Cytosolic Peroxiredoxin Research Articles

Kinetic and structural assessment of the reduction of human 2-Cys peroxiredoxins by thioredoxins.

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105 -106 m-1 s-1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins.

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.

Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in Saccharomyces cerevisiae.

Heat stress is one of the abiotic stresses that leads to oxidative stress. To protect themselves, yeast cells activate the antioxidant response, in which cytosolic peroxiredoxin Tsa1 plays an important role in hydrogen peroxide removal. Concomitantly, the activation of the heat shock response (HSR) is also triggered. Nitro-fatty acids are signaling molecules generated by the interaction of reactive nitrogen species with unsaturated fatty acids. These molecules have been detected in animals and plants. They exert their signaling function mainly through a post-translational modification called nitroalkylation. In addition, these molecules are closely related to the induction of the HSR. In this work, the endogenous presence of nitro-oleic acid (NO2-OA) in Saccharomyces cerevisiae is identified for the first time by LC-MS/MS. Both hydrogen peroxide levels and Tsa1 activity increased after heat stress with no change in protein content. The nitroalkylation of recombinant Tsa1 with NO2-OA was also observed. It is important to point out that cysteine 47 (peroxidatic) and cysteine 171 (resolving) are the main residues responsible for protein activity. Moreover, the in vivo nitroalkylation of Tsa1 peroxidatic cysteine disappeared during heat stress as the hydrogen peroxide generated in this situation caused the rupture of the NO2-OA binding to the protein and, thus, restored Tsa1 activity. Finally, the amino acid targets susceptible to nitroalkylation and the modulatory effect of this PTM on the enzymatic activity of Tsa1 are also shown in vitro and in vivo. This mechanism of response was faster than that involving the induction of genes and the synthesis of new proteins and could be considered as a key element in the fine-tuning regulation of defence mechanisms against oxidative stress in yeast.

Spatial and temporal control of mitochondrial H2O2 release in intact human cells

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high‐affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria‐released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2‐mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

Dietary manganese levels influence growth, manganese bioaccumulation and expression of genes involved in antioxidant response of swimming crab ( Portunus trituberculatus )

An 8-week feeding trial was conducted to investigate the effects of dietary manganese (Mn) levels on growth performance, Mn bioaccumulation in tissues and antioxidant capacity in juvenile swimming crab (Portunus trituberculatus). Six semipurified experimental diets were formulated to contain graded levels of Mn (manganese sulphate monohydrate as Mn source); the analysed Mn concentrations were 9.8, 20.2, 29.7, 38.2, 65.6 and 123.6 mg/kg. The percent weight gain (PWG) and specific growth rate (SGR) were significantly affected by dietary Mn levels; the highest PWG and SGR were found in crabs fed with the diet containing 38.2 mg/kg Mn. Crabs fed with the diet containing 123.6 mg/kg Mn had higher Mn concentration in muscle and carapace than those fed with the other diets. However, the highest Mn concentration in hepatopancreas was recorded in crabs fed with the diet containing 65.6 mg/kg Mn. Moreover, the lowest activities of manganese superoxide dismutase (Mn-SOD), catalase (CAT) and total antioxidant capacity (T-AOC) occurred at crabs fed with the dietary 9.8 mg/kg Mn. Crabs fed with the 9.8 mg/kg Mn diet exhibited lower relative expression of genes involved into antioxidant ability such as cytosolic manganese superoxide dismutase (cMnsod), glutathione peroxidase (gpx), peroxiredoxin (prx) and catalase (cat) in hepatopancreas than those fed with the other diets, and the highest expression were recorded in crabs fed with the diet containing 38.2 mg/kg Mn. Based on two slope broken-line and quadratic regression analysis of PWG against dietary Mn levels, the optimum dietary Mn requirement was estimated to be 48.02 and 53.30 mg/kg for juvenile Portunus trituberculatus. The present study provided further insight into the function of dietary Mn in crustacean.

Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors

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

The Mitochondria-to-Cytosol H2O2 Gradient Is Caused by Peroxiredoxin-Dependent Cytosolic Scavenging.

Fluorescent protein-based reporters used to measure intracellular H2O2 were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular H2O2, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible H2O2 in the cytosol from mitochondrial origin unless the main H2O2 scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and ∆tpx1 cells. We conclude that there is a bidirectional flux of H2O2 across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.

Comparison of the protective effect of cytosolic and mitochondrial Peroxiredoxin 5 against glutamate-induced neuronal cell death

ABSTRACT Objectives: Although glutamate is an essential factor in the neuronal system, excess glutamate can produce excitotoxicity. We previously reported that Peroxiredoxin 5 (Prx5) protects neuronal cells from glutamate toxicity via its antioxidant effects. However, it is unclear whether cytosolic or mitochondrial Prx5 provides greater neuroprotection. Here, we investigated differences in the neuroprotective effects of cytosolic and mitochondrial Prx5. Methods: We analyzed patterns of cytosolic and mitochondrial H2O2 generation in glutamate toxicity using HyPer protein. And then, we confirmed the change of intracellular ROS level and apoptosis with respective methods. The mitochondrial dynamics was assessed with confocal microscope imaging and western blotting. Results: We found that the level of mitochondrial H2O2 greatly increased compared to cytosolic H2O2 and it affected cytosolic H2O2 generation after glutamate treatment. In addition, we confirmed that mitochondrial Prx5 provides more effective neuroprotection than cytosolic Prx5. Discussion: Overall, our study reveals the mechanisms of cytosolic and mitochondrial ROS in glutamate toxicity. Our findings suggest that mitochondrial ROS and Prx5 are attractive therapeutic targets and that controlling these factors be useful for the prevention of neurodegenerative diseases.

Tolerance to nascent protein misfolding stress requires fine-tuning of the cAMP/PKA pathway

Protein aggregation is the abnormal association of misfolded proteins into larger, often insoluble structures that can be toxic during aging and in protein aggregation-associated diseases. Previous research has established a role for the cytosolic Tsa1 peroxiredoxin in responding to protein misfolding stress. Tsa1 is also known to downregulate the cAMP/protein kinase A (PKA) pathway as part of the response to hydrogen peroxide stress. However, whether the cAMP/PKA pathway is involved in protein misfolding stress is not known. Using transcriptomics, we examined the response to protein misfolding stress and found upregulation of numerous stress gene functions and downregulation of many genes related to protein synthesis and other growth-related processes consistent with the well-characterized environmental stress response. The scope of the transcriptional response is largely similar in wild-type and tsa1 mutant strains, but the magnitude is dampened in the strain lacking Tsa1. We identified a direct protein interaction between Tsa1 and the Bcy1 regulatory subunit of PKA that is present under normal growth conditions and explains the observed differences in gene expression profiles. This interaction is increased in a redox-dependent manner in response to nascent protein misfolding, via Tsa1-mediated oxidation of Bcy1. Oxidation of Bcy1 causes a reduction in cAMP binding by Bcy1, which dampens PKA pathway activity, leading to a targeted reprogramming of gene expression. Redox regulation of the regulatory subunit of PKA provides a mechanism to mitigate the toxic consequences of protein misfolding stress that is distinct to stress caused by exogenous sources of reactive oxygen species.

Miro1-mediated mitochondrial positioning supports subcellular redox status

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.

Wine yeast peroxiredoxin TSA1 plays a role in growth, stress response and trehalose metabolism in biomass propagation.

Peroxiredoxins are a family of peroxide-degrading enzymes for challenging oxidative stress. They receive their reducing power from redox-controlling proteins called thioredoxins, and these, in turn, from thioredoxin reductase. The main cytosolic peroxiredoxin is Tsa1, a moonlighting protein that also acts as protein chaperone a redox switch controlling some metabolic events. Gene deletion of peroxiredoxins in wine yeasts indicate that TSA1, thioredoxins and thioredoxin reductase TRR1 are required for normal growth in medium with glucose and sucrose as carbon sources. TSA1 gene deletion also diminishes growth in molasses, both in flasks and bioreactors. The TSA1 mutation brings about an expected change in redox parameters but, interestingly, it also triggers a variety of metabolic changes. It influences trehalose accumulation, lowering it in first molasses growth stages, but increasing it at the end of batch growth, when respiratory metabolism is set up. Glycogen accumulation at the entry of the stationary phase also increases in the tsa1Δ mutant. The mutation reduces fermentative capacity in grape juice, but the vinification profile does not significantly change. However, acetic acid and acetaldehyde production decrease when TSA1 is absent. Hence, TSA1 plays a role in the regulation of metabolic reactions leading to the production of such relevant enological molecules.

Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A.

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

The Use of Specific Serological Biomarkers to Detect CaniLeish Vaccination in Dogs.

Canine leishmaniosis (CanL) prevention in the Mediterranean basin is considered essential to stop human zoonotic visceral leishmaniasis. In this context, vaccination of dogs is expected to have a significant impact in disease control. CaniLeish® (Virbac Animal Health) is one of a few CanL vaccines that are at this moment licensed in Europe. This vaccine contains purified excreted-secreted proteins of Leishmania having several antigens/immunogens with potential to influence serological response. Therefore, it is important to know if CaniLeish vaccination increased the diagnostic challenges associated with conventional serology, limiting the value of some antigens. To address this 20 dogs from a cohort of 35 healthy dogs that were vaccinated, maintained indoor for 1 month and then returned to their natural domiciles for 2 years. After this period, they were re-called to evaluate their clinical/parasitological condition and assess the evolution of seroreactivity against different antigens: soluble promastigote Leishmania antigens (SPLA), recombinant protein Leishmania infantum cytosolic peroxiredoxin, recombinant protein K39 (rK39), recombinant protein K28 and recombinant kinesin degenerated derived repeat using ELISA. Two years after vaccination all vaccinated non-infected animals were seropositive for SPLA. For the other antigens the serological profile was indistinguishable from non-infected animals. Moreover, vaccinated animals presented a characteristic relative serological profile, with higher normalized serological response to SPLA than rK39. This fact enabled to distinguish with sensitivity 92.3% and specificity 95.4%, vaccinated non-infected dogs from infected and non-infected dogs. Ultimately, relative serological profile enabled the detection of healthy vaccinated animals enabling more accurate serological surveys.

A role for peroxiredoxins in H2O2- and MEKK-dependent activation of the p38 signaling pathway

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the cellular response to various stresses and its deregulation accompanies pathological conditions such as cancer and chronic inflammation. Hydrogen peroxide (H2O2) is a well-established activator of the p38 MAPK signaling pathway. However, the mechanisms of H2O2-induced p38 activation are not yet fully understood. In Drosophila cells, we find that H2O2-induced activation of p38 depends on the MAPK kinase kinase (MAP3K) Mekk1. In line with the emerging role of peroxiredoxins as H2O2 sensors and signal transmitters we observe an H2O2-dependent interaction between Mekk1 and the cytosolic peroxiredoxin of Drosophila, Jafrac1. In human cells, MEKK4 (the homologue of Mekk1) and peroxiredoxin-2 (Prx2) interact in a similar manner, suggesting an evolutionarily conserved mechanism. In both organisms, H2O2 induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin. We propose that these conjugates represent the relaying of oxidative equivalents from H2O2 to the MAP3K and that the oxidation of Mekk1/MEKK4 leads to the downstream activation of p38 MAPK. Indeed, the depletion of cytosolic 2-Cys peroxiredoxins in human cells diminished H2O2-induced activation of p38 MAPK.

Aberrant expression of redox regulatory proteins in patients with concomitant primary Sclerosing cholangitis/inflammatory bowel disease

ObjectivePrimary Sclerosing Cholangitis (PSC) is a severe cholestatic liver disease characterized by progressive peri-biliary tract inflammation, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%–80% of PSC patients (PSC/IBD). We previously reported dysregulation of key anti-oxidant pathways in PSC/IBD. The objective of this study was to expand previous data by examining the abundance of thioredoxins (Trx) in PSC/IBD. MethodsUsing hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, the protein abundance of thioredoxin, thioredoxin reductase (TrxR1), and their downstream substrates peroxiredoxins was assessed. ResultsWestern blot analyses of thioredoxin and peroxiredoxin abundance revealed significant increases in abundance of Trx1 and TrxR1 whereas expression of thioredoxin-interacting protein was significantly decreased in PSC/IBD. Concurrently, abundance of cytosolic peroxiredoxins was not significantly impacted. The abundance of mitochondrial Trx2, along with peroxiredoxins 3, 5 and 6 were significantly decreased by concurrent PSC/IBD. Histological staining of Trx1/TrxR1 revealed elevated nuclear Trx1 and TrxR1 staining within cholangiocytes as well as an overall periportal increase in expression in PSC/IBD. An examination of additional anti-oxidant responses reveal suppression of gamma-glutamylcysteine synthetase and heme oxygenase (HO-1) whereas expression of the protein chaperone glucose regulated protein 78 increased suggesting elevated cellular stress in PSC/IBD. ConclusionsResults herein suggest that in addition to severe dysregulation of anti-oxidant responses, cholestasis impacts both cytosolic/nuclear (Trx1) as well as mitochondrial (Trx2) redox signaling and control pathways.

P-260 - Peroxiredoxin expression restores infectivity in avirulent Trypanosoma cruzi strains: role of pathogen antioxidant enzymes as virulence factors

Chagas’ disease (CD), caused by the protozoan Trypanosoma cruzi, remains a major public health concern. During invasion, T. cruzi must deal with the oxidative environment found inside the mammalian host in order to spread and persist in the infected tissues for years. The parasite antioxidant armamentarium is critical for parasite survival either in cell (macrophage/cardiomyocyte) or animal models. In this work, we used a human isolated pathogenic T. cruzi clone (C8C3) that was maintained for years by regular mice passages preserving its virulence (C8C3hvir) or, in axenic culture, turning it avirulent (C8C3lvir). To study if the antioxidant armamentarium influences this virulence behaviour, we searched for the expression of different antioxidant enzymes. Specifically, we found that the virulent cell line expresses higher levels of mitochondrial (TcMPX) and cytosolic (TcCPX) peroxiredoxins (Prx) respect to the avirulent cell line. To determine if these changes in Prx expression may be involved in parasite virulence, we genetically-modified the avirulent cell line in order to overexpress TcCPX. Avirulent parasites overexpressing TcCPX restored infectivity to macrophages and cardiomyocytes in culture and, importantly, in the mouse model of CD.

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.

A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation.

Hydrogen peroxide (H2O2) acts as a signaling messenger by triggering the reversible oxidation of redox-regulated proteins. It remains unclear how proteins can be oxidized by signaling levels of H2O2 in the presence of peroxiredoxins, which are highly efficient peroxide scavengers. Here we show that the rapid formation of disulfide bonds in cytosolic proteins is enabled, rather than competed, by cytosolic 2-Cys peroxiredoxins. Under the conditions tested, the combined deletion or depletion of cytosolic peroxiredoxins broadly frustrated H2O2-dependent protein thiol oxidation, which is the exact opposite of what would be predicted based on the assumption that H2O2 oxidizes proteins directly. We find that peroxiredoxins enable rapid and sensitive protein thiol oxidation by relaying H2O2-derived oxidizing equivalents to other proteins. Although these findings do not rule out the existence of Prx-independent H2O2 signaling mechanisms, they suggest a broader role for peroxiredoxins as sensors and transmitters of H2O2 signals than hitherto recognized.

Trypanosoma cruzi tryparedoxin II interacts with different peroxiredoxins under physiological and oxidative stress conditions

Trypanosoma cruzi, the etiologic agent of Chagas disease, has to cope with reactive oxygen and nitrogen species during its life cycle in order to ensure its survival and infection. The parasite detoxifies these species through a series of pathways centered on trypanothione that depend on glutathione or low molecular mass dithiol proteins such as tryparedoxins. These proteins transfer reducing equivalents to peroxidases, including mitochondrial and cytosolic peroxiredoxins, TcMPx and TcCPx, respectively. In T. cruzi two tryparedoxins have been identified, TXNI and TXNII with different intracellular locations. TXNI is a cytosolic protein while TXNII due to a C-terminal hydrophobic tail is anchored in the outer membrane of the mitochondrion, endoplasmic reticulum and glycosomes. TXNs have been suggested to be involved in a majority of biological processes ranging from redox mechanisms to protein translation. Herein, a comparison of the TXNII interactomes under physiological and oxidative stress conditions was examined. Under physiological conditions, apart from the proteins with unknown biological process annotation, the majority of the identified proteins are related to cell redox homeostasis and biosynthetic processes, while under oxidative stress conditions, are involved in stress response, cell redox homeostasis, arginine biosynthesis and microtubule based process. Interestingly, although TXNII interacts with both peroxiredoxins under physiological conditions, upon oxidative stress, TcMPx interaction prevails. The relevance of the interactions is discussed opening a new perspective of TXNII functions.

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.