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

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

The molecular mechanism of high-temperature stress (HTS) response, in plants, has so far been investigated using transcriptomics, while the dynamics of HTS-responsive proteome remain unexplored. We examined the adaptive responses of the resilient wheat cultivar 'Unnat Halna' and dissected the HTS-responsive proteome landscape. This led to the identification of 55 HTS-responsive proteins (HRPs), which are predominantly involved in metabolism and defense pathways. Interestingly, HRPs included a 2-cysteine peroxiredoxin (2CP), designated Ta2CP, presumably involved in stress perception and adaptation. Complementation of Ta2CP in yeast and heterologous expression in Arabidopsis demonstrated its role in thermotolerance. Both Ta2CP silencing and overexpression inferred the involvement of Ta2CP in plant growth and chlorophyll biosynthesis. We demonstrated that Ta2CP interacts with protochlorophyllide reductase b, TaPORB. Reduced TaPORB expression was found in Ta2cp-silenced plants, while upregulation was observed in Ta2CP-overexpressed plants. Furthermore, the downregulation of Ta2CP in Taporb-silenced plants and reduction of protochlorophyllide in Ta2cp-silenced plants suggested the key role of Ta2CP in chlorophyll metabolism. Additionally, the transcript levels of AGPase1 and starch were increased in Ta2cp-silenced plants. More significantly, HTS-treated Ta2cp-silenced plants showed adaptive responses despite increased reactive oxygen species and peroxide concentrations, which might help in rapid induction of high-temperature acclimation.

Neoporphyra haitanensis is an economically important red seaweed that inhabits upper intertidal zones. The thallus tolerates extreme fluctuating environmental stresses (e.g., surviving more than 80% water loss during low tides). To elucidate the global molecular responses relevant to this outstanding desiccation tolerance, a quantitative proteomics analysis of N. haitanensis under different desiccation treatments as well as rehydration was performed. According to the clustering of expression patterns and the functional interpretation of the 483 significantly differentially expressed proteins, a three-stage cellular response to desiccation stress and subsequent rehydration was proposed. Stage I: at the beginning of water loss, multiple signal transduction pathways were triggered including lipid signaling, protein phosphorylation cascades, and histone acetylation controlling acetate biosynthesis to further modulate downstream hormone signaling. Protein protection by peptidyl-prolyl isomerase and ROS scavenging systems were also immediately switched on. Stage II: with the aggravation of stress, increases in antioxidant systems, the accumulation of LEA proteins, and the temporary biosynthesis of branched starch were observed. Multiple enzymes involved in redox homeostasis, including peroxiredoxin, thioredoxin, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, were hypothesized to function in specific cellular compartments. Stage III: when the desiccated thalli had rehydrated for 30 mins, photosynthesis and carbon fixation were recovered, and antioxidant activities and protein structure protection were maintained at a high level. This work increases the understanding of the molecular responses to environmental stresses via a proteomic approach in red seaweeds and paves the way for further functional studies and genetic engineering.

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

Background Radioresistance of some non-small cell lung cancer (NSCLC) types increases the risk of recurrence or metastasis in afflicted patients, following radiotherapy. As such, further improvements to NSCLC radiotherapy are needed. The expression of oncogene TP53-regulated inhibitor of apoptosis 1 (TRIAP1) in NSCLC is increased following irradiation. Furthermore, gene set enrichment analysis (GSEA) has suggested that TRIAP1 might be involved in maintaining redox homeostasis. This in turn might enhance cell radioresistance. Methods In this study we irradiated human NSCLC cell lines (A549 and H460), while knocking down TRIAP1, to determine whether a disrupted redox homeostasis could attenuate radioresistance. Results Irradiation notably increased both mRNA and protein levels of TRIAP1. In addition, TRIAP1 knockdown decreased the expression of several antioxidant proteins, including thioredoxin-related transmembrane protein (TMX) 1, TMX2, thioredoxin (TXN), glutaredoxin (GLRX) 2, GLRX3, peroxiredoxin (PRDX) 3, PRDX4, and PRDX6 in A549 and H460 cells. In addition, silencing TRIAP1 impaired the radiation-induced increase of the aforementioned proteins. Continuing along this line, we observed a radiation-induced reduction of cell viability and invasion, as well as increased apoptosis and intracellular reactive oxygen species following TRIAP1 knockdown. Conclusions In summary, we identified TRIAP1 as a key contributor to the radioresistance of NSCLC by maintaining redox homeostasis.

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

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

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 co-cultured 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.

Contents Summary 1197 I. Introduction 1198 II. Measurement and imaging of H2 O2 1198 III. H2 O2 and O2 ·- toxicity 1199 IV. Production of H2 O2 : enzymes and subcellular locations 1200 V. H2 O2 transport 1205 VI. Control of H2 O2 concentration: how and where? 1205 VII. Metabolic functions of H2 O2 1207 VIII. H2 O2 signalling 1207 IX. Where next? 1209 Acknowledgements 1209 References 1209 SUMMARY: Hydrogen peroxide (H2 O2 ) is produced, via superoxide and superoxide dismutase, by electron transport in chloroplasts and mitochondria, plasma membrane NADPH oxidases, peroxisomal oxidases, type III peroxidases and other apoplastic oxidases. Intracellular transport is facilitated by aquaporins and H2 O2 is removed by catalase, peroxiredoxin, glutathione peroxidase-like enzymes and ascorbate peroxidase, all of which have cell compartment-specific isoforms. Apoplastic H2 O2 influences cell expansion, development and defence by its involvement in type III peroxidase-mediated polymer cross-linking, lignification and, possibly, cell expansion via H2 O2 -derived hydroxyl radicals. Excess H2 O2 triggers chloroplast and peroxisome autophagy and programmed cell death. The role of H2 O2 in signalling, for example during acclimation to stress and pathogen defence, has received much attention, but the signal transduction mechanisms are poorly defined. H2 O2 oxidizes specific cysteine residues of target proteins to the sulfenic acid form and, similar to other organisms, this modification could initiate thiol-based redox relays and modify target enzymes, receptor kinases and transcription factors. Quantification of the sources and sinks of H2 O2 is being improved by the spatial and temporal resolution of genetically encoded H2 O2 sensors, such as HyPer and roGFP2-Orp1. These H2 O2 sensors, combined with the detection of specific proteins modified by H2 O2 , will allow a deeper understanding of its signalling roles.

Background and purpose Antioxidants provide a promising therapeutic effect for the cardiovascular disease. Luteolin, a polyphenolic bioflavonoid, is known to confer cardioprotection, although the underlying mechanisms, especially the role of luteolin on the antioxidant enzymes, such as the peroxiredoxin family, remain unknown. Experimental approach We measured the effects of luteolin on myocardial ischaemia/reperfusion (MI/R) injury in vivo (Sprague-Dawley rats) and in vitro, together with the underlying mechanisms, with a focus on signalling by peroxiredoxins. H9c2 cells were used to assess the changes in peroxiredoxins and the other antioxidant enzymes. Oxidative stress, cardiac function, LDH release, ROS and infarct size were also assayed. Key results Luteolin exerted significant cardioprotective effects in vivo and in vitro via improving cardiac function, increasing the expression of anti-apoptotic protein Bcl-2 and decreasing the pro-apoptotic protein Bax and active caspases 3 and 9, associated with MI/R. Mechanistically, luteolin markedly enhanced expression of peroxiredoxin II, without significant effects on other forms of peroxiredoxin, catalase or SOD1. Molecular docking showed that luteolin could indeed bind to the enzymic active pocket of peroxiredoxin II. Furthermore, down-regulation of peroxiredoxin II by peroxiredoxin II-antisense, administered by adenovirus infection of H9c2 cardiomyocytes, and inhibition of peroxiredoxin II in vivo significantly reversed the cardioprotective effects of luteolin. Conclusions and implications Our findings, for the first time, demonstrate that luteolin protects against MI/R injury through promoting signalling through the endogenous antioxidant enzyme, peroxiredoxin II, indicating the important beneficial role of this antioxidant system in the heart.

Casticin, a compound purified from the Chinese herb Viticis Fructus, has been proven effective in preventing tumor progression in previous studies. Ulcerative colitis (UC) is a common inflammatory bowel disease that affects millions of people worldwide, but no effective and safe drugs are available. In this study, we aimed to study how did casticin affect UC by evaluating its effects on dextran sulfate sodium (DSS)-induced colitis in mice. Our data suggested that casticin attenuated body weight loss, colon length shortening, and pathological damage in the colon of DSS-treated mice. Casticin decreased reactive oxygen species level and chemocytokines (IL-1β, IL-6, TNF-α) productions in colon tissue. The decreased reactive oxygen species level and suppressed proinflammatory cytokines productions were also confirmed in casticin-treated LPS-stimulated RAW264.7 cells and hydrogen peroxide-treated CACO-2 cells in vitro. Mechanistically, casticin treatment prevented the profound activation of AKT signaling caused by DSS administration. And casticin inhibited the productions of proinflammatory chemocytokines through downregulating AKT/NF-κB pathway in macrophages. Meanwhile, data revealed that casticin increased expressions of endogenous antioxidants peroxiredoxin 3 and MnSOD were through activation in FOXO3α signaling by downregulating AKT signaling in colon epithelium cells. Our findings demonstrated that casticin alleviated DSS-induced UC by increasing the antioxidant enzyme peroxiredoxin 3 and MnSOD expressions, and decreasing the production of proinflammatory chemocytokines through inhibition of AKT signaling.

Objective Hepatocellular carcinoma (HCC) is a high-burden disease. Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family of antioxidant enzymes. The aim of this study was to assess the value of PRDX1 for predicting HCC recurrence after curative resection and to explore the role of PRDX1 in HCC cell migration and invasion. Methods Data of patients with HCC who had undergone complete resection between 2002 and 2006 were collected. Immunohistochemical detection of PRDX1 in HCC tissue and adjacent non-cancerous tissue was conducted. Kaplan-Meier survival estimate and log-rank test were used to assess the relationship between PRDX1 expression and prognostic significance. HCC cell migration and invasion together with the interaction between PRDX1 and ubiquitin C-terminal hydrolase 37 (UCH37) were studied in vitro. Results PRDX1 was expressed at lower levels in HCC tissues than in adjacent non-cancerous tissues, and PRDX1 was found to be an independent risk factor for disease-free survival and overall survival. PRDX1 restrained cell migration and invasion in vitro. PRDX1 was found to interact with UCH37 to affect HCC cell migration and invasion. Conclusion PRDX1 restrains cell migration and invasion in HCC cell lines and that may be involved in a UCH37-relevant pathway, suggesting that PRDX1 may be a new marker for HCC recurrence after surgery.

The peroxiredoxin (PRDX) family, a new family of proteins with a pivotal antioxidative function, is ubiquitously synthesized and abundantly identified in various organisms. In contrast to the intracellular localization of other family members (PRDX1/2/3/5/6), PRDX4 is the only known secretory form and protects against oxidative damage by scavenging reactive oxygen species in both the intracellular (especially the endoplasmic reticulum) compartments and the extracellular space. We generated unique human PRDX4 (hPRDX4) transgenic (Tg) mice on a C57BL/6J background and investigated the critical and diverse protective roles of PRDX4 against diabetes mellitus, atherosclerosis, insulin resistance, and nonalcoholic fatty liver disease (NAFLD) as well as evaluated its role in the intestinal function in various animal models. Our published data have shown that PRDX4 helps prevent the progression of metabolic syndrome by reducing local and systemic oxidative stress and synergistically suppressing steatosis, inflammatory reactions, and/or apoptotic activity. These observations suggest that Tg mice may be a useful animal model for studying the relevance of oxidative stress on inflammation and the dysregulation of lipid/bile acid/glucose metabolism upon the progression of human metabolic syndrome, and that specific accelerators of PRDX4 may be useful as therapeutic agents for ameliorating various chronic inflammatory diseases.

Brassinosteroids (BRs) regulate plant development and stress response. Although much has been learned about their roles in plant development, the mechanisms by which BRs regulate plant stress tolerance remain unclear. Chilling is a major stress that adversely affects plant growth. Here, we report that BR positively regulates chilling tolerance in tomato. BR partial deficiency aggravated chilling-induced oxidized protein accumulation, membrane lipid peroxidation, and decrease of maximum quantum efficiency of photosystem II (Fv/Fm). By contrast, overexpression of BR biosynthetic gene Dwarf or treatment with 24-epibrassinolide (EBR) attenuated chilling-induced oxidative damages and resulted in an increase of Fv/Fm. BR increased transcripts of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and GLUTAREDOXIN (GRX) genes, and BR-induced chilling tolerance was associated with an increase in the ratio of reduced/oxidized 2-cysteine peroxiredoxin (2-Cys Prx) and activation of antioxidant enzymes. However, RBOH1-RNAi plants failed to respond to EBR as regards to the induction of GRX genes, activation of antioxidant capacity, and attenuation of chilling-induced oxidative damages. Furthermore, silencing of GRXS12 and S14 compromised EBR-induced increases in the ratio of reduced/oxidized 2-Cys Prx and activities of antioxidant enzymes. Our study suggests that BR enhances chilling tolerance through a signalling cascade involving RBOH1, GRXs, and 2-Cys Prx in tomato.

Genetic signaling and redox homeostasis are required for proper growth of blood vessels. Here, we report a novel function of peroxiredoxin1 (Prdx1) in vascular development in zebrafish. Knockdown of prdx1 impairs the growth of intersegmental vessel and caudal vein plexus (CVP), and reduces the expression of vascular markers, thus suggesting a role for prdx1 in vasculature and indicating that the antioxidant function of prdx1 is important. We found that H2 O2 -treated embryos also have CVP defects and observed synergistic effects when prdx1 knockdown was combined with H2 O2 treatment. Moreover, N-acetyl-cysteine treatment rescues the vascular defects in prdx1 morphants. These results suggest that oxidative stress disturbs vascularization. Furthermore, we show that the regulation of prdx1 is mediated by Notch and BMP signals.

Identification of arsenic-binding proteins is important for understanding arsenic health effects and for developing arsenic-based therapeutics. We report here a strategy for the capture and identification of arsenic-binding proteins in living cells. We designed an azide-labeled arsenical, p-azidophenylarsenoxide (PAzPAO), to serve bio-orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic-binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic-binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde-3-phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic-induced health effects and in cancer treatment.

Seed longevity, the maintenance of viability during storage, is a major factor for conservation of genetic resources and biodiversity. Seed longevity is an important trait of agriculture crop and is impaired by reactive oxygen species (ROS) during seed desiccation, storage and germination (C. R. Biol., 331, 2008 and 796). Seeds possess a wide range of systems (protection, detoxification, repair) allowing them to survive during storage and to preserve a high germination ability. In many plants, 1-cys peroxiredoxin (1-Cys Prx, also named PER1) is a seed-specific antioxidant which eliminates ROS with cysteine residues. Here we identified and characterized a seed-specific PER1 protein from seeds of sacred lotus (Nelumbo nucifera Gaertn.). Purified NnPER1 protein protects DNA against the cleavage by ROS in the mixed-function oxidation system. The transcription and protein accumulation of NnPER1 increased during seed desiccation and imbibition and under abiotic stress treatment. Ectopic expression of NnPER1 in Arabidopsis enhanced the seed germination ability after controlled deterioration treatment (CDT), indicating that NnPER1 improves seed longevity of transgenic plants. Consistent with the function of NnPER1 on detoxifying ROS, we found that the level of ROS release and lipid peroxidation was strikingly lower in transgenic seeds compared to wild-type with or without CDT. Furthermore, transgenic Arabidopsis seeds ectopic-expressing NnPER1 displayed enhanced tolerance to high temperature and abscisic acid (ABA), indicating that NnPER1 may participate in the thermotolerance and ABA signaling pathway.

Objective Damage-associated molecular patterns (DAMP) and cytokines can play a crucial role in inflammation at neuromuscular junctions in myasthenia gravis (MG). However, the relationship between DAMP and cytokine levels in MG pathogenesis remains unknown. To clarify this, we examined the relationship between serum levels of DAMP and cytokines in MG patients. Methods Using serum data from 26 patients with anti-acetylcholine receptor antibody-positive MG, we investigated the relationship between the levels of DAMP (high-mobility group box 1 [HMGB1] and peroxiredoxin 5) and cytokines (interleukin [IL]-4, IL-15, IL-19, IL-20, IL-28A, IL-35, a proliferation-inducing ligand [APRIL] and vascular endothelial growth factor), which were reported to be significantly elevated in MG. Results Serum levels of APRIL (r = 0.4789, P = 0.0133), IL-19 (r = 0.5496, P = 0.0036) and IL-35 (r = 0.5396, P = 0.0044) were correlated with those of HMGB1, but no cytokine levels were correlated with peroxiredoxin 5 levels. When we carried out multivariate analyses, only APRIL (P = 0.0244) and IL-19 (P = 0.0009) levels were correlated with HMGB1 levels. Conclusions HMGB1 might be related with upregulation of IL-19 and APRIL levels in MG immunopathogenesis. These molecules could play a key role in controlling autoimmune inflammatory response, and represent a potent therapeutic target in MG.

Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.

Peroxiredoxins (Prxs) play an important role in the protection of insects against the toxicity of reactive oxygen species. Here, we identified and characterized a novel, atypical 2-cysteine (Cys) peroxiredoxin (BmPrx3) from an expressed sequence tag database in a lepidopteran insect, Bombyx mori. The BmPrx3 cDNA contained an open reading frame of 684 bp that encodes a 228-amino-acid protein with a calculated molecular mass of 25 kDa. Sequence comparison revealed that BmPrx3 belongs to the atypical 2-Cys Prxs. Quantitative real-time PCR revealed that BmPrx3 can be detected in all tissues and developmental stages. Recombinant BmPrx3 purified from Escherichia coli exhibited antioxidant activity that removed hydrogen peroxide and protected DNA from oxidative damage. Disc diffusion and viability assays revealed that recombinant BmPrx3 increased bacterial survival under H2 O2 -mediated oxidative stress. In addition, quantitative real-time PCR analysis indicated that BmPrx3 transcription levels were significantly increased in response to various oxidative stresses. Furthermore, BmPrx3 transcription levels in the midgut were regulated by bacterial infection. Taken together, these results suggest that BmPrx3 acts as an antioxidant enzyme to protect the silkworm from various oxidative stresses.

There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra-low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long-term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2-Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin-secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post-thaw viability, good adhesion properties, and well-maintained cell-specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols. Biotechnol. Bioeng. 2016;113: 1511-1521. © 2015 Wiley Periodicals, Inc.

We have previously shown that immunization of outbred rodents with cysteine peptidases-based vaccine elicited type 2-biased immune responses associated with consistent and reproducible protection against challenge Schistosoma mansoni. We herein start to elucidate the molecular basis of C57BL/6 mouse resistance to S. mansoni following treatment with the cysteine peptidase, papain. We evaluated the early cytokine signals delivered by epidermal, dermal, and draining lymph node cells of naive, and S. mansoni -infected mice treated 1 day earlier with 0 or 50 μg papain, or immunized twice with papain only (10 μg/mouse), papain-free recombinant S. mansoni glyceraldehyde 3-phosphate dehydrogenase and 2-Cys peroxiredoxin peptide (10 and 15 μg/mouse, respectively = antigen Mix), or papain-adjuvanted antigen Mix. Schistosoma mansoni infection induced epidermal and lymph node cells to release type 1, type 2 and type 17 cytokines, known to counteract each other. Expectedly, humoral immune responses were negligible until patency. Papain pretreatment or papain-based vaccination diminished or shut off S. mansoni infection early induction of type 1, type 17 and type 2 cytokines except for thymic stromal lymphopoietin and programmed the immune system towards a polarized type 2 immune milieu, associated with highly significant (P < 0.005

Peroxiredoxins are ubiquitous thioredoxin-dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox-dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2-Cys peroxiredoxin (2-Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin-related electron donors, but not on sulfiredoxin, the enzyme reducing the 2-Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2-Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water-stressed Arabidopsis, barley and potato plants, whereas species-dependent up- and down-variations in overoxidation were observed. In contrast, both 2-Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2-Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2-Cys PRX are discussed.

Alkyl hydroperoxide reductase genes (ahpCF) from the soft coral associated Bacillus aquimaris MKSC6.2 have been isolated. The cloned 546 bp ahpC gene encodes a 181 amino acid residues polypeptide. The AhpC belongs to typical 2-Cys peroxiredoxin (Prx) containing conserved peroxidatic cysteine residue (C46 ) required for hydroperoxide reduction and conserved resolving cysteine (C166 ). The isolated 1530 bp ahpF gene encodes a polypeptide of 509 amino acid residues with two conserved C128 HNC131 and C337 PHC340 catalytic residues required for reduction of oxidized-AhpC during catalytic turnover. A survival study with Escherichia coli showed that overexpression of AhpC and AhpF resulted in a total protection against 0.16 mM t-butyl hydroperoxide.

Objective Peroxiredoxin 6 plays important and complex roles in the process of inflammation, but its role in the development of rheumatoid arthritis (RA) remains unclear. We undertook this study to investigate the roles and mechanisms of peroxiredoxin 6 in the development of collagen antibody-induced arthritis (CAIA) and antigen-induced arthritis (AIA) in peroxiredoxin 6-overexpressing transgenic mice, in peroxiredoxin 6-transfected RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients. Methods CAIA and AIA were induced using standard methods. Peroxiredoxin 6-transfected RAW 264.7 cells, macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and synoviocytes from arthritis patients were used to study proinflammatory responses and mechanisms. Clinical scores and histopathologic changes were determined in peroxiredoxin 6-overexpressing transgenic mice and wild-type (WT) mice with CAIA or AIA. Generation of nitric oxide (NO), expression of inducible NO synthase and cyclooxygenase 2, and activity of NF-κB and activator protein 1 (AP-1) were determined in cultured macrophages and synoviocytes as well as in joint tissue from mice by Western blotting, electrophoretic mobility shift assay, and immunohistochemical analysis. Results Development of CAIA and AIA and proinflammatory responses were more exacerbated in peroxiredoxin 6-overexpressing transgenic mice than in WT mice. Overexpression of peroxiredoxin 6 increased lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells, in macrophages isolated from peroxiredoxin 6-overexpressing transgenic mice, and in synoviocytes from arthritis patients, and this was accompanied by up-regulation of the JNK pathway. Moreover, a JNK inhibitor completely blocked RA development and proinflammatory responses. Conclusion Our findings suggest that overexpression of peroxiredoxin 6 might promote development of RA through NF-κB and AP-1 activity via the JNK pathway.

Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone.

Novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being clinically evaluated by several groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this paper have the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.

Aims To investigate redox-regulating enzymes that may have a special role in melanoma pathogenesis due to continuous exposure to microenvironment-produced and ultraviolet radiation-induced oxidative stress. Methods and results We assessed immunohistochemically the expression of antioxidant enzymes peroxiredoxins (Prxs) I–IV, sulfiredoxin (Srx) and redox-regulated proto-oncogene DJ-1 in material consisting of 30 benign naevi, 14 lentigo malignas and 67 malignant melanomas. Evaluation of immunostaining was performed with special attention paid to protein expression in different tumour compartments. In particular, the expression patterns of nuclear Prx I and Prx II and cytoplasmic DJ-1 were decreased significantly in melanomas compared with dysplastic and benign naevi. In multivariate analysis, several prognostic factors were identified: Prx III expression in the cytoplasm of stromal fibroblasts was associated with shortened melanoma-specific survival [hazard ratio (HR) 6.730; 95% confidence interval (CI) 1.579–28.689], while cytoplasmic Prx IV expression in endothelial cells (HR 6.563; 95% CI 1.750–24.620) and Srx expression in the cytoplasm of keratinocytes (HR 6.988; 95% CI 1.559–31.324) were associated with better prognosis independently of ulceration, thickness of melanoma or its diagnostic type. Conclusions Redox-regulating enzymes have the potential to serve as novel prognostic factors and targeting them may offer new therapeutic options in malignant melanoma.

Peroxiredoxins (Prx) catalyse the reduction of hydrogen peroxide (H2O2) and, in association with catalases and other peroxidases, may participate in signal transduction by regulating intercellular H2O2 concentration that in turn can control gene transcription and cell signaling. Using virus-induced-gene-silencing (VIGS), 2-Cys Peroxiredoxin (2CysPrx) family and type-II Peroxiredoxin B (PrxIIB) gene were silenced in Nicotiana benthamiana, to study the impact that the loss of function of each Prx would have in the antioxidant system under control (22 °C) and severe heat stress conditions (48 °C). The results showed that both Prxs, although in different organelles, influence the regeneration of ascorbate to a significant extent, but with different purposes. 2CysPrx affects abscisic acid (ABA) biosynthesis through ascorbate, while PrxIIB does it probably through the xanthophyll cycle. Moreover, 2CysPrx is key in H2O2 scavenging and in consequence in the regulation of ABA signaling downstream of reactive oxygen species and PrxIIB provides an important assistance for H2O2 peroxisome scavenges.

Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system.

The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities is fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA), which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat2;1, cyp20-3 and 2-cysprxA/B, and wild-type (WT). Leaf transcriptomes were analyzed 6 h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared with WT. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism, and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling.

Scope Phosphoserine-containing peptides have been shown to exert antioxidative stress effects, by lowering lipid peroxidation, increasing intracellular glutathione, and increasing the expression of antioxidant enzymes in human intestinal epithelial cells. However, the role of phosphoserine residues in antioxidative stress activity, and their mechanism of action, remains unknown. Methods and results The antioxidative stress activity of phosphoserine and phosphoserine peptides was examined using an in vitro model of hydrogen peroxide (H2O2)-induced oxidative stress in Caco-2 cells. Phosphoserine dimers (2PS) reduced IL-8 secretion in H2O2-treated Caco-2 cells, and reduced H2O2-induced expression of genes involved in inflammation and generation of reactive oxygen species (ROS), including chemokine (C-C motif) ligand 5 (CCL5), lactoperoxidase (LPO), myeloperoxidase (MPO), neutrophil cytosolic factor 1/2 (NCF1/2), and nitric oxide synthase 2A (NOS2), and upregulated metallothionein 3 (MT3), peroxiredoxin 3 (PRDX3), and surfactant, pulmonary-associated protein D (SFTPD), which are involved in protection against oxidative stress and activation of the Nrf2 signaling pathway. At the protein level, 2PS reduced H2O2-induced phosphorylation of the ERK1/2 and JNK MAPKs, and increased Nrf2 expression. Moreover, the ability of 2PS to reduce H2O2-induced IL-8 secretion, a marker of inflammation and oxidative stress, was abrogated in Nrf2 knockdown cells. Conclusion These results suggest that 2PS reduce H2O2-induced oxidative stress via the Nrf2 signaling pathway, and reveal a potential mechanism for the antioxidative stress activity of phosphoserine-containing peptides.

To investigate the relationship between oxidative stress and gastric carcinogenesis of poorly differentiated adenocarcinoma in young patients, we analyzed the surgically resected specimens of 22 young patients (21-30 years) and 29 older patients (41-72 years) with intramucosal gastric cancer of the poorly differentiated type. We used immunohistochemical staining to evaluate the expression of 8-hydroxydeoxyguanosine (8OHdG), induced nitric oxide synthetase (iNOS), and antioxidant enzymes (thioredoxin [TRX] and peroxiredoxin [PRDX1, 2 and 3]). We assessed these proteins in the cancer, noncancerous gastric foveolar epithelium and noncancerous mucosal neck. In both the young and older patient groups, the 8OHdG and TRX expressions were gradually increased in cancer cells compared with the noncancerous foveolar epithelial cells and the noncancerous mucosal neck cells (P < 0.001). Although the iNOS and PRDXs expressions were increased in the noncancerous mucosal neck cells compared with the noncancerous foveolar epithelial cells, regardless of age (P < 0.001), the iNOS and PRDX2 expression in the cancer cells were significantly reduced in the young patients compared with the older patients (P < 0.001, P < 0.05). In conclusion, the reduced expression of iNOS or PRDX2 may play an important role in the carcinogenesis of gastric cancer associated with Helicobacter pylori-induced chronic active gastritis in young patients.

Summary The main peroxiredoxin in Schizosaccharomyces pombe, Tpx1, is important to sustain aerobic growth, and cells lacking this protein are only able to grow on solid plates under anaerobic conditions. We have found that deletion of the gene coding for thioredoxin reductase, trr1, is a suppressor of the sensitivity to aerobic growth of Δtpx1 cells, so that cells lacking both proteins are able to grow on solid plates in the presence of oxygen. We have investigated this suppression effect, and determined that it depends on the presence of catalase, which is constitutively expressed in Δtrr1 cells in a transcription factor Pap1-dependent manner. A complete characterization of the repertoire of hydrogen peroxide scavenging activities in fission yeast suggests that Tpx1 is the only enzyme with sufficient sensitivity for peroxides and cellular abundance as to control the low levels produced during aerobic growth, catalase being the next barrier of detoxification when the steady-state levels of peroxides are increased in Δtpx1 cells. Gpx1, the only glutathione peroxidase encoded by the S. pombe genome, only has a minor secondary role when extracellular peroxides are added. Our study proposes non-overlapping roles for the different hydrogen peroxide scavenging activities of this eukaryotic organism.

Silkworm (Bombyx mori) larvae are widely used to express exogenous proteins. Moreover, some silkworm pupal proteins can be used as drug-loading materials for selfexpressed oral tolerance drugs. However, several proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals. Interestingly, some baculovirus vectors have been shown to alter the host gene and its expression in insect cells, but this has not been confirmed in silkworm. Here, we analyzed the effects of infection with an empty B. mori baculovirus (BmNPV) vector on silkworm pupal protein expression. Using a proteomics approach, the allergens thiol peroxiredoxin (Jafrac1), 27-kDa glycoprotein (p27k), arginine kinase, and paramyosin as well as 32 additional differentially expressed proteins were identified. Downregulation of the messenger RNA expression of the four known allergens was observed after BmNPV infection; subsequent changes in protein expression were confirmed by the western blot analysis using polyclonal antibodies prepared with recombinant proteins of the four allergens. Collectively, these data indicate that the four known allergens of silkworm pupae can be reduced by infection ith an empty BmNPV vector to increase the safety of silkworm pupa-based exogenous protein expression and drug delivery of oral pharmaceuticals. In addition, the four recombinant allergen proteins may contribute to the diagnosis of allergic diseases of silkworm pupa.

Mechanisms of oxidative stress resistance are crucial virulence factors for survival and proliferation of fungal pathogens within the human host. In this study we have identified and functionally characterized the role of sulfiredoxin, Srx1, in oxidative stress resistance of Cryptococcus neoformans causing fungal meningoencephalitis and regulation of peroxiredoxins, Tsa1 and Tsa3, and thioredoxins, Trx1 and Trx2. The C. neoformans HOG (High Osmolarity Glycerol) pathway was essential for the transcriptional regulation of SRX1 under peroxide stress conditions. A gene deletion study revealed that Srx1 was required for cells to counteract peroxide stress, but not other oxidative damaging agents. HOG1 was found to be essential for the induction of adaptive response to peroxide stress with concurrent repression of ergosterol biosynthesis in an SRX1-independent manner. Consistent with this, phosphorylation of C. neoformans Hog1 was modulated by both low and high doses of exogenous hydrogen peroxide treatment. Immunoblot analysis using the C. neoformans Tsa1 specific antibody revealed that both Srx1 and Trx1 were essential for recycling of oxidized Tsa1. In addition to its role in peroxide sensing and response C. neoformans Srx1 was also found to be required for a peroxiredoxin-independent function in promoting fungicide-dependent cell swelling and growth arrest. Finally we showed the importance of C. neoformans Srx1 in fungal pathogenesis by demonstrating its requirement for full virulence using a mouse infection model.

The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed.

Peroxiredoxins (Prxs) are ubiquitous and efficient antioxidant enzymes crucial for redox homeostasis in most organisms, and are of special importance for disease-causing parasites that must protect themselves against the oxidative weapons of the human immune system. Here, we describe reanalyses of crystal structures of two Prxs from malaria parasites. In addition to producing improved structures, we provide normalizing explanations for features that had been noted as unusual in the original report of these structures (Qiu et al., BMC Struct Biol 2012;12:2). Most importantly, we provide evidence that the unusual octameric assembly seen for Plasmodium yoelii Prx1a is not physiologically relevant, but arises because the structure is not of authentic P. yoelii Prx1a, but a variant we designate PyPrx1aN* that has seven native N-terminal residues replaced by an affinity tag. This N-terminal modification disrupts a previously unrecognized, hydrophobic “ball-and-socket” interaction conserved at the B-type dimer interface of Prx1 subfamily enzymes, and is accommodated by a fascinating two-residue “β-slip” type register shift in the β-strand association at a dimer interface. The resulting change in the geometry of the dimer provides a simple explanation for octamer formation. This study illustrates how substantive impacts can occur in protein variants in which native residues have been altered.

Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S-glutathionylation. The formation of an S-glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S-glutathionylation. In pathophysiological settings, S-glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S-glutathionylation in situ, which will enable further discovery into the role of S-glutathionylation in biology and disease. J. Cell. Biochem. 114: 1962-1968, 2013.