Prokaryotic Peroxiredoxins Can Be Sensitive♦ : Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes. Cyanobacterial 2-Cys Peroxiredoxins Sensitive to Oxidative Stress
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PROVIDER: S-EPMC2966139 | biostudies-literature | 2010 Nov
REPOSITORIES: biostudies-literature
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Project description:In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.
| S-EPMC2966063 | biostudies-literature
Project description:Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H(2)O(2)) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.
| S-EPMC3077232 | biostudies-literature
Project description:Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.
| S-EPMC4423512 | biostudies-literature
Project description:Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H2O2). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light-dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.
| S-EPMC6126724 | biostudies-literature
Project description:Peroxiredoxins are abundant cellular antioxidant proteins that help to control intracellular peroxide levels. These proteins may also function, in part, through an evolved sensitivity of some peroxiredoxins towards peroxide-mediated inactivation in hydrogen peroxide signaling in eukaryotes. This review summarizes recent progress in our understanding of the catalytic and regulatory mechanisms of 'typical 2-Cys' peroxiredoxins and of the biological roles played by these important enzymes in oxidative stress and nonstress-related cellular signaling. New evidence suggests localized peroxide buildup plays a role in nonstress-related signaling.
| S-EPMC2747500 | biostudies-literature
Project description:2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.
| S-EPMC6066324 | biostudies-literature
Project description:Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C(P) and C(R), but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C(P)-SO(3)H specific immunoblotting demonstrated overoxidation of C(P) to C(P)-SO(2)H (or C(P)-SO(3)H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C(P) was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H(2)O(2) and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H(2)O(2). A mutagenesis study indicated that lack of Prx2 accumulated sufficient H(2)O(2) to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H(2)O(2), whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H(2)O(2), and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.
| S-EPMC3522253 | biostudies-literature
Project description:Peroxiredoxins (Prxs) are crucially involved in maintaining intracellular H2O2 homeostasis via their peroxidase activity. However, more recently, this class of proteins was found to also transmit oxidizing equivalents to selected downstream proteins, which suggests an important function of Prxs in the regulation of cellular protein redox relays. Using a pull-down assay based on mixed disulfide fishing, we characterized the thiol-dependent interactome of cytosolic Prx1a and mitochondrial Prx1m from the apicomplexan malaria parasite Plasmodium falciparum (Pf). Here, 127 cytosolic and 20 mitochondrial proteins that are components of essential cellular processes were found to interact with PfPrx1a and PfPrx1m, respectively. Notably, our data obtained with active-site mutants suggests that reducing equivalents might also be transferred from Prxs to target proteins. Initial functional analyses indicated that the interaction with Prx can strongly impact the activity of target proteins. The results provide initial insights into the interactome of Prxs at the level of a eukaryotic whole cell proteome. Furthermore, they contribute to our understanding of redox regulatory principles and thiol-dependent redox relays of Prxs in subcellular compartments.
| S-EPMC6753162 | biostudies-literature
Project description:The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease that has devastated pine forests in Asia. Parasitic nematodes are known to have evolved antioxidant stress responses that defend against host plant defenses. In this study, the infestation of whitebark pine, Pinus bungean, with B. xylophilus led to a significant increase in plant hydrogen peroxide (H2O2) and salicylic acid levels. Correspondingly, the expression of an antioxidative enzyme, 2-Cysteine peroxiredoxin (BxPrx), was elevated in B. xylophilus following the H2O2 treatments. Recombinant BxPrx, a thermal stabile and pH tolerant enzyme, exhibited high level of antioxidant activity against H2O2, suggesting that it is capable of protecting cells from free radical attacks. Immunohistochemical localization study showed that BxPrx was broadly expressed across different tissues and could be secreted outside the nematode. Finally, the number of BxPrx homologs in both dauer-like and fungi-feeding B. xylophilus were comparable based on bioinformatics analysis of existing EST libraries, indicating a potential role of BxPrx in both propagative and dispersal nematodes. These combined results suggest that BxPrx is a key genetic factor facilitating the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease.
| S-EPMC4895224 | biostudies-literature
Project description:Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.
| S-EPMC2592686 | biostudies-literature