The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was found to be evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulfide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (the typical 2-Cys-Peroxiredoxin from Anabaena), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulfide bond between the two active site cysteines (Cys-38 and Cys-41). However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulfide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulfide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

ROS production and signalling in chloroplasts: cornerstones and evolving concepts.

Reactive oxygen species (ROS) such as singlet oxygen, superoxide (O2●- ) and hydrogen peroxide (H2 O2 ) are the markers of living cells. Oxygenic photosynthesis produces ROS in abundance, which act as a readout of a functional electron transport system and metabolism. The concept that photosynthetic ROS production is a major driving force in chloroplast to nucleus retrograde signalling is embedded in the literature, as is the role of chloroplasts as environmental sensors. The different complexes and components of the photosynthetic electron transport chain (PETC) regulate O2●- production in relation to light energy availability and the redox state of the stromal Cys-based redox systems. All of the ROS generated in chloroplasts have the potential to act as signals and there are many sulphhydryl-containing proteins and peptides in chloroplasts that have the potential to act as H2 O2 sensors and function in signal transduction. While ROS may directly move out of the chloroplasts to other cellular compartments, ROS signalling pathways can only be triggered if appropriate ROS-sensing proteins are present at or near the site of ROS production. Chloroplast antioxidant systems serve either to propagate these signals or to remove excess ROS that cannot effectively be harnessed in signalling. The key challenge is to understand how regulated ROS delivery from the PETC to the Cys-based redox machinery is organised to transmit redox signals from the environment to the nucleus. Redox changes associated with stromal carbohydrate metabolism also play a key role in chloroplast signalling pathways.

Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells.

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.

Studying the Human Microbiota: Advances in Understanding the Fundamentals, Origin, and Evolution of Biological Timekeeping.

The recently observed circadian oscillations of the intestinal microbiota underscore the profound nature of the human-microbiome relationship and its importance for health. Together with the discovery of circadian clocks in non-photosynthetic gut bacteria and circadian rhythms in anucleated cells, these findings have indicated the possibility that virtually all microorganisms may possess functional biological clocks. However, they have also raised many essential questions concerning the fundamentals of biological timekeeping, its evolution, and its origin. This narrative review provides a comprehensive overview of the recent literature in molecular chronobiology, aiming to bring together the latest evidence on the structure and mechanisms driving microbial biological clocks while pointing to potential applications of this knowledge in medicine. Moreover, it discusses the latest hypotheses regarding the evolution of timing mechanisms and describes the functions of peroxiredoxins in cells and their contribution to the cellular clockwork. The diversity of biological clocks among various human-associated microorganisms and the role of transcriptional and post-translational timekeeping mechanisms are also addressed. Finally, recent evidence on metabolic oscillators and host-microbiome communication is presented.

Changes of the Protein CoAlation Pattern in Response to Oxidative Stress and Capacitation in Human Spermatozoa.

The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H2O2), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H2O2 and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation.

Minocycline pre-treatment up-regulates antioxidant enzymes and enhances the regenerative potential of MSCs in rat myocardial infarction model.

Objectives: To determine the effect of the pre-treatment of mesenchymal stem cells (MSCs) with minocycline on the expression of antioxidant genes and cardiac repair post myocardial infarction (MI) in rats. METHODS: Rat bone marrow derived MSCs were used in the study. Cytotoxicity of minocycline in MSCs was determined using JC1 assay to identify a safe drug dose for further experiments. The MSCs were pre-treated with 1.0 µM minocycline for 24 hours and then treated with hydrogen peroxide (H2O2), after that mRNA was isolated and the expression levels of antioxidant genes including peroxiredoxin, glutathione peroxidase, and superoxide dismutase were determined. Finally, minocycline pre-treated MSCs were used to treat rats induced with MI by the ligation of left anterior descending coronary artery. The cardiac function was evaluated at two and four weeks post MI using echocardiography. RESULTS: At 1.0 µM concentration, minocycline was found to be safe for MSCs and used for subsequent experiments. Minocycline pre-treatment was found to up regulate several antioxidant genes in oxidatively stressed MSCs. Furthermore, minocycline pre-treated MSCs displayed greater improvement in cardiac left ventricular function at two and four-weeks post MI as compared to untreated rats. CONCLUSIONS: Pre-treatment of MSCs with minocycline enhances the expression of antioxidant genes and promotes their capability to repair cardiac function after MI.

A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae.

Genetically encoded fluorescent H2O2 probes continue to advance the field of redox biology. Here, we compare the previously established peroxiredoxin-based H2O2 probe roGFP2-Tsa2ΔCR with the newly described OxyR-based H2O2 probe HyPer7, using yeast as the model system. Although not as sensitive as roGFP2-Tsa2ΔCR, HyPer7 is much improved relative to earlier HyPer versions, most notably by ratiometric pH stability. The most striking difference between the two probes is the dynamics of intracellular probe reduction. HyPer7 is rapidly reduced, predominantly by the thioredoxin system, whereas roGFP2-Tsa2ΔCR is reduced more slowly, predominantly by the glutathione system. We discuss the pros and cons of each probe and suggest that future side-by-side measurements with both probes may provide information on the relative activity of the two major cellular reducing systems.

Thiol-based redox-active proteins as cardioprotective therapeutic agents in cardiovascular diseases.

Thiol-based redox compounds, namely thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs), stand as a pivotal group of proteins involved in antioxidant processes and redox signaling. Glutaredoxins (Grxs) are considered as one of the major families of proteins involved in redox regulation by removal of S-glutathionylation and thereby reactivation of other enzymes with thiol-dependent activity. Grxs are also coupled to Trxs and Prxs recycling and thereby indirectly contribute to reactive oxygen species (ROS) detoxification. Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases, which play an essential role in the detoxification of hydrogen peroxide, aliphatic and aromatic hydroperoxides, and peroxynitrite. The Trxs, Grxs and Prxs systems, which reversibly induce thiol modifications, regulate redox signaling involved in various biological events in the cardiovascular system. This review focuses on the current knowledge of the role of Trxs, Grxs and Prxs on cardiovascular pathologies and especially in cardiac hypertrophy, ischemia/reperfusion (I/R) injury and heart failure as well as in the presence of cardiovascular risk factors, such as hypertension, hyperlipidemia, hyperglycemia and metabolic syndrome. Further studies on the roles of thiol-dependent redox systems in the cardiovascular system will support the development of novel protective and therapeutic strategies against cardiovascular diseases.

Integrative analysis the characterization of peroxiredoxins in pan-cancer.

BACKGROUND: Peroxiredoxins (PRDXs) are an antioxidant enzymes protein family involved in several biological functions such as differentiation, cell growth. In addition, previous studies report that PRDXs play critical roles in the occurrence and development of carcinomas. However, few studies have conducted systematic analysis of PRDXs in cancers. Therefore, the present study sought to explore the molecular characteristics and potential clinical significance of PRDX family members in pan cancer and further validate the function of PRDX6 in bladder urothelial carcinoma (BLCA). METHODS: A comprehensive analysis of PRDXs in 33 types of cancer was performed based on the TCGA database. This involved an analysis of mRNA expression profiles, genetic alterations, methylation, prognostic values, potential biological pathways and target drugs. Moreover, both the gain and loss of function strategies were used to assess the importance and mechanism of PRDX6 in the cell cycle of BLCA. RESULT: Analysis showed abnormal expression of PRDX1-6 in several types of cancer compared to normal tissues. Univariate Cox proportional hazard regression analysis showed that expression levels of PRDX1, PRDX4 and PRDX6 were mostly associated with poor survival of OS, DSS and PFI, and PRDX2 and PRDX3 with favorable survival. In addition, the expression of PRDX genes were positively correlated with CNV and negatively with methylation. Moreover, analysis based on PharmacoDB dataset showed that the augmented levels of PRDX1, PRDX3 and PRDX6 were significantly correlated with EGFR/VEGFR inhibitor drugs. Furthermore, knocking down of PRDX6 inhibited growth of cancer cells through the JAK2-STAT3 in bladder cell lines. CONCLUSIONS: PRDXs are potential biomarkers and therapeutic targets for several carcinomas, especially for BLCA. In addition, PRDX6 could regulate proliferation of cancer cell via JAK2-STAT3 pathway and involve into the process of cell cycle in BLCA.

Expression patterns of peroxiredoxin genes in bronchial epithelial cells exposed to diesel exhaust particles.

Several mechanisms have been suggested to explain the adverse effects of air pollutants on airway cells. One such explanation is the presence of high concentrations of oxidants and pro-oxidants in environmental pollutants. All animal and plant cells have developed several mechanisms to prevent damage by oxidative molecules. Among these, the peroxiredoxins (PRDXs) are of interest due to a high reactivity with reactive oxygen species (ROS) through the functioning of the thioredoxin/thioredoxin reductase system. This study aimed to verify the gene expression patterns of the PRDX family in bronchial epithelial airway cells (BEAS-2B) cells exposed to diesel exhaust particles (DEPs) at a concentration of 15 µg/mL for 1 or 2 h because this it is a major component of particulate matter in the atmosphere. There was a significant decrease in mRNA fold changes of PRDX2 (0.43 ± 0.34; *p = 0.0220), PRDX5 (0.43 ± 0.34; *p = 0.0220), and PRDX6 (0.33 ± 0.25; *p = 0.0069) after 1 h of exposure to DEPs. The reduction in mRNA levels may consequently lead to a decrease in the levels of PRDX proteins, increasing oxidative stress in bronchial epithelial cells BEAS-2B and thus, negatively affecting cellular functions.

Relevance of peroxiredoxins in pathogenic microorganisms.

The oxidative and nitrosative responses generated by animals and plants are important defenses against infection and establishment of pathogenic microorganisms such as bacteria, fungi, and protozoa. Among distinct oxidant species, hydroperoxides are a group of chemically diverse compounds that comprise small hydrophilic molecules, such as hydrogen peroxide and peroxynitrite, and bulky hydrophobic species, such as organic hydroperoxides. Peroxiredoxins (Prx) are ubiquitous enzymes that use a highly reactive cysteine residue to decompose hydroperoxides and can also perform other functions, like molecular chaperone and phospholipase activities, contributing to microbial protection against the host defenses. Prx are present in distinct cell compartments and, in some cases, they can be secreted to the extracellular environment. Despite their high abundance, Prx expression can be further increased in response to oxidative stress promoted by host defense systems, by treatment with hydroperoxides or by antibiotics. In consequence, some isoforms have been described as virulence factors, highlighting their importance in pathogenesis. Prx are very diverse and are classified into six different classes (Prx1-AhpC, BCP-PrxQ, Tpx, Prx5, Prx6, and AhpE) based on structural and biochemical features. Some groups are absent in hosts, while others present structural peculiarities that differentiate them from the host's isoforms. In this context, the intrinsic characteristics of these enzymes may aid the development of new drugs to combat pathogenic microorganisms. Additionally, since some isoforms are also found in the extracellular environment, Prx emerge as attractive targets for the production of diagnostic tests and vaccines. KEY POINTS: • Peroxiredoxins are front-line defenses against host oxidative and nitrosative stress. • Functional and structural peculiarities differ pathogen and host enzymes. • Peroxiredoxins are potential targets to microbicidal drugs.

Role of Glutathione Peroxidases and Peroxiredoxins in Free Radical-Induced Pathologies.

In this review, we discuss the pathogenesis of some socially significant diseases associated with the development of oxidative stress, such as atherosclerosis, diabetes, and radiation sickness, as well as the possibilities of the therapeutic application of low-molecular-weight natural and synthetic antioxidants for the correction of free radical-induced pathologies. The main focus of this review is the role of two phylogenetically close families of hydroperoxide-reducing antioxidant enzymes peroxiredoxins and glutathione peroxidases - in counteracting oxidative stress. We also present examples of the application of exogenous recombinant antioxidant enzymes as therapeutic agents in the treatment of pathologies associated with free-radical processes and discuss the prospects of the therapeutic use of exogenous antioxidant enzymes, as well as the ways to improve their therapeutic properties.

Hydroperoxide-Reducing Enzymes in the Regulation of Free-Radical Processes.

The review presents current concepts of the molecular mechanisms of oxidative stress development and describes main stages of the free-radical reactions in oxidative stress. Endogenous and exogenous factors of the oxidative stress development, including dysfunction of cell oxidoreductase systems, as well as the effects of various external physicochemical factors, are discussed. The review also describes the main components of the antioxidant defense system and stages of its evolution, with a special focus on peroxiredoxins, glutathione peroxidases, and glutathione S-transferases, which share some phylogenetic, structural, and catalytic properties. The substrate specificity, as well as the similarities and differences in the catalytic mechanisms of these enzymes, are discussed in detail. The role of peroxiredoxins, glutathione peroxidases, and glutathione S-transferases in the regulation of hydroperoxide-mediated intracellular and intercellular signaling and interactions of these enzymes with receptors and non-receptor proteins are described. An important contribution of hydroperoxide-reducing enzymes to the antioxidant protection and regulation of such cell processes as growth, differentiation, and apoptosis is demonstrated.

Effect of 2-Cys Peroxiredoxins Inhibition on Redox Modifications of Bull Sperm Proteins.

Sperm peroxiredoxins (PRDXs) are moonlighting proteins which, in addition to their antioxidant activity, also act as redox signal transducers through PRDX-induced oxidative post-translational modifications of proteins (oxPTMs). Despite extensive knowledge on the antioxidant activity of PRDXs, the mechanisms related to PRDX-mediated oxPTMs are poorly understood. The present study aimed to investigate the effect of bull sperm 2-Cys PRDX inhibition by Conoidin A on changes in oxPTM levels under control and oxidative stress conditions. The results showed that a group of sperm mitochondrial (LDHAL6B, CS, ACO2, SDHA, ACAPM) and actin cytoskeleton proteins (CAPZB, ALDOA, CCIN) is oxidized due to the action of 2-Cys PRDXs under control conditions. In turn, under oxidative stress conditions, 2-Cys PRDX activity seems to be focused on antioxidant function protecting glycolytic, TCA pathway, and respiratory chain enzymes; chaperones; and sperm axonemal tubulins from oxidative damage. Interestingly, the inhibition of PRDX resulted in oxidation of a group of rate-limiting glycolytic proteins, which is known to trigger the switching of glucose metabolism from glycolysis to pentose phosphate pathway (PPP). The obtained results are expected to broaden the knowledge of the potential role of bull sperm 2-Cys in both redox signal transmission and antioxidant activity.

Hydrogen peroxide reactivity and specificity in thiol-based cell signalling.

Reversible oxidation of thiol proteins is an important cell signalling mechanism. In many cases, this involves generation or exposure of the cells to H2O2, and oxidation of proteins that are not particularly H2O2-reactive. There is a conundrum as to how these proteins are oxidised when other highly reactive proteins such as peroxiredoxins are present. This article discusses potential mechanisms, focussing on recent evidence for oxidation being localised within the cell, redox relays involving peroxiredoxins operating in some signalling pathways, and mechanisms for facilitated or directed oxidation of specific targets. These findings help define conditions that enable redox signalling but there is still much to learn regarding mechanisms.

Redox regulation by peroxiredoxins is linked to their thioredoxin-dependent oxidase function.

The chloroplast contains three types of peroxiredoxins (PRXs). Recently, 2-CysPRX was associated with thioredoxin (TRX) oxidation-dependent redox regulation. Here, this analysis was expanded to include PRXQ and PRXIIE. Oxidized PRXQ was able to inactivate NADPH malate dehydrogenase and fructose-1,6-bisphosphatase most efficiently in the presence of TRX-m1 and TRX-m4. The inactivation ability of TRXs did not entirely match their reductive activation efficiency. PRXIIE was unable to function as TRX oxidase in enzyme regulation. This conclusion was further supported by the observation that PRXQ adopts the oxidized form by about 50% in leaves, supporting a possible function as a TRX oxidase similar to 2-CysPRX. Results on the oxidation state of photosystem I (P700), plastocyanin, and ferredoxin in intact leaves indicate that each type of PRX has distinct regulatory functions, and that both 2-CysPRX and PRXQ conditionally assist in adjusting the redox state of target proteins for proper activity.

Protective role of peroxiredoxin-4 in heart failure.

Recently, we have read with great interest the article published by Ibarrola et al. (Clin. Sci. (Lond.) (2018) 132, 1471-1485), which used proteomics and immunodetection methods to show that Galectin-3 (Gal-3) down-regulated the antioxidant peroxiredoxin-4 (Prx-4) in cardiac fibroblasts. Authors concluded that 'antioxidant activity of Prx-4 had been identified as a protein down-regulated by Gal-3. Moreover, Gal-3 induced a decrease in total antioxidant capacity which resulted in a consequent increase in peroxide levels and oxidative stress markers in cardiac fibroblasts.' We would like to point out some results stated in the article that need further investigation and more detailed discussion to clarify certain factors involved in the protective role of Prx-4 in heart failure.

(Letter to the Editor) Response to: protective role of peroxiredoxin-4 in heart failure.

We thank Ahmed et al. for their letter regarding our study 'Galectin-3 down-regulates antioxidant peroxiredoxin-4 in human cardiac fibroblasts' [1]. As emphasized by Ahmed et al., Prx-4 levels decrease [2] whereas MFN-2, OPA-1 and PGC-1α levels increase [3] in dilated cardiomyopathy (DCM). Moreover, Gal-3 expression is also increased in DCM [4]. In our study, we showed in vitro that Gal-3 decreased Prx-4 without modifying MFN-2 or PGC-1α levels in human cardiac fibroblasts. Although cardiac Prx-4 decrease could be a direct consequence of Gal-3 effects on cardiac fibroblasts, we cannot exclude the possibility that other factors increase MFN-2, OPA-1 and PGC-1α levels in both cardiac fibroblasts or cardiomyocytes in the context of DCM. Further studies are needed to clarify the association between Prx-4 decrease and the increase in other mitochondrial proteins in DCM.

Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis.

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p < .05). The H2O2 concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p < .05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.

Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism.

The FUR (Ferric Uptake Regulator) family in Anabaena sp. PCC 7120 consists of three paralogs named FurA (Fur), FurB (Zur) and FurC (PerR). furC seems to be an essential gene in the filamentous nitrogen-fixing strain Anabaena sp. PCC 7120, suggesting that it plays a fundamental role in this organism. In order to better understand the functions of FurC in Anabaena, the phenotype of a derivative strain that overexpresses this regulator (EB2770FurC) has been characterized. The furC-overexpressing variant presented alterations in growth rate, morphology and ultrastructure, as well as higher sensitivity to peroxide than Anabaena sp. PCC 7120. Interestingly, the overexpression of furC led to reduced photosynthetic O2 evolution, increased respiratory activity, and had a significant influence in the composition and efficiency of both photosystems. Comparative transcriptional analyses, together with electrophoretic mobility shift assays allowed the identification of different genes directly controlled by FurC, and involved in processes not previously related to PerR proteins, such as the cell division gene ftsZ and the major thylakoid membrane protease ftsH. The rise in the transcription of ftsH in EB2770FurC cells correlated with reduced levels of the D1 protein, which is involved in the PSII repair cycle. Deregulation of the oxidative stress response in EB2770FurC cells led to the identification of novel FurC targets involved in the response to H2O2 through different mechanisms. These results, together with the effect of furC overexpression on the composition, stability and efficiency of the photosynthetic machinery of Anabaena, disclose novel links between PerR proteins, cell division and photosynthesis in filamentous cyanobacteria.