosynthesis deactivation at night. Their functions rely on the redox status of their two cysteines and on the enzyme quaternary structure, knowledge of which remains poor in plant cells. Using ex vivo and biochemical approaches, we thoroughly characterized the 2-CysPRX dimer/monomer distribution, hyperoxidation level, and thiol content in Arabidopsis, barley, and potato in relation to the light cycle. Our data reveal that the enzyme hyperoxidization level and its distribution as a dimer and monomer vary through the light cycle in a species-dependent manner. A differential susceptibility to hyperoxidation was observed for the two Arabidopsis 2-CysPRX isoforms and among the proteins of the three species, and was associated to sequence variation in hyperoxidation resistance motifs. Alkylation experiments indicate that only a minor fraction of the 2-CysPRX pool carries one free thiol in the three species, and that this content does not change during the light period. We conclude that most plastidial 2-CysPRX forms are oxidized and propose that there is a species-dependent variability in their functions since dimer and hyperoxidized forms fulfill distinct roles regarding direct oxidation of partners and signal transmission. [ABSTRACT FROM AUTHOR]

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, 107 - 108 M−1s−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 various 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. Image 1 • Tyrosine nitration of hPrx2 renders a peroxidase more resistant to hyperoxidation. • Nitro Y193 at C-term YF motif increases the rate of disulfide formation. • Side-chain at position 193 affects the rate of the resolution step. • C hyp1% calculated from rate constants correlates well with resolution kinetics. • Rate of resolution step impacts on the fate of Prx2 in redox signaling. [ABSTRACT FROM AUTHOR]

Redox enzymes modulate intracellular redox balance and are secreted in response to cellular oxidative stress, potentially modulating systemic inflammation. Both aerobic and resistance exercise are known to cause acute systemic oxidative stress and inflammation; however, how redox enzyme concentrations alter in extracellular fluids following bouts of either type of exercise is unknown. Recreationally active men (n = 26, mean ± SD: age 28 ± 8 yr) took part in either: 1) two separate energy-matched cycling bouts: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE) or 2) an eccentricbased resistance exercise protocol (RES). Alterations in plasma (study 1) and serum (study 2) peroxiredoxin (PRDX)-2, PRDX-4, superoxide dismutase-3 (SOD3), thioredoxin (TRX-1), TRX-reductase and interleukin (IL)-6 were assessed before and at various timepoints after exercise. There was a significant increase in SOD3 (+1.5 ng/mL) and PRDX-4 (+5.9 ng/mL) concentration following HIIE only, peaking at 30- and 60-min post-exercise respectively. TRX-R decreased immediately and 60 min following HIIE (-7.3 ng/mL) and MOD (-8.6 ng/mL), respectively. In non-resistance trained men, no significant changes in redox enzyme concentrations were observed up to 48 h following RES, despite significant muscle damage. IL-6 concentration increased in response to all trials, however there was no significant relationship between absolute or exercise-induced changes in redox enzyme concentrations. These results collectively suggest that HIIE, but not MOD or RES increase the extracellular concentration of PRDX-4 and SOD3. Exercise-induced changes in redox enzyme concentrations do not appear to directly relate to systemic changes in IL-6 concentration. NEW & NOTEWORTHY Two studies were conducted to characterize changes in redox enzyme concentrations after single bouts of exercise to investigate the emerging association between extracellular redox enzymes and inflammation. We provide evidence that SOD3 and PRDX-4 concentration increased following high-intensity aerobic but not eccentric-based resistance exercise. Changes were not associated with IL-6. The results provide a platform to investigate the utility of SOD3 and PRDX-4 as biomarkers of oxidative stress following exercise. [ABSTRACT FROM AUTHOR]

One of the former definitions of "obligate anaerobiosis" was based on three main criteria: 1) it occurs in organisms, so-called obligate anaerobes, which live in environments without oxygen (O 2), 2) O 2 -dependent (aerobic) respiration, and 3) antioxidant enzymes are absent in obligate anaerobes. In contrast, aerobes need O 2 in order to grow and develop properly. Obligate (or strict) anaerobes belong to prokaryotic microorganisms from two domains, Bacteria and Archaea. A closer look at anaerobiosis covers a wide range of microorganisms that permanently or in a time-dependent manner tolerate different concentrations of O 2 in their habitats. On this basis they can be classified as obligate/facultative anaerobes, microaerophiles and nanaerobes. Paradoxically, O 2 tolerance in strict anaerobes is usually, as in aerobes, associated with the activity of the antioxidant response system, which involves different antioxidant enzymes responsible for removing excess reactive oxygen species (ROS). In our opinion, the traditional definition of "obligate anaerobiosis" loses its original sense. Strict anaerobiosis should only be restricted to the occurrence of O 2 -independent pathways involved in energy generation. For that reason, a term better than "obligate anaerobes" would be O 2 /ROS tolerant anaerobes, where the role of the O 2 /ROS detoxification system is separated from O 2 -independent metabolic pathways that supply energy. Ubiquitous key antioxidant enzymes like superoxide dismutase (SOD) and superoxide reductase (SOR) in contemporary obligate anaerobes might suggest that their origin is ancient, maybe even the beginning of the evolution of life on Earth. It cannot be ruled out that c. 3.5 Gyr ago, local microquantities of O 2 /ROS played a role in the evolution of the last universal common ancestor (LUCA) of all modern organisms. On the basis of data in the literature, the hypothesis that LUCA could be an O 2 /ROS tolerant anaerobe is discussed together with the question of the abiotic sources of O 2 /ROS and/or the early evolution of cyanobacteria that perform oxygenic photosynthesis. Image 1 • Obligate anaerobes live in the absence of O 2 that can be toxic for them. • O 2 tolerance in strict anaerobes is based on the activity of antioxidant enzymes. • SOD and SOR are key antioxidant enzymes that commonly occur in strict anaerobes. • O 2 and ROS might played a role in the evolution of primordial organisms on Earth. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism. [ABSTRACT FROM AUTHOR]

Seeds are the basis of propagation for the common beech (Fagus sylvatica L.), but the seed set of the beech is unsteady, with 5–10 years between abundant crops. Beech seeds are very difficult to store and lose their viability quickly even in optimum storage conditions. To date, it has not been possible to determine factors indicative of the aging process and the loss of viability of beech seeds during storage. To address this important economic challenge and interesting scientific problem, we analyzed the adjustment of the redox state during the development and storage of seeds. Many metabolic processes are based on reduction and oxidation reactions. Thiol proteins control and react to the redox state in the cells. The level of thiol proteins increased during seed maturation and decreased during storage. Gel-based redox proteomics identified 17 proteins in beech seeds during development. The proteins could be assigned to processes like metabolism and antioxidant functions. During storage, the number of proteins decreased to only six, i.e., oxidoreductases, peptidases, hydrolases and isomerases. The occurrence of peroxiredoxins (PRX) as thiol peroxidases and redox regulators indicates an important role of cytosolic 1CysPRX and PRXIIC, mitochondrial PRXIIF, and plastidic PRXIIE, 2CysPRX, and PRXQ in beech seeds during development and storage. Particularly, 2CysPRX was present in beech seeds during development and storage and may perform an important function in regulation of the redox state during both seed development and storage. The role of thiol proteins in the regulation of the redox state during the development and storage of beech seeds is discussed. [ABSTRACT FROM AUTHOR]

Similar to humans, the normal aged rat population is not homogeneous in terms of cognitive function. Two distinct subpopulations of aged Sprague–Dawley rats can be identified on the basis of spatial memory performance in the hole-board paradigm. It was the aim of the study to reveal protein changes relevant to aging and spatial memory performance. Aged impaired (AI) and unimpaired (AU) male rats, 22–24 months old were selected from a large cohort of 160 animals; young animals served as control. Enriched synaptosomal fractions from dentate gyrus from behaviorally characterized old animals were used for isobaric tags labeling based quantitative proteomic analysis. As differences in peroxiredoxin 6 (PRDX6) levels were a pronounced finding, PRDX6 levels were also quantified by immunoblotting. AI showed impaired spatial memory abilities while AU performed comparably to young animals. Our study demonstrates substantial quantitative alteration of proteins involved in energy metabolism, inflammation and synaptic plasticity during aging. Moreover, we identified protein changes specifically coupled to memory performance of aged rats. PRDX6 levels clearly differentiated AI from AU and levels in AU were comparable to those of young animals. In addition, it was observed that stochasticity in protein levels increased with age and discriminate between AI and AU groups. Moreover, there was a significantly higher variability of protein levels in AI. PRDX6 is a member of the PRDX family and well-defined as a cystein-1 PRDX that reduces and detoxifies hydroxyperoxides. It is well-known and documented that the aging brain shows increased active oxygen species but so far no study proposed a potential target with antioxidant activity that would discriminate between impaired and unimpaired memory performers. Current data, representing so far the largest proteomics data set in aging dentate gyrus (DG), provide the first evidence for a probable role of PRDX6 in memory performance. [ABSTRACT FROM AUTHOR]

The chloroplastic thioredoxins (Trxs), a family of thiol-disulphide oxidoreductases, are reduced by either ferredoxin (Fd)-dependent Trx reductase (FTR) or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (NTR). Two Trx systems are present in chloroplasts including Trxs, Trx-like proteins, and reductase FTR and NTRC. FTR is the main reductant for Trxs in chloroplasts, while the flavoprotein NTRC integrates NTR and Trx activity, and plays multiple roles in the Calvin cycle, the oxidative pentose phosphate pathway (OPPP), anti-peroxidation, tetrapyrrole metabolism, ATP and starch synthesis, and photoperiodic regulation. In addition, not only there exists a reduction potential transfer pathway across the thylakoid membrane, but also FTR and NTRC coordinate with each other to regulate chloroplast redox homeostasis. Herein, we summarise the physiological functions of these two Trx reduction systems, discuss how both regulate redox homeostasis in plant plastids, and emphasize the significance of chloroplast thioredoxin systems in maintaining photosynthetic efficiency in plants. • Two thioredoxins (Trxs) systems, Fd/FTR/Trxs and NTRC/Trxs, are present in chloroplasts. • FTR is the main reductant for Trxs in chloroplasts. • NTRC plays multiple roles in redox signalling of chloroplasts. • As Trxs reductase, FTR and NTRC coordinately regulate chloroplast redox homeostasis. [ABSTRACT FROM AUTHOR]

Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration. [ABSTRACT FROM AUTHOR]

Abstract Peroxiredoxin (Prx, EC 1.11.1.15) is a family of the thiol-dependent antioxidant enzyme. In this study, a cold-adapted Prx gene from Antarctic psychrophilic bacterium Psychrobacter sp. ANT206 (PsPrx) consisted of an open reading frame (ORF) of 567 bp was cloned. Amino acid sequence analysis revealed that PsPrx contained one catalytic site (Thr45, Cys48 and Arg121) and could be categorized as a typical 2-Cys Prx. Compared with the mesophilic StPrx, PsPrx with a reduced amount of hydrogen bonds and salt bridges and other characteristics, may be responsible for its enzymatic stability and flexibility at low temperature. The recombinant PsPrx (rPsPrx) was purified to homogeneity by Ni-NTA and its enzymatic characterization was described. Interestingly, rPsPrx exhibited the maximum activity at 30 °C and remained 42.6% of its maximum activity at 0 °C. rPsPrx was a salt-tolerance enzyme that showed 42.2% of its maximum activity under 2.5 M NaCl. The kinetic parameters of different substrates revealed that it could efficiently catalyze the peroxides, especially H 2 O 2 and t -BOOH (tert ‑butyl hydroperoxide). Moreover, rPsPrx exhibited the ability to protect super-coiled DNA from oxidative damage. These results indicated that rPsPrx has special catalytic properties and may be a promising candidate for food and industrial applications. [ABSTRACT FROM AUTHOR]

As a component of the photosynthetic apparatus in cyanobacteria, the phycobilisome (PBS) plays an important role in harvesting and transferring light energy to the core photosynthetic reaction centers. The size, composition (phycobiliprotein and chromophore), and assembly of PBSs can be dynamic to cope with tuning photosynthesis and associated cellular fitness in variable light environments. Here, we explore the role of PBS-related stress responses by analyzing deletion mutants of cpcF or cpcG1 genes in Synechocystis sp. PCC 6803. The cpcF gene encodes a lyase that links the phycocyanobilin (PCB) chromophore to the alpha subunit of phycocyanin (PC), a central phycobiliprotein (PBP) in PBSs. Deletion of cpcF (i.e., Δ cpcF strain) resulted in slow growth, reduced greening, elevated reactive oxygen species (ROS) levels, together with an elevated accumulation of a stress-related Peroxiredoxin protein (Sll1621). Additionally, Δ cpcF exhibited reduced sensitivity to a photosynthesis-related stress inducer, methyl viologen (MV), which disrupts electron transfer. The cpcG1 gene encodes a linker protein that serves to connect PC to the core PBP allophycocyanin. A deletion mutant of cpcG1 (i.e.,Δ cpcG1) exhibited delayed growth, a defect in pigmentation, reduced accumulation of ROS, and insensitivity to MV treatment. By comparison, Δ cpcF and Δ cpcG1 exhibited similarity in growth, pigmentation, and stress responses; yet, these strains showed distinct phenotypes for ROS accumulation, sensitivity to MV and Sll1621 accumulation. Our data emphasize an importance of the regulation of PBS structure in ROS-mediated stress responses that impact successful growth and development in cyanobacteria. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes. Image 1 • Differentiation of HL-60 cells decreases their peroxiredoxin content. • Stimulation of HL-60 cells by PMA or bacteria causes Prx oxidation. • Prxs 1&2 are oxidized in neutrophils despite the presence of reduced thioredoxin. • Neutrophils appear unable to regenerate reduced Prx1 and Prx2. [ABSTRACT FROM AUTHOR]

Inflammation is associated with production of reactive oxygen species (ROS) and results in the induction of thioredoxin (TXN) and peroxiredoxins (PRDXs) and activation of nuclear factor-like 2 (Nrf2). In this study we have used the mouse RAW 264.7 macrophage and the human THP-1 monocyte cell line to investigate the pattern of expression of three Nrf2 target genes, PRDX1, TXN reductase (TXNRD1) and heme oxygenase (HMOX1), by activation of different Toll-like receptors (TLRs). We found that, while the TLR4 agonist lipopolysaccharide (LPS) induces all three genes, the pattern of induction with agonists for TLR1/2, TLR3, TLR2/6 and TLR7/8 differs depending on the gene and the cell line. In all cases, the extent of induction was HMOX1>TXNRD1>PRDX1. Since LPS was a good inducer of all genes in both cell lines, we studied the mechanisms mediating LPS induction of the three genes using mouse RAW 264.7 cells. To assess the role of ROS we used the antioxidant N-acetylcysteine (NAC). Only LPS induction of HMOX1 was inhibited by NAC while that of TXNRD1 and PRDX1 was unaffected. These three genes were also induced by phorbol myristate acetate (PMA), a ROS-inducer acting by activation of protein kinase C (PKC). The protein kinase inhibitor staurosporine inhibited the induction of all three genes by PMA but only that of HMOX1 by LPS. This indicates that activation of these genes by inflammatory agents is regulated by different mechanisms involving either ROS or protein kinases, or both. Image 1 • Inflammation induces Nrf2 target genes Prdx1, Txnrd1 and Hmox1. • Different Toll-like receptors agonists induce these genes differently. • Induction of Prdx1 and Txnd1 by TLR4 is ROS-independent, Hmox1 is protein kinase-dependent. • Assuming that inflammation activates Nrf2 via ROS is an oversimplification. [ABSTRACT FROM AUTHOR]

The Lasbcp (CLIBASIA_RS00445) 1-Cys peroxiredoxin gene is conserved among all 13 sequenced strains of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing or "citrus greening" disease. LasBCP was previously characterized as a secreted peroxiredoxin with substrate specificity for organic peroxides, and as a potential pathogenicity effector. Agrobacterium -mediated transient expression of LasBCP in citrus leaves provided significant protection against peroxidation of free and membrane-bound lipids, thereby preserving the molecular integrity of the chlorophyll apparatus and reducing accumulation of lipid peroxidation products (oxylipins) following exposure to tert -butyl hydroperoxide (t BOOH, an organic peroxide). Oxylipins extracted from GUS-expressing citrus leaves reduced viability of L. crescens , the only Liberibacter species cultured to date. However, similar extracts obtained from LasBCP-expressing leaves were less inhibitory to L. crescens growth and viability in culture. Quantitative RT-PCR analyses showed coordinated transcriptional downregulation of oxylipin biosynthetic (CitFAD , CitLOX , CitAOS and CitAOC), and jasmonic acid (JA) (CitJAR1 , CitCOI1 and CitJIN1) and salicylic acid (SA) (CitPAL , CitICS and CitPR1) signaling pathway genes in citrus leaves expressing LasBCP and treated with t BOOH. The negative response regulator of jasmonic acid CitJAZ1 was upregulated in LasBCP-expressing citrus leaves under similar conditions. These data clearly demonstrated a protective role of secreted LasBCP in favor of Las survival and colonization by alleviating ROS-induced lipid peroxidation in citrus host, preventing accumulation of antimicrobial oxylipins, and suppressing both localized and systemic immune responses in planta. [ABSTRACT FROM AUTHOR]

B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR‐ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP‐ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP‐ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP‐ALL cell lines and pediatric and adult BCP‐ALL primary cells, including primary cells cocultured with bone marrow‐derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL‐rearranged patient‐derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP‐ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti‐BCP‐ALL drugs should be continued. [ABSTRACT FROM AUTHOR]

Oligomeric proteins are abundant in nature and are useful for a range of nanotechnological applications; however, a key requirement in using these proteins is controlling when and how they form oligomeric assemblies. Often, protein oligomerisation is triggered by various cellular signals, allowing for controllable oligomerisation. An example of this is human peroxiredoxin 3 (Prx), a stable protein that natively forms dimers, dodecameric rings, stacks, and tubes in response to a range of environmental stimuli. Although we know the key environmental stimuli for switching between different oligomeric states of Prx, we still have limited molecular knowledge and control over the formation and size of the protein's stacks and tubes. Here, we have generated a range of Prx mutants with either a decreased or knocked out ability to stack, and used both imaging and solution studies to show that Prx stacks through electrostatic interactions that are stabilised by a hydrogen bonding network. Furthermore, we show that altering the length of the polyhistidine tag will alter the length of the Prx stacks, with longer polyhistidine tags giving longer stacks. Finally, we have analysed the effect a variety of heavy metals have on the oligomeric state of Prx, wherein small transition metals like nickel enhances Prx stacking, while larger positively charged metals like tungstate ions can prevent Prx stacking. This work provides further structural characterisation of Prx, to enhance its use as a platform from which to build protein nanostructures for a variety of applications. • Peroxiredoxin mutants made with a decreased or knocked out ability to form stacks. • Altering the length of the polyhistidine tag changes the length of stacks. • Phosphotungstate ions alter peroxiredoxin oligomerisation properties. • Nickel ions stabilise peroxiredoxin oligomeric assemblies. [ABSTRACT FROM AUTHOR]

Light is probably the most important environmental stimulus for plant development. As sessile organisms, plants have developed regulatory mechanisms that allow the rapid adaptation of their metabolism to changes in light availability. Redox regulation based on disulfide-dithiol exchange constitutes a rapid and reversible post-translational modification, which affects protein conformation and activity. This regulatory mechanism was initially discovered in chloroplasts when it was identified that enzymes of the Calvin-Benson cycle (CBC) are reduced and active during the day and become rapidly inactivated by oxidation in the dark. At present, the large number of redox-sensitive proteins identified in chloroplasts extend redox regulation far beyond the CBC. The classic pathway of redox regulation in chloroplasts establishes that ferredoxin (Fdx) reduced by the photosynthetic electron transport chain fuels reducing equivalents to the large set of thioredoxins (Trxs) of this organelle via the activity of a Fdx-dependent Trx reductase (FTR), hence linking redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. The presence in chloroplasts of this NADPH-dependent redox system raises the question of the functional relationship between NTRC and the Fdx-FTR-Trx pathways. Here, we update the current knowledge of these two redox systems focusing on recent evidence showing their functional interrelationship through the action of the thiol-dependent peroxidase, 2-Cys peroxiredoxin (2-Cys Prx). The relevant role of 2-Cys Prxs in chloroplast redox homeostasis suggests that hydrogen peroxide may exert a key function to control the redox state of stromal enzymes. Indeed, recent reports have shown the participation of 2-Cys Prxs in enzyme oxidation in the dark, thus providing an explanation for the long-lasting question of photosynthesis deactivation during the light-dark transition. [ABSTRACT FROM AUTHOR]