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Oxidation of thiol proteins and redox signaling occur in cells exposed to H 2 O 2 but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H 2 O 2 with CO 2 /bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with H 2 O 2 were measured by isotopically labeling reduced thiols and analysis by mass spectrometry. The effects of bicarbonate and of knocking out either Prdx1 or Prdx2 were examined. Approximately 14,000 Cys-peptides were detected, of which ∼1 % underwent 2–10 fold loss in thiol content with H 2 O 2. Those showing the most oxidation were not affected by the presence of bicarbonate or knockout of either Prdx. Consistent with previous evidence that bicarbonate potentiates inactivation of glyceraldehyde-3-phosphate dehydrogenase, the GAPDH active site Cys residues were significantly more sensitive to H 2 O 2 when bicarbonate was present. Several other proteins were identified as promising candidates for further investigation. Although we identified some potential protein candidates for Prdx-dependent oxidation, most of the significant differences between KO and WT cells were seen in proteins for which H 2 O 2 unexpectedly increased their CysSH content over untreated cells. We conclude that facilitation of protein oxidation by bicarbonate or Prdx-mediated relays is restricted to a small number of proteins and is insufficient to explain the majority of the oxidation of the cell thiols that occured in response to H 2 O 2. [Display omitted] • The effect of 100 μM H 2 O 2 (20 nM/106 cells) on the reduced thiol proteome of Jurkat cells was determined. • More than 2-fold loss in -SH was seen for ∼1 % of 14,000 Cys-peptides. • Most changes were independent of bicarbonate. • Oxidation of GAPDH and several other proteins was enhanced by bicarbonate. • Proteins showing the most oxidation were not affected by knockout of Prdx1 or Prdx2. • No compelling evidence for new Prdx-mediated redox relays was revealed. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are important regulators of cellular peroxide metabolism. As antioxidants, they restrict oxidation of other cell proteins, but as signaling molecules they can act as sensors and promote thiol protein oxidation via a redox relay mechanism. The presence of peroxiredoxins could therefore influence other thiol proteins, even in cells experiencing endogenous redox activity. To investigate this for the two cytoplasmic peroxiredoxins, Prdx1 and Prdx2, we have compared the thiol proteome of wildtype Jurkat cells with cells in which either one was knocked out. Using mass spectrometry and isotope tagging, approximately 10,000 common CysSH-containing peptides were detected for each WT/KO comparison. Knockout of Prdx1 or Prdx2 resulted in a change in redox state of a small selection of Cys residues, with less than 100 giving more than a 2-fold difference. Strikingly, a large proportion of these, including those that showed the greatest change, were common to both KOs. Some Cys residues showed more oxidation in the knockouts, whereas others showed less. The candidate proteins have diverse functions and have not been known to be oxidant sensitive. No differences were seen in redox state of Cys residues of other Prdxs and oxidant sensitive proteins. A change in expression in Prdx2 knockout cells was indicated for seven cytoskeletal or regulatory thiol proteins, three of which were tested and validated by western blotting. Little firm evidence was found for thiol redox changes dependent on either Prdx that could be attributed to oxidation via a relay mechanism. [Display omitted] • Redox proteomics was used to compare levels of reduced Cys residues in wildtype and Prdx1 or Prdx knockout Jurkat cells. • Less than 1 % of the 10,000 Cys-containing peptides detected showed changes. • A large proportion of changes were common to both knockouts. • No strong evidence for knockout of one of the Prdxs affecting the redox state of other Cys proteins. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the consequences of deleting the glutaredoxin-2 gene (Glrx2 −/− ) on the development of non-alcoholic fatty liver disease (NAFLD) in male and female C57BL6N mice fed a control (CD) or high-fat diet (HFD). We report that the HFD induced a significant increase in body mass in the wild-type (Wt) and Glrx2 −/− male, but not female, mice, which was associated with the hypertrophying of the abdominal fat. Interestingly, while the Wt male mice fed the HFD developed NAFLD, the deletion of the Glrx2 gene mitigated vesicle formation, intrahepatic lipid accumulation, and fibrosis in the males. The protective effect associated with ablating the Glrx2 gene in male mice was due to enhancement of mitochondrial redox buffering capacity. Specifically, liver mitochondria from male Glrx2 −/− fed a CD or HFD produced significantly less hydrogen peroxide (mtH 2 O 2), had lower malondialdehyde levels, greater activities for glutathione peroxidase and thioredoxin reductase, and less protein glutathione mixed disulfides (PSSG) when compared to the Wt male mice fed the HFD. These effects correlated with the S-glutathionylation of α-ketoglutarate dehydrogenase (KGDH), a potent mtH 2 O 2 source and key redox sensor in hepatic mitochondria. In comparison to the male mice, both Wt and Glrx2 −/− female mice displayed almost complete resistance to HFD-induced body mass increases and the development of NAFLD, which was attributed to the superior redox buffering capacity of the liver mitochondria. Together, our findings show that modulation of mitochondrial S-glutathionylation signaling through Glrx2 augments resistance of male mice towards the development of NAFLD through preservation of mitochondrial redox buffering capacity. Additionally, our findings demonstrate the sex dimorphisms associated with the manifestation of NAFLD is related to the superior redox buffering capacity and modulation of the S-glutathionylome in hepatic mitochondria from female mice. [Display omitted] • Glrx2 gene knockout prevents NAFLD manifestation in male mice. • Glrx2 gene knockout increases redox buffering in liver mitochondria. • Female mice resistant to NAFLD due to increased redox buffering capacity. • Succinate, aspartate, GSSG new sex-dependent circulating markers for NAFLD. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are antioxidant proteins that detoxify peroxynitrite, hydrogen peroxide, and organic hydroperoxides, impacting various physiological processes such as immune responses, apoptosis, cellular homeostasis, and so on. In the present study, we identified and characterized peroxiredoxin 1 from Antheraea pernyi (thereafter designated as ApPrx-1) that encodes a predicted 195 amino acid residue protein with a 21.8 kDa molecular weight. Quantitative real-time PCR analysis revealed that the mRNA level of ApPrx-1 was highest in the hemocyte, fat body, and midgut. Immune-challenged larval fat bodies and hemocytes showed increased ApPrx-1 transcript. Moreover, ApPrx-1 expression was induced in hemocytes and the whole body of A. pernyi following exogenous H 2 O 2 administration. A DNA cleavage assay performed using recombinant ApPrx-1 protein showed that rApPrx-1 protein manifests the ability to protect supercoiled DNA damage from oxidative stress. To test the rApPrx-1 protein antioxidant activity, the ability of the rApPrx-1 protein to remove H 2 O 2 was assessed in vitro using rApPrx-1 protein and DTT, while BSA + DDT served as a control group. The results revealed that ApPrx-1 can efficiently remove H 2 O 2 in vitro. In the loss of function analysis, we found that ApPrx-1 significantly increased the levels of H 2 O 2 in ApPrx-1 -depleted larvae compared to the control group. We also found a significantly lower survival rate in the larvae in which ApPrx-1 was knocked down. Interestingly, the antibacterial activity was significantly higher in the ApPrx-1 depleted larvae, compared to the control. Collectively, evidence strongly suggests that ApPrx-1 may regulate physiological activities and provides a reference for further studies to validate the utility of the key genes involved in reliving oxidative stress conditions and regulating the immune responses of insects. • Prx-1 gene was identified from the Chinese oak silkworm, A. pernyi. • Microbial challenges modulate the expression patterns of ApPrx-1 in immune tissues. • Recombinant ApPrx-1 protein protects supercoiled DNA damage from oxidative stress. • Silencing of ApPrx-1 strongly affected the H 2 O 2 and bacterial clearance in A. pernyi. [ABSTRACT FROM AUTHOR]

In addition to their role in the breakdown of H 2 O 2 , some peroxiredoxins (Prxs) have chaperone and H 2 O 2 sensing functions. Acting as an H 2 O 2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA , ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H 2 O 2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H 2 O 2 , indicating its critical role in H 2 O 2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development. [ABSTRACT FROM AUTHOR]

The redox state of proteins is essential for their function and guarantees cell fitness. Peroxiredoxins protect cells against oxidative stress, maintain redox homeostasis, act as chaperones, and transmit hydrogen peroxide signals to redox regulators. Despite the profound structural and functional knowledge of peroxiredoxins action, information on how the different functions are concerted is still scarce. Using global proteomic analyses, we show here that the yeast peroxiredoxin Tsa1 interacts with many proteins of essential biological processes, including protein turnover and carbohydrate metabolism. Several of these interactions are of a covalent nature, and we show that failure of peroxiredoxinylation of Gnd1 affects its phosphogluconate dehydrogenase activity and impairs recovery upon stress. Thioredoxins directly remove TSA1-formed mixed disulfide intermediates, thus expanding the role of the thioredoxin-peroxiredoxin redox cycle pair to buffer the redox state of proteins. [Display omitted] • The peroxiredoxin Tsa1 interacts with a large fraction of the proteome in a redox manner • Covalent Tsa1 attachment to target proteins (peroxiredoxinylation) increases upon stress • Metabolic enzymes and proteins involved in translation are targeted by Tsa1 • Stress survival requires peroxiredoxinylation of the 6-phosphogluconate dehydrogenase In redox signaling, peroxiredoxins are key regulators modulating cysteine oxidation on target proteins. Here, Seisenbacher et al. report a redox-sensitive interactome of the S. cerevisiae peroxiredoxin Tsa1 and show that the covalent attachment of Tsa1 to target proteins protects cellular processes and contributes to survival upon environmental stress. [ABSTRACT FROM AUTHOR]

The effects of overexpression of the thioredoxin-like protein CDSP32 (Trx CDSP32) on reactive oxygen species (ROS) metabolism in tobacco leaves exposed to cadmium (Cd) were studied by combining physiological measures and proteomics technology. Thus, the number of differentially expressed proteins (DEPs) in plants overexpressing the Trx CDSP32 gene in tobacco (OE) was observed to be evidently lower than that in wild-type (WT) tobacco under Cd exposure, especially the number of down-regulated DEPs. Cd exposure induced disordered ROS metabolism in tobacco leaves. Although Cd exposure inhibited the activities of superoxide dismutase (SOD), catalase (CAT), and l -ascorbate peroxidase (APX) and the expression of proteins related to the thioredoxin-peroxiredoxin (Trx-Prx) pathway, the increase in the activities of peroxidase (POD), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S -transferase (GST) and their protein expression levels played an important role in the physiological response to Cd exposure. Notably, Trx CDSP32 was observed to alleviate the decrease in the expression and activities of SOD and CAT caused by Cd exposure and enhance the function of POD. Trx CDSP32 was observed to increase the H 2 O 2 scavenging capacity of the ascorbic acid-glutathione (AsA-GSH) cycle and Trx-Prx pathway under Cd exposure, and it can especially regulate 2-Cys peroxiredoxin (2-Cys Prx) protein expression and thioredoxin peroxidase (TPX) activity. Thus, overexpression of the Trx CDSP32 gene can alleviate the oxidative damage that occurs in tobacco leaves under Cd exposure by modulating antioxidant defense systems. • Trx CDSP32 alleviated Cd-induced oxidative damage in tobacco leaves. • Trx CDSP32 enhanced the function of antioxidant enzymes such as SOD, POD and CAT. • Trx CDSP32 increased the ability of AsA-GSH cycle and Trx-Prx pathway to remove H 2 O 2. [ABSTRACT FROM AUTHOR]

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

Although microbial pathogens utilize various strategies to evade plant immunity, host plants have evolved powerful defense mechanisms that can be activated in preparation for threat by infective organisms. Here, we identified one 24 kDa alkyl hydroperoxide reductase C (AhpC) from the culture supernatant of Ralstonia solanacearum strain FQY-4 (denoted RsAhpC) in the presence of host roots. RsAhpC contributes to H 2 O 2 detoxification and the pathogenicity of R. solanacearum. However, the introduction of RsAhpC into the apoplast could activate immune defense, leading to suppression of pathogen colonization in both Nicotiana benthamiana and the Honghua Dajinyuan (HD) cultivar of N. tabacum. Consequently, overexpression of RsAhpC in the HD cultivar enhanced the resistance of tobacco to bacterial wilt disease caused by FQY-4. Overall, this study provides insight into the arms race between pathogens and their plant hosts. Specifically, it is firstly reported that plants can sense pathogen-derived AhpC to activate defenses, in addition to the role of AhpC in pathogen ROS detoxification. Therefore, the macromolecule AhpC produced by Ralstonia solanacearum has the ability to enhance plant defense as an elicitor, which provides a practical strategy for disease resistance breeding. [ABSTRACT FROM AUTHOR]

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii , shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis. By using an array of chemical biology techniques, the direct target of celastrol and its molecular mechanisms of action are explored as the treatment of liver fibrosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically.Objectives: We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.Methods: A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0-200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.Results: Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 μM, 4 hours; P < 0.05).Conclusions: These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency. [ABSTRACT FROM AUTHOR]

H 2 O 2 signals trigger adaptive responses affecting cell division, differentiation, migration, and survival. These signals are transduced by selective oxidation of cysteines on specific target proteins, with redox-sensitive cysteines now identified in many proteins, including both kinases and phosphatases. Assessing the contribution of these oxidation events to cell signalling presents several challenges including understanding how and when the selective oxidation of specific proteins takes place in vivo. In recent years, a combination of biochemical, structural, genetic, and computational approaches in fungi, plants, and animals have revealed different ways in which thiol peroxidases (peroxiredoxins) are bypassed or utilised in relaying these signals. Together, these mechanisms provide a conceptual framework for selectively oxidising proteins that will further advance understanding of how redox modifications contribute to health and disease. [Display omitted] [ABSTRACT FROM AUTHOR]

• The mechanisms underlying the diabetogenic effects of POPs remain poorly known. • POPs reduce insulin production and secretion of β-cells. • POPs impair the oxidant/antioxidant balance of β-cells. • NAC treatment restores the oxidant/antioxidant balance of POP-treated β-cells. • NAC treatment allows POP-treated β-cells to normally produce and secrete insulin. Persistent organic pollutants (POPs) have emerged as potent diabetogenic agents, but their mechanisms of action remain poorly identified. In this study, we aim to determine the mechanisms regulating the damaging effects of POPs in pancreatic β-cells, which have a central role in the development of diabetes. We treated INS-1E pancreatic β-cells with PCB-153, p,p'-DDE, PCB-126, or TCDD at doses ranging from 1 × 10-15 to 5 × 10-6 M. We measured insulin content and secretion, cell viability and assessed the mRNA expression of the xenobiotic nuclear receptors Nr1i2 and Nr1i3 , and the aryl hydrocarbon receptor (Ahr). In addition, we evaluated the antioxidant defense and production of reactive oxygen species (ROS). Finally, we studied the ability of the antioxidant N -acetyl-L-cysteine (NAC) to counteract the effects of POPs in INS-1E cells. When exposed to environmental POP levels, INS-1E cells had impaired production and secretion of insulin. These defects were observed for all tested POPs and were paralleled by reduced Ins1 and Ins2 mRNA expression. While POP treatment for 3 days did not affect INS-1E cell viability, longer treatment progressively killed the cells. Furthermore, we found that the xenobiotic detoxification machinery is poorly expressed in the INS-1E cells, as characterized by the absence of Nr1i2 and Nr1i3 and their respective downstream targets Cyp3a1 / Cyp3a2 and Cyp2b1 / Cyp2b3 , and the weak functionality of the Ahr / Cyp1a1 signaling. Interestingly, POPs dysregulated key antioxidant enzymes such as glutathione peroxidases, peroxiredoxins, thioredoxins, and catalases. In parallel, the production of intracellular ROS, including superoxide anion (O 2 •−) and hydrogen peroxide (H 2 O 2), was increased by POP exposure. Improving the oxidant scavenging capacity of INS-1E cells by NAC treatment restored the production and secretion of insulin. By promoting oxidative stress and impairing the ability of INS-1E cells to produce and secrete insulin, this study reveals how POPs can mechanistically act as diabetogenic agents, and provides new scientific evidence supporting the concept that POPs are fueling the diabetes epidemics. [ABSTRACT FROM AUTHOR]

Aerobic organisms possess numerous antioxidant enzymatic families, including catalases, superoxide dismutases (SODs), peroxiredoxins (PRDXs), and glutathione peroxidases (GPXs), which work cooperatively to protect cells from an excess of reactive oxygen species (ROS) derived from endogenous metabolism or external microenvironment. Catalase, as well as other antioxidant enzymes, plays an important dichotomous role in cancer. Therefore, therapies aimed at either reverting the increased or further escalating catalase levels could be effective, depending on the metabolic landscape and on the redox status of cancer cells. This dichotomous role of catalase in cancers highlights the importance to deepen comprehensively the role and the regulation of this crucial antioxidant enzyme. The present review highlights the role of catalase in cancer and provides a comprehensive description of the molecular mechanisms associated with the multiple levels of catalase regulation. [Display omitted] • Highlights the role of catalase in cancer. • Provides comprehensive review of data from the literature on alterations of catalase in various cancer types. • Provides a comprehensive description of the molecular mechanisms associated with the multiple levels of catalase regulation. • Provides a detailed review on mechanisms controlling catalase expression with a specific focus on dysregulations in cancer. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (PRDXs) are abundant peroxidases present in all kingdoms of life. Recently, they have been shown to also carry out additional roles as molecular chaperones. To address this emerging supplementary function, this review focuses on structural studies of 2-Cys PRDX systems exhibiting chaperone activity. We provide a detailed understanding of the current knowledge of structural determinants underlying the chaperone function of PRDXs. Specifically, we describe the mechanisms which may modulate their quaternary structure to facilitate interactions with client proteins and how they are coordinated with the functions of other molecular chaperones. Following an overview of PRDX molecular architecture, we outline structural details of the presently best-characterized peroxiredoxins exhibiting chaperone function and highlight common denominators. Finally, we discuss the remarkable structural similarities between 2-Cys PRDXs, small HSPs, and J-domain-independent Hsp40 holdases in terms of their functions and dynamic equilibria between low- and high-molecular-weight oligomers. [Display omitted] Troussicot et al. review high-molecular-weight structures and structural determinants of chaperone function in typical 2-cysteine peroxiredoxin antioxidants, tumor suppressors, and signaling modulators. Structure-function relationships of the three currently best understood 2-cysteine peroxiredoxin chaperones are highlighted and compared with small heat shock proteins and J-domain-independent Hsp40 holdases. [ABSTRACT FROM AUTHOR]

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

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H 2 O 2 and t -BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H 2 O 2 better than t -BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at K mapp = 11.7 mM , V maxapp = 10.9 IU/μg TrGPx, k cat = 19 s−1 and a catalytic efficiency of 1.6 mM−1 s−1 to H 2 O 2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0–12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications. [ABSTRACT FROM AUTHOR]

Skeletal muscle generates superoxide during contractions which is rapidly converted to H 2 O 2. This molecule has been proposed to activate signalling pathways and transcription factors that regulate key adaptive responses to exercise but the concentration of H 2 O 2 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 H 2 O 2 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 H 2 O 2 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. Image 1 • Hydrogen peroxide is generated by skeletal muscle during contractions. • Peroxiredoxins (Prx) react with H 2 O 2 at the physiological levels generated during contractions. • Prx 1–3 are all oxidised by H 2 O 2 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. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) is a thiol-based peroxidase that eliminates reactive oxygen species to avoid oxidative damage. Alkyl hydroperoxide reductase Ahp1 is a novel and specific typical 2-cysteine Prx. Here, we present the crystal structure of sulfonic Ahp1 complexed with thioredoxin Trx2 at 2.12 Å resolution. This structure implies that the transient Ahp1-Trx2 complex during the catalytic cycle already have an ability to decompose the peroxides. Structural analysis reveals that the segment glutamine23–lysine32 juxtaposed to the resolving cysteine (C R) of Ahp1 moves inward to generate a compact structure upon peroxidatic cysteine (C P) overoxidation, resulting in the breakdown of several conserved hydrogen bonds formed by Ahp1-Trx2 complex interaction. Structural comparisons suggest that the structure of sulfonic Ahp1 represents a novel conformation of Ahp1, which can mimic a conformational intermediate between the reduced and oxidized forms. Therefore, this study may provide a new structural insight into the intermediate state in which the segment glutamine23–lysine32 juxtaposed to the cysteine31 (C R) undergoes a conformational change upon cysteine62 (C P) oxidation to prepare for the formation of an intermolecular C P -C R disulfide bond during Ahp1 catalytic cycle. • 2.12 Å resolution crystal structure of sulfonic Ahp1 in complex with Trx2 is provided. • Transient Ahp1-Trx2 complex already possess an ability to decompose the peroxides. • Segment Gln23–Lys32 of Ahp1 moves inward upon peroxidatic cysteine overoxidation. • Conserved hydrogen bonds breakdown of Ahp1-Trx2 caused by movement of Gln23–Lys32. • Provided structure can mimic an Ahp1 intermediate between reduced and oxidized forms. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 6 (Prdx6) is a unique enzyme among mammalian peroxiredoxins as it lacks resolving cysteine. It is found to be involved in number of different diseases including tumours and its expression level is highest in lungs as compared to other organs. It has been found that Prdx6 plays a significant role different metabolic diseases, ocular damage, neurodegeneration and male infertility. It is a bifunctional protein having phospholipase A 2 and peroxidase (also has the ability to reduce phospholipid hydroperoxides) activities. In order to complete the peroxidise reaction cycle it requires glutathione catalyzed by glutathione S-transferase. Equilibrium unfolding and conformational stability of Prdx6 was studied by using urea as a chemical denaturant to understand the changes it goes under cellular stress conditions. Three different spectroscopic methods were employed to monitor urea-induced denaturation. From the results obtained, it was found that the urea denaturation of Prdx6 follows a variable two state process due to non-coincidence of the normalized transition curves obtained from different optical probes. The different denaturation curves were normalized and thermodynamic parameters, Δ G D o, Gibbs free energy change related to the urea-induced denaturation, midpoint of denaturation (C m), and m = (δΔ G D / [urea]) were obtained. The structural information of Prdx6 were further analysed by several parameters obtained by 100 ns MD simulation. The results of MD simulation clearly favour the outcome of spectroscopic studies. [ABSTRACT FROM AUTHOR]

p53 is a tumor suppressor protein with a very low content in the basal condition, but the content rapidly rises during stress conditions including ischemia-reperfusion. An increase in p53 content increases cardiac injury during ischemia-reperfusion. Since mitochondrial damage plays a key role in cardiac injury during ischemia-reperfusion, we asked if genetic ablation of p53 decreases cardiac injury by protecting mitochondria. Isolated, perfused hearts from cardiac specific p53 deletion or wild type underwent 25 min global ischemia at 37 °C and 60 min reperfusion. At the end of reperfusion, hearts were harvested for infarct size measurement. In separate groups, cardiac mitochondria were isolated at 30 min reperfusion. Time control hearts were buffer-perfused without ischemia. Compared to wild type, deletion of p53 improved cardiac functional recovery and decreased infarct size following ischemia-reperfusion. Oxidative phosphorylation was improved in p53 deletion mitochondria following ischemia-reperfusion compared to wild type. The net release of ROS generation from wild type but not in p53 deletion mitochondria was increased following ischemia-reperfusion. Peroxiredoxin 3 (PRDX 3) content was higher in p53 deletion than that in wild type, indicating that p53 deletion increases a key antioxidant. Ischemia-reperfusion led to increased spectrin cleavage (a marker of cytosolic calpain1 activation) in wild type but not in p53 deletion mice. Ischemia-reperfusion increased the truncation of mature AIF (apoptosis inducing factor, an indicator of mitochondrial calpain1 activation) in wild type but not in p53 deletion mice. The loss of cytochrome c from mitochondria was also decreased in p53 deletion following ischemia-reperfusion. Bcl-2 content was decreased in wild type but not in p53 deletion following reperfusion, suggesting that depletion of bcl-2 contributes to permeabilization of the mitochondrial outer membrane. Thus, deletion of p53 decreases cardiac injury by protecting mitochondria through attenuation of oxidative stress and calpain activation during ischemia-reperfusion. Image 1 • Cardiomyocyte specific deletion of p53 decreases cardiac injury in mouse hearts following ischemia-reperfusion by protecting mitochondria. • Cardiomyocyte specific deletion of p53 maintains bcl-2 content during ischemia-reperfusion with enhanced retention of cytochrome c by mitochondria. • p53 knockout increased peroxiredoxin 3 content and decreased ROS generation from mitochondria. • Deletion of p53 in cardiac myocytes decreased cytosolic and mitochondrial calpain 1 activation during ischemia-reperfusion. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana atypical Cys His-rich thioredoxins (ACHTs) are a small class of atypical thioredoxins (TRXs) located in chloroplasts thylakoids and are characterized by a noncanonical motif at their redox active site, C (G/S)(S/G)C. Previous studies have reported that ACHT1 can interact with A. thaliana 2-Cys peroxiredoxins (2-Cys Prxs, including PrxA and PrxB) to transmit oxidation signals in response to illumination with normal light intensity. In this study, we reported the crystal structure of ACHT1 and show that ACHT1 adopts a canonical TRX fold. Comparison of the structures of ACHT1 in both reducing and oxidizing environments revealed that while the redox environment did not influence the overall structure of ACHT1, it did change the conformation of its catalytic residues. We found that the catalytic C125 of ACHT1 is the target residue for PrxA in vitro. In addition, we found that ACHT1 can reduce the peroxidase activity of PrxA, and further confirmed that the ability of ACHT1 to restore the peroxidase function of PrxA was due to the interaction between the two. Our results provide a structural basis for studying the function of atypical TRXs and the oxidative regulation mechanism of ACHT1 and 2-Cys Prxs in chloroplasts. • The ACHT1 structure provides the first atomic information on chloroplast TRX. • The catalytic C125 of ACHT1 is the target residue for 2-Cys PrxA. • ACHT1 forms intermolecular disulfide bonds with 2-Cys PrxA. • Interaction with ACHT1 restores the peroxidase activity of 2-Cys PrxA. • The function of chloroplast TRXs mainly depends on the TRX fold. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, Tt Bcp, was biochemically characterized. Tt Bcp 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 Tt Bcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (Tt PDO) 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. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are an unusual family of thiol-specific peroxidases that possess a binding site for H 2 O 2 and rely on a conserved cysteine residue for rapid reaction with H 2 O 2. Among 6 mammalian isoforms (Prx I to VI), Prx I and Prx II are mainly found in the cytosol and nucleus. Prx I and Prx II function as antioxidant enzymes and protein chaperone under oxidative distress conditions. Under oxidative eustress conditions, Prx I and Prx II regulate the levels of H 2 O 2 at specific area of the cells as well as sense and transduce H 2 O 2 signaling to target proteins. Prx I and Prx II are known to be covalently modified on multiple sites: Prx I is hyperoxidized on Cys52; phosphorylated on Ser32, Thr90, and Tyr194; acetylated on Lys7, Lys16, Lys27, Lys35, and Lys197; glutathionylated on Cys52, Cys83, and Cys173; and nitrosylated on Cys52 and Cys83, whereas Prx II is hyperoxidized on Cys51; phosphorylated on Thr89, Ser112, and Thr182; acetylated on Ala2 and Lys196; glutathionylated on Cys51 and Cys172; and nitrosylated on Cys51 and Cys172. In this review, we describe how these post-translational modifications affect various functions of Prx I and Prx II. Image 1 • Prxs (Prx I and II) reduce H 2 O 2 using a conserved Cys and Trx in peroxidase cycle. • Cys–SOH intermediate of the cycle occasionally undergoes hyperoxidation to Cys–SO 2 H. • Hyperoxidation of Prxs converts them to peroxidase-independent protein chaperone. • Peroxidase and chaperone functions of Prxs are regulated via covalent modifications. • Which include phosphorylation, acetylation, glutathionylation, and nitosylation. [ABSTRACT FROM AUTHOR]

• Vaccine comprised gut cysteine peptidases and peroxiredoxin-1-derived peptides. • Vaccine/alum elicited significant reduction in challenge Schistosoma mansoni burden. • Vaccination was associated with tissues worm egg burden similar to control mice. • Increase in lung cytokines and reactive oxygen species content led to larval death. • Increase in lung lipids induced surviving worms to resist liver immune effectors. The study aimed to elicit protective immune responses against murine schistosomiasis mansoni at the parasite lung- and liver stage. Two peptides showing amino acid sequence similarity to gut cysteine peptidases, which induce strong memory immune effectors in the liver, were combined with a peptide based on S. mansoni thioredoxin peroxidase (TPX), a prominent lung-stage schistosomula excretory-secretory product, and alum as adjuvant. Only one of the 2 cysteine peptidases-based peptides in a multiple antigenic peptide construct (MAP-3 and MAP-4) appeared to adjuvant protective immune responses induced by the TPX peptide in a MAP form. Production of TPX MAP-specific IgG1 serum antibodies, and increase in lung interleukin-1 (IL-1), uric acid, and reactive oxygen species (ROS) content were associated with significant (P < 0.05) 50 % reduction in recovery of lung-stage larvae. Increase in lung triglycerides and cholesterol levels appeared to provide the surviving worms with nutrients necessary for a stout double lipid bilayer barrier at the parasite-host interface. Surviving worms-released products elicited memory responses to the MAP-3 immunogen, including production of specific IgG1 antibodies and increase in liver IL-33 and ROS. Reduction in challenge worm burden recorded 45 days post infection did not exceed 48 % associated with no differences in parasite egg counts in the host liver and small intestine compared to unimmunized adjuvant control mice. Alum adjuvant assisted the second peptide, MAP-4, in production of IgG1, IgG2a, IgG2b and IgA specific antibodies and increase in liver ROS, but with no protective potential, raising doubt about the necessity of adjuvant addition. Accordingly, different vaccine formulas containing TPX MAP and 1, 2 or 3 cysteine peptidases-derived peptides with or without alum were used to immunize parallel groups of mice. Compared to unimmunized control mice, significant (P < 0.05 to < 0.005) 22 to 54 % reduction in worm burden was recorded in the different groups associated with insignificant changes in parasite egg output. The results together indicated that a schistosomiasis vaccine able to entirely prevent disease and halt its transmission still remains elusive. [Display omitted] [ABSTRACT FROM AUTHOR]

The health benefits of lipoic acid (LA) are generally attributed to mitigating the harmful effects of reactive oxygen species (ROS). ROS are chemically similar to reactive sulfur species (RSS) and signal through identical mechanisms. Here we examined the effects of LA on RSS in HEK293 cells using H 2 S and polysulfide (PS) specific fluorophores, AzMC and SSP4. We show that LA concentration-dependently increased both H 2 S and PS. Physioxia (5% O 2) augmented the effects of LA on H 2 S production but decreased PS production. Thiosulfate, a known substrate for reduced LA, and an intermediate in the catabolism of H 2 S enhanced the effects of LA on H 2 S and PS production. Inhibiting peroxiredoxins with conoidin A and gluraredoxins with tiopronin augmented the effects of LA on PS and H 2 S, respectively while decreasing glutathione with buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased the stimulatory effect of LA on H 2 S production but augmented LA's effect on PS. Aminooxyacetate (AOA) and propargylglycine (PPG), inhibitors of H 2 S production from cysteine partially inhibited LA augmentation of H 2 S production and further decreased the LA effect when applied concurrently with BSO and DEM. The selective and cell-permeable H 2 S scavenger, SS20, inhibited the effects of LA on cellular H 2 S. Estimates of single-cell H 2 S production suggest that 0.1–0.2% of O 2 consumption is used to metabolize H 2 S and these requirements may increase to 1–2% with 1 mM LA. Collectively, these results suggest that LA rescues H 2 S from irreversible oxidation and that the effects of LA on RSS directly confer antioxidant, anti-inflammatory and cytoprotective responses. They also suggest that TS may be an effective supplement to increase the efficacy of LA in clinical settings. Protective effects of lipoic acid through rescue of reactive sulfur species (RSS). H 2 S derived from glutathione (GSH), cysteine (Cys) and protein catabolism is normally oxidized to sulfate (SO 4 2-) and excreted with thiosulfate (S 2 O 3 2-) as an intermediate in oxidation. Exogenous lipoic acid (LA) taken up by the cell is reduced to dihydrolipoic acid (DHLA) by thioredoxin (Trx), glutaredoxin (Grx) or peroxiredoxin (Prx) utilizing NADH as the reductant. DHLA reduces thiosulfate regenerating H 2 S which is then oxidized to persulfide (H 2 S 2), possibly by LA as the oxidant, although a catalyst for this reaction has not been identified. Persulfide is not only a strong antioxidant but it persulfidates regulatory cysteines that initiate a variety of transcriptional and post-transcriptional antioxidant, anti-inflammatory and cytoprotective defenses. Many of the "antioxidant" properties of LA can be explained by these RSS metabolic pathways. Image 1 [ABSTRACT FROM AUTHOR]

Hydrogen peroxide undergoes an equilibrium reaction with bicarbonate/CO 2 to produce peroxymonocarbonate (HCO 4 -). Peroxymonocarbonate is more reactive with thiols than H 2 O 2 but it makes up only a small fraction of the H 2 O 2 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/CO 2 on the oxidation of peroxiredoxins (Prdxs) 2 and 3. Using an assay in which reduced Prdx2 inhibits oxidation of horseradish peroxidase by H 2 O 2 , 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 H 2 O 2 , and prevents its condensation to a disulfide. Using LC/MS analysis, we determined that the presence of 25 mM bicarbonate/CO 2 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. Image 1 • Excess H 2 O 2 causes hyperoxidation of mammalian 2Cys peroxiredoxins. • Prdx2 and Prxd3 are more readily hyperoxidized in presence of CO 2 /bicarbonate. • Peroxymonocarbonate formed in equilibrium with H 2 O 2 can account for enhancement. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress. To understand this, we used oxidative-stress sensitive tsa1tsa2Δ yeast strain to express hPRXI or hPRXII. We found that hPrxI in yeast exists as a mixture of disulfide-linked dimer and reduced monomer but becomes hyperoxidized upon elevated oxidative stress as analyzed under denaturing conditions (SDS-PAGE). In contrast, hPrxII was present predominantly as the disulfide in unstressed cells and readily converted to its hyperoxidized, peroxidase-inactive form even with mild oxidative stress. Interestingly, we found that plant extracts containing polyphenol antioxidants provided further protection against the growth defects of the tsa1tsa2Δ strain expressing hPrx and preserved the peroxidase-active forms of the Prxs. The extracts also helped to protect against hyperoxidation of hPrxs in HeLa cells. Based on these findings we can conclude that resistance to oxidative stress of yeast cells expressing individual hPrxs requires the hPrx to be maintained in a redox state that permits redox cycling and peroxidase activity. Peroxidase activity decreases as the hPrx becomes hyperoxidized and the limited protection by hPrxII compared with hPrxI can be explained by its greater sensitivity to hyperoxidation. Image 1 • Disulfide:Hyperoxidized states ratio of hPrxs vary in yeast under redox stress (RS). • Peroxidase activity of hPrxI/II leads to hyperoxidation and sensitivity of cells to RS. • hPrxII is more sensitive than hPrxI and becomes hyperoxidized with mild oxidative stress. • Polyphenol antioxidants of plants preserved the peroxidase-active forms of hPrxI/II. [ABSTRACT FROM AUTHOR]

• The protein identification analysis identified five differentially expressed proteins in the OTA-GES-1 T cells group. • The expressions of Annexin A3 both at protein and mRNA levels increased in OTA-GES-1 T cells group. • Annexin A3 siRNA transfection could inhibit the proliferation of OTA-GES-1 T cells. • Annexin A3 siRNA transfection could lead cells to be arrested at G0/G 1 phase in OTA-GES-1 T cells group. • Inhibition Annexin A3 by siRNA effectively prevented the migration and invasion abilities of OTA-GES-1 T cells. Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1 T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen β). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1 T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1 T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity. [ABSTRACT FROM AUTHOR]

2-Cys peroxiredoxins (Prxs) rapidly reduce H2O2, thereby acting as antioxidants and also as sensors and transmitters of H2O2 signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H2O2 levels. Under these conditions, H2O2 oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (CPSOH) to the sulfinate (CPSO2-) and sulfonated (CPSO3-) forms, redirecting the CPSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the CPSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H2O2 and was selected here to investigate the effect of a physiological concentration of HCO3-/CO2 (25 mM) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO3-/CO2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H2O2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO3-/CO2 was due to HCO4-, a peroxide present in equilibrated solutions of H2O2 and HCO3-/CO2. Indeed, additional experiments and calculations uncovered that HCO4- oxidizes CPSOH to CPSO2- with a second-order rate constant 2 orders of magnitude higher than that of H2O2 ((1.5 ± 0.1) x 105 and (2.9 ± 0.2) x 10³ M-1⋅s-1, respectively) and that HCO4- is 250 times more efficient than H2O2 at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO3-/CO2 pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity. [ABSTRACT FROM AUTHOR]

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-molecularweight thiol in M. tuberculosis, mycothiol.H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic 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-molecularweight 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 thiolMtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity. [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]

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis , its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr. [ABSTRACT FROM AUTHOR]

• Antigen B (AgB) and thioredoxin peroxidase (TPx) from Taenia multiceps were cloned. • The antigens were evaluated as agents for serological diagnosis of coenurosis. • Tm-AgB shows high sensitivity and thermal stability. • Tm-AgB is an excellent candidate antigen for coenurosis diagnosis kits. Coenurosis is a serious parasitic disease of herbivorous animals caused by the metacestode of Taenia multiceps (Coenurus cerebralis). Accordingly, a significant amount of research is currently dedicated to the development of appropriate antigens for use in rapid and accurate coenurosis diagnosis kits. In the present study, antigen B (AgB) and thioredoxin peroxidase (TPx) from T. multiceps were cloned and expressed using a prokaryotic system, molecular characterization of Tm -AgB was determined by bioinformatical analyses. The serological diagnostic potentials of r Tm -AgB and r Tm -TPx were evaluated by indirect ELISA and compared with those of previously reported r Tm -AnxB2, r Tm -HSP70, and r Tm -GST. The results showed that Tm -AgB is a specific lipoprotein of cestodes with good thermal stability. The ELISA assay showed that r Tm -AgB exhibited a sensitivity of 95.8% and a specificity of 87.5%, indicating its strong potential for serological diagnosis of T. multiceps. [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]

The vancomycin-resistant Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (Ef AhpC) and AhpF (Ef AhpF) is of paramount importance to restore redox homeostasis. Therefore, knowledge about this defense system is essential to understand its antibiotic-resistance and survival in hosts. Recently, we described the crystallographic structures of Ef AhpC, the two-fold thioredoxin-like domain of Ef AhpF, the novel phenomenon of swapping of the catalytic domains of Ef AhpF as well as the unique linker length, connecting the catalytically active N-and C-terminal domains of Ef AhpF. Here, using mutagenesis and enzymatic studies, we reveal the effect of an additional third cysteine (C503) in Ef AhpF, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions. The crystal structure and solution NMR data of the engineered C503A mutant of the thioredoxin-like domain of Ef AhpF were used to describe alterations in the environment of the additional cysteine residue during modulation of the redox-state. To glean insight into the epitope and mechanism of Ef AhpF and -AhpC interaction as well as the electron transfer from the thioredoxin-like domain of Ef AhpF to AhpC, NMR-titration experiments were performed, showing a coordinated disappearance of peaks in the thioredoxin-like domain of Ef AhpF in the presence of full length Ef AhpC, and indicating a stable Ef AhpF-AhpC-complex. Combined with docking studies, the interacting residues of Ef AhpF were identified and a mechanism of electron transfer of the Ef AhpF donor to the electron acceptor Ef AhpC is described. Image 1 • Residue C503 is important in catalysis of Enterococcus faecalis AhpF (Ef AhpF). • Crystallographic structure of the Ef AhpF mutant C503A is presented. • Ef AhpF residues identified to be essential for redox-modulation. • A long-lived E. faecalis AhpF-AhpC-complex formation has been identified. • The binding epitopes of the Ef AhpC-AhpF machinery is well established. [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]

Hydrogen peroxide (H 2 O 2), a natural metabolite commonly found in aerobic organisms, plays a crucial role in numerous cellular signaling processes. One of the key organelles involved in the cell's metabolism of H 2 O 2 is the peroxisome. In this review, we first provide a concise overview of the current understanding of H 2 O 2 as a molecular messenger in thiol redox signaling, along with the role of peroxisomes as guardians and modulators of cellular H 2 O 2 balance. Next, we direct our focus toward the recently identified primary protein targets of H 2 O 2 originating from peroxisomes, emphasizing their importance in unraveling the complex interplay between peroxisomal H 2 O 2 and cell signaling. We specifically focus on three areas: signaling through peroxiredoxin redox relay complexes, calcium signaling, and phospho-signaling. Finally, we highlight key research directions that warrant further investigation to enhance our comprehension of the molecular and biochemical mechanisms linking alterations in peroxisomal H 2 O 2 metabolism with disease. [Display omitted] • H 2 O 2 is a crucial molecular messenger in thiol redox signaling. • Peroxisomes play a vital role in cellular H 2 O 2 metabolism. • Peroxisome-derived H 2 O 2 targets important regulators of key cellular processes. • Peroxisomal H 2 O 2 signaling is largely an uncharted frontier. • Peroxisomal H 2 O 2 signaling may involve thiol-disulfide oxidoreductase relays. [ABSTRACT FROM AUTHOR]

Peroxiredoxins have been shown to protect insects from oxidative damage and to play a role in the immune system. In the present study, we cloned and characterized the Antheraea pernyi peroxiredoxin 2 (ApPrx-2) gene, then assessed its functional roles. The ApPrx-2 gene has a 687 bp open reading frame that encodes a protein with 288 amino acid residues. Quantitative real-time PCR analysis revealed that the mRNA levels of ApPrx-2 were highest in the hemocytes. Immune challenge assay revealed that ApPrx-2 transcription could be induced after microbial challenge. A DNA cleavage assay employing recombinant ApPrx-2 protein and a metal-catalyzed oxidation system showed that rApPrx-2 protein could protect supercoiled DNA against oxidative stress. The protein antioxidant activity of rApPrx-2 was examined, and it was found that rApPrx-2 exhibited a high level of antioxidant activity by removing H 2 O 2. In addition, ApPrx-2 knockdown larvae had higher H 2 O 2 levels and a lower survival rate when compared to controls. Interestingly, the antibacterial activity was significantly higher in ApPrx-2 depleted larvae compared with control. Overall, our findings indicate that ApPrx-2 may be involved in a range of physiological functions of A. pernyi , as it protects supercoiled DNA from oxidative stress and regulates antibacterial activity. [ABSTRACT FROM AUTHOR]

Abstract Background The aim of present study was to investigate the effects and mechanisms of peroxiredoxin (Prdx) 6 on cecal ligation and puncture (CLP) induced acute lung injury (ALI) in mice. Methods The cecal of male Prdx 6 knockout and wildtype C57BL/6J mice were ligated and perforated. Stool was extruded to ensure wound patency. Two hours, 4 h, 8 h and 16 h after stimulation, the morphology, wet/dry ratio, protein concentration in bronchial alveolar lavage fluid (BALF) were measured to evaluate lung injury. Myeloperoxidase (MPO) activity, hydrogen peroxide (H 2 O 2), malondialdehyde (MDA), total superoxide dismutase (SOD), xanthine oxidase (XOD), CuZn-SOD, total anti-oxidative capability (TAOC), glutathione peroxidase (GSH-PX), catalase (CAT) in lungs were measured by assay kits. The mRNA expression of lung tumor necrosis factor (TNF-α), interleukin (IL)-1β, and matrix metalloproteinases (MMP) 2 and 9 were tested by real-time RT-PCR. The nuclear factor (NF)-κB activity was measured by TransAM kit. Results CLP-induced ALI was characterized by inflammation in morphology, increased wet/dry ratio, elevated protein concentration in BALF and higher level of MPO activity. The levels of H 2 O 2 , MDA, and XOD were significantly increased and SOD, CuZn-SOD, GSH-PX, CAT, and T-AOC were significantly decreased in lungs after CLP. The activity of NF-κB was significantly increased and subsequently, the mRNA expression of TNF-α, IL-1β and MMP2 and MMP9 were significantly increased after CLP. Those above injury parameters were more severe in Prdx 6 knockout mice than those in wildtype mice. Conclusions Prdx 6 knockout aggravated the CLP induced lung injury by augmenting oxidative stress, inflammation and matrix degradation partially through NF-κB pathway. Highlights • ALI can be caused by sepsis and CLP is an ideal model for sepsis. • Prdx6 can protect lung against ALI. • Prdx6 knock out worsens the CLP-induced ALI. • NF-κB activation, increased ROS and MMPs may play roles in the mechanism of Prdx6. [ABSTRACT FROM AUTHOR]

Abstract Chondrocytes are responsible for the maintenance of the articular cartilage. A loss of homeostasis in cartilage contributes to the development of osteoarthritis (OA) when the synthetic capacity of chondrocytes is overwhelmed by processes that promote matrix degradation. There is evidence for an age-related imbalance in reactive oxygen species (ROS) production relative to the anti-oxidant capacity of chondrocytes that plays a role in cartilage degradation as well as chondrocyte cell death. The ROS produced by chondrocytes that have received the most attention include superoxide, hydrogen peroxide, the reactive nitrogen species nitric oxide, and the nitric oxide derived product peroxynitrite. Excess levels of these ROS not only cause oxidative-damage but, perhaps more importantly, cause a disruption in cell signaling pathways that are redox-regulated, including Akt and MAP kinase signaling. Age-related mitochondrial dysfunction and reduced activity of the mitochondrial superoxide dismutase (SOD2) are associated with an increase in mitochondrial-derived ROS and are in part responsible for the increase in chondrocyte ROS with age. Peroxiredoxins (Prxs) are a key family of peroxidases responsible for removal of H 2 O 2 , as well as for regulating redox-signaling events. Prxs are inactivated by hyperoxidation. An age-related increase in chondrocyte Prx hyperoxidation and an increase in OA cartilage has been noted. The finding in mice that deletion of SOD2 or the anti-oxidant gene transcriptional regulator nuclear factor-erythroid 2- related factor (Nrf2) result in more severe OA, while overexpression or treatment with mitochondrial targeted anti-oxidants reduces OA, further support a role for excessive ROS in the pathogenesis of OA. Therefore, new therapeutic strategies targeting specific anti-oxidant systems including mitochondrial ROS may be of value in reducing the progression of age-related OA. Graphical abstract fx1 Highlights • An age-related increase in chondrocyte reactive oxygen species (ROS) levels exists. • Mitochondrial dysfunction contributes to oxidative stress in osteoarthritis. • Excessive ROS levels alter signaling to promote cartilage matrix destruction. [ABSTRACT FROM AUTHOR]

Abstract Chronic kidney disease (CKD) has become epidemic worldwide. Mitochondrial reactive oxygen species (ROS)-induced oxidative stress is an important mediator of CKD, and Prx3 plays a critical role in maintenance of mitochondrial ROS. The present study examined the role of Prx3 in the context of fibrosis, a common feature of CKD, using Prx3 KO mice under obstructive and diabetic stress. Prx3 deficiency accelerated fibrosis and inflammation accompanied by mitochondrial oxidative stress in obstructed and diabetic kidneys as well as in proximal tubular epithelial (mProx) cells. In addition, Prx3 deficiency induced Raw264.7 macrophages activation, leading to upregulation of proinflammatory cytokines. Conditioned media from LPS-stimulated Prx3 deficient macrophages accelerated proinflammatory and profibrotic cytokines in mProx cells. Interestingly, Prx3 deficiency induced most inflammatory and fibrotic cytokines at basal condition in both tissues and cells. Taken together, these results demonstrate that Prx3 deficiency can accelerate CKD through interactions between macrophages and tubular epithelial cells. Graphical abstract fx1 Highlights • Prx3 deficiency accelerates chronic kidney diseases (CKD) in mice. • Prx3 deficiency increases macrophages activation in CKD. • Prx3 deficiency upregulates interactions between macrophages and epithelial cells. • Prx3 deficiency increases mitochondrial ROS leading to kidney fibrosis. [ABSTRACT FROM AUTHOR]

Abstract The events that trigger early onset of atherosclerotic lesion formation are poorly understood. Initially, microscopic atherosclerotic lesions appear in the aortic root in 10-week-old apoE-knockout mice that are fed normal chow. Using proteome and immunohistochemical analyses, we investigated proteins in aortic media whose expression changes in athero-prone regions at the beginning of lesion formation. Protein profiles of the root/arch and thoracic/abdominal regions of aortas in 10-week-old apoE-knockout mice were analyzed using 2D-gel electrophoresis. Proteins in 81 spots with different abundance were identified. Among them, we focused on proteins related to oxidative stress and smooth muscle cells (SMCs). The level of peroxiredoxin 2 (Prx2), a major cellular antioxidant enzyme that reduces hydrogen peroxide, was lower in aortic root/arch compared with thoracic/abdominal aorta. Immunohistochemical staining demonstrated that Prx2 expression in SMCs in the aortic root was high at 4 weeks and decreased at 10 weeks in apoE-knockout mice, while Prx2 expression in the aorta was unchanged in wild-type mice. The level of Prx2 expression correlated positively with the SMC differentiation markers, α-smooth muscle actin and transgelin, suggesting that a decline in Prx2 expression accompanies SMC dedifferentiation. Accumulated acrolein-modified proteins and the infiltration of macrophages in aortic media were observed in areas with low Prx2 expression. These results showed that Prx2 expression declines in athero-prone aortic root before lesion formation, and this reduction in Prx2 expression correlates with lipid peroxidation, SMC dedifferentiation, and macrophage recruitment. Graphical abstract fx1 Highlights • Peroxiredoxin 2 (Prx2) decreased at the beginning of atherosclerotic lesion formation in aortic media in apoE-KO mice. • Local aortic regions with low Prx2 expression accumulate oxidation products. • The concomitant reduction of Prx2, α-SMA and transgelin expressions suggests Prx2 relates to SMC dedifferentiation. • Infiltration of macrophages into media is observed at the site of Prx2 reduction. • Prx2 reduction at small regions is a potential triggering event at the early onset of atherogenesis. [ABSTRACT FROM AUTHOR]

Abstract Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO 2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria. Graphical abstract fx1 Highlights • Human Prx3 rapidly reduces peroxynitrite. • Prx3 intrinsic fluorescence presents biphasic changes upon oxidation. • Peroxynitrite in excess leads to Prx3 nitration and hyperoxidation. • Both Prx3 and Prx5 are predicted to be main targets for mitochondrial peroxynitrite. [ABSTRACT FROM AUTHOR]

Abstract Protein aggregation, oxidative and nitrosative stress are etiological factors common to all major neurodegenerative disorders. Therefore, identifying proteins that function at the crossroads of these essential pathways may provide novel targets for therapy. Oxidation resistance 1 (Oxr1) is a protein proven to be neuroprotective against oxidative stress, although the molecular mechanisms involved remain unclear. Here, we demonstrate that Oxr1 interacts with the multifunctional protein, peroxiredoxin 2 (Prdx2), a potent antioxidant enzyme highly expressed in the brain that can also act as a molecular chaperone. Using a combination of in vitro assays and two animal models, we discovered that expression levels of Oxr1 regulate the degree of oligomerization of Prdx2 and also its post-translational modifications (PTMs), specifically suggesting that Oxr1 acts as a functional switch between the antioxidant and chaperone functions of Prdx2. Furthermore, we showed in the Oxr1 knockout mouse that Prdx2 is aberrantly modified by overoxidation and S-nitrosylation in the cerebellum at the presymptomatic stage; this in-turn affected the oligomerization of Prdx2, potentially impeding its normal functions and contributing to the specific cerebellar neurodegeneration in this mouse model. Graphical abstract fx1 Highlights • Oxr1 interacts with the multifunctional antioxidant enzyme, Prdx2. • Oxr1 modulates the oligomerization and post-translational modifications of Prdx2. • Oxr1 acts as a functional switch between Prdx2 antioxidant and chaperone functions. • Loss of Oxr1 leads to aberrantly modified Prdx2 that contributes to neurodegeneration. [ABSTRACT FROM AUTHOR]

Abstract Hydrogen peroxide (H 2 O 2) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H 2 O 2 because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H 2 O 2 and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H 2 O 2. Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H 2 O 2 caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H 2 O 2 (k~1 M−1s−1). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H 2 O 2 did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay. Highlights • Cytosolic peroxiredoxins constitutively interact with CRMP2 in several different cell types. • Addition of H 2 O 2 to Jurkat T lymphoma cells results in CRMP2 oxidation. • Recombinant CRMP2 has low reactivity with H 2 O 2. • CRMP2 remained sensitive to oxidation in single peroxiredoxin knockout Jurkat cells. • The data suggest a cell-type specific mechanism to increase the sensitivity of CRMP2 to oxidation. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Abstract Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H 2 O 2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H 2 O 2 in eukaryotic cells. The genome of Aquifex aeolicus , a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8 Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine C P 49 was found to be hyperoxidized to the sulfonic acid (SO 3 H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins. Highlights • AhpC2 is arranged as a circular dodecamer both in crystals and in solution. • Tube-like higher-order assemblies in solution were observed. • Peroxidatic Cys49 was hyperoxidized. • Cys212 and Cys218 form an intramonomeric disulfide bond. • The C-terminal domain of AhpC2 adopts an opposite orientation. [ABSTRACT FROM AUTHOR]