Biochemical and biophysical characterization of a peroxiredoxin selected from the Delftia sp., a polyethylene-associated bacterium.

Rubrerythrins are non-haem iron proteins that have been implicated in oxidative stress protection in anaerobic bacteria and archaea. However, up to now, this role has not been confirmed directly by inactivation of a rubrerythrin gene. Here we report generation of an rbr- mutant of Porphyromonas gingivalis, an obligately anaerobic gingival pathogenic bacterium. Characterization of the rbr- strain clearly showed that P. gingivalis produces a rubrerythrin-like protein that is absent in the rbr- strain, and that the P. gingivalis rbr- strain is more dioxygen- and hydrogen peroxide-sensitive than the wild type. The latter conclusion is based on two independent results, namely, deeper no-growth zones upon diffusion of the oxidants through soft agar culture tubes and growth impairment of liquid cultures exposed to the oxidants. A same-site rbr+ revertant showed increased hydrogen peroxide and dioxygen resistance relative to the rbr- strain. Transcription of the P. gingivalis rubrerythrin gene is induced above its constitutive anaerobic level in response to dioxygen or hydrogen peroxide exposures. Purified rubrerythrins from other organisms have been shown to catalyse reduction of hydrogen peroxide, while showing relatively sluggish reaction with dioxygen and little or no catalase or superoxide dismutase activities. Porphyromonas gingivalis contains a superoxide dismutase but lacks catalase and haem peroxidases. We therefore suggest that rubrerythrin provides oxidative stress protection via catalytic reduction of intracellular hydrogen peroxide.

The organosulfur compound dimethylsulfoniopropionate (DMSP) has key roles in stress protection, global carbon and sulfur cycling, chemotaxis, and is a major source of climate-active gases. Saltmarshes are global hotspots for DMSP cycling due to Spartina cordgrasses that produce exceptionally high concentrations of DMSP. Here, in Spartina anglica, we identify the plant genes that underpin high-level DMSP synthesis: methionine S-methyltransferase (MMT), S-methylmethionine decarboxylase (SDC) and DMSP-amine oxidase (DOX). Homologs of these enzymes are common in plants, but differences in expression and catalytic efficiency explain why S. anglica accumulates such high DMSP concentrations and other plants only accumulate low concentrations. Furthermore, DMSP accumulation in S. anglica is consistent with DMSP having a role in oxidative and osmotic stress protection. Importantly, administration of DMSP by root uptake or over-expression of Spartina DMSP synthesis genes confers plant tolerance to salinity and drought offering a route for future bioengineering for sustainable crop production.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ΔgshA, ΔgshB, and double ΔgshAΔgshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ΔgshA, ΔgshB and double ΔgshAΔgshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa.

Extra-mitochondrial Cu/Zn superoxide dismutase (Sod1) is dispensable for protection against oxidative stress but mediates peroxide signaling in Saccharomyces cerevisiae

Oxidative stress results in protein oxidation and is implicated in cerebral disease, such as Parkinson's disease, Alzheimer's disease, and ischemic stroke. Sulfiredoxin-1 (Srxn1) is an endogenous antioxidant protein that has neuroprotective effects. The mechanisms of Srxn1 in oxidative stress have not been well studied, however. This study used 180 μM H2 O2 exposure for 24 hr to model oxidative stress. This experimental design allowed us to explore the protective effects and underlying mechanisms of Srxn1 in PC12 cells. To investigate Srxn1's role in oxidative stress protection, transient knockdowns of Srxn1 in PC12 cells were performed prior to treatment with 180 μM H2 O2 for 24 hr. Knockdown of Srxn1 resulted in decreased cell viability and increased cellular damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehyrogenase analysis, respectively. Intracellular superoxide dismutase and glutathione are important indexes of oxidative stress; these were reduced in Srxn1 knockdown PC12. We further found that the decreased Srxn1 correlated with a reduction in 2-Cys Prdxs activity. Moreover, 2-Cys Prdxs protein levels were increased in the H2 O2 -dosed cells, as measured by RT-PCR and immunoblot analysis. These results suggested that Srxn1 can protect PC12 cells from H2 O2 -induced oxidative stress and are involve in Prdxs activity. Srxn1 play a protective role against oxidative injury and demonstrates potential as a target for neuroprotective intervention in oxidative stress.

Seipin is a widely expressed protein but with highest levels found in the brain and testes. Seipin function is not yet completely understood, therefore the aim of this study was to evaluate the expression of BSCL2 transcripts in the central nervous system (CNS) of humans and investigate the effect of their overexpression on a neuron model and their relationship with oxidative stress protection, as well as shed light on the pathogenic mechanisms of Celia’s Encephalopathy. We analyzed the expression of BSCL2 transcripts using real-time RT–PCR in samples across the brain regions of subjects who underwent necropsy and from a case with Celia’s Encephalopathy. The transcript encoding the long seipin isoform (BSCL2-203, 462 aa) is expressed primarily in the brain and its expression is inversely correlated with age in the temporal lobe, amygdala, and hypothalamus. Strong positive correlations were found between BSCL2 expression and some genes encoding protective enzymes against oxidative stress including SOD1 and SOD2, as well as peroxisome proliferator-activated receptor gamma (PPARG) in the amygdala. These results were experimentally corroborated by overexpressing BSCL2 transcripts in SH-SY5Y cells with lentiviral transduction and assessing their effects on neuron differentiated cells. Confocal microscopy studies showed that both seipin and PEX16 are closely expressed in the hypothalami of healthy human brains, and PEX16 was absent in the same region of the PELD case. We hypothesize that seipin has specific CNS functions and may play a role in peroxisome biogenesis.

A Role for Rubredoxin in Oxidative Stress Protection in Desulfovibrio vulgaris: Catalytic Electron Transfer to Rubrerythrin and Two-Iron Superoxide Reductase

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

Study question Can metabolomic analysis identify and validate biomarkers associated with human seminal quality? Summary answer Targeted metabolomic analysis confirmed that DHA, cholesterol, carnitine, and betaine are enriched in high-quality semen samples, supporting their potential as biomarkers for assessing semen quality. What is known already Male infertility accounts for approximately 50% of infertility cases and is frequently associated with poor semen quality. Conventional semen analysis assess parameters such as sperm concentration, motility, and morphology, but lacks molecular insights into sperm function. Metabolomic profiling has revealed distinct biochemical differences between samples from men diagnosed with infertility and those from healthy individuals with good semen quality, suggesting that specific metabolites may serve as biomarkers of semen quality. However, few studies have validated these metabolites as reliable indicators of male reproductive potential. Study design, size, duration This metabolomic study was conducted in two phases. First, a cross-sectional analysis included 100 males: 55 donors from CEIFER-NextClinics Biobank (Granada, Spain) with high-quality semen and 45 patients from the Reproductive Unit at Hospital Virgen de las Nieves (Granada, Spain) with low-quality semen. In a second validation study, 50 donors were equally classified into high- and low-quality groups based on sperm concentration (>100 million/mL vs. <45 million/mL) and progressive motility (>40% vs. <40%). Participants/materials, setting, methods Semen samples were collected following WHO guidelines, with 3–5 days of abstinence. Samples were snap-frozen in liquid nitrogen and stored at − 80 °C. Untargeted metabolomics using UPLC-MS was conducted in 100 males. Metabolites showing significant differences and supported by the literature were validated using targeted metabolomics with a WATERS XEVO TQ-S QqQ-MS in a cohort of 50 sperm donors. Statistical analyses, adjusted for age and BMI, compared metabolite levels between high- and low-quality semen groups. Main results and the role of chance A total of 695 metabolites were identified through untargeted metabolomics. Targeted validation confirmed that DHA, cholesterol, carnitine, and betaine exhibited the strongest associations with semen quality (p < 0.05). DHA, an essential omega-3 polyunsaturated fatty acid, was significantly higher in high-quality semen, correlating with sperm concentration and motility. Its role in membrane fluidity and oxidative stress protection suggests a crucial function in sperm viability. Cholesterol, a key component of sperm membranes, was also elevated in high-quality samples, reinforcing its role in membrane stability and capacitation, essential for fertilization. Sperm capacitation has been linked to cholesterol efflux, emphasizing its importance in maturation. Carnitine, known for its role in mitochondrial energy metabolism, was significantly associated with sperm motility. As sperm rely on mitochondrial ATP production for movement, this correlation supports carnitine’s role in functionality and energy regulation. Betaine, a known osmoprotectant and methyl donor, exhibited higher concentrations in high-quality semen, suggesting its function in cellular homeostasis and oxidative stress protection. Its presence may help maintain sperm membrane integrity, contributing to overall viability. These findings highlight the biological relevance of these metabolites in sperm function and confirm their potential as molecular biomarkers for assessing male reproductive health. Limitations, reasons for caution While this study validates key biomarkers, larger multi-center cohorts are required to confirm diagnostic accuracy. Metabolite levels may be influenced by lifestyle factors (e.g., diet, smoking), requiring further adjustment. Longitudinal studies are also needed to assess their predictive value for fertility outcomes and clinical applicability. Wider implications of the findings These findings suggest that DHA, cholesterol, carnitine, and betaine could enhance clinical fertility diagnostics by providing metabolomic insights into male reproductive health. Integrating targeted metabolomic assays in andrology labs may complement conventional semen analysis, enabling a more functional assessment of semen quality and improving infertility diagnosis and treatment strategies. Trial registration number No

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.

NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in the glycolytic pathway. It has been widely demonstrated that mammalian GAPDH, in addition to its role in glycolysis, fulfills alternative functions mainly linked to its susceptibility to oxidative posttranslational modifications. Here, we investigated the responses of Arabidopsis (Arabidopsis thaliana) cytosolic GAPDH isoenzymes GAPC1 and GAPC2 to cadmium-induced stress in seedlings roots. GAPC1 was more responsive to cadmium than GAPC2 at the transcriptional level. In vivo, cadmium treatments induced different concomitant effects, including (1) nitric oxide accumulation, (2) cytosolic oxidation (e.g. oxidation of the redox-sensitive Green fluorescent protein2 probe), (3) activation of the GAPC1 promoter, (4) GAPC1 protein accumulation in enzymatically inactive form, and (5) strong relocalization of GAPC1 to the nucleus. All these effects were detected in the same zone of the root tip. In vitro, GAPC1 was inactivated by either nitric oxide donors or hydrogen peroxide, but no inhibition was directly provided by cadmium. Interestingly, nuclear relocalization of GAPC1 under cadmium-induced oxidative stress was stimulated, rather than inhibited, by mutating into serine the catalytic cysteine of GAPC1 (C155S), excluding an essential role of GAPC1 nitrosylation in the mechanism of nuclear relocalization, as found in mammalian cells. Although the function of GAPC1 in the nucleus is unknown, our results suggest that glycolytic GAPC1, through its high sensitivity to the cellular redox state, may play a role in oxidative stress signaling or protection in plants.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a recognized periodontopathogen. It exhibits a high degree of aerotolerance and is able to survive in host cells, indicating that efficient oxidative stress protection mechanisms must be present in this organism. Manganese homeostasis plays a major role in oxidative stress protection in a variety of organisms; however, the transport and role of this metal in P. gingivalis is not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of a ferrous iron transport protein, FeoB1 and FeoB2. FeoB2 has been implicated in manganese accumulation in P. gingivalis. We sought to determine the role of the FeoB2 protein in metal transport as well as its contribution to resistance to oxygen radicals. Quantitative reverse transcriptase PCR analyses demonstrated that expression of feoB2 is induced in the presence of oxygen. The role of FeoB2 was investigated using an isogenic mutant strain deficient in the putative transporter. We characterized the FeoB2-mediated metal transport using (55)Fe(2+) and (54)Mn(2+). The FeoB2-deficient mutant had dramatically reduced rates of manganese uptake (0.028 pmol/min/10(7) bacteria) compared with the parental strain (0.33 pmol/min/10(7) bacteria) (after 20 min of uptake using 50 nM of (54)Mn(2+)). The iron uptake rates, however, were higher in the mutant strain (0.75 pmol/min/10(7) bacteria) than in the wild type (0.39 pmol/min/10(7) bacteria). Interestingly, reduced survival rates were also noted for the mutant strain after exposure to H(2)O(2) and to atmospheric oxygen compared to the parental strain cultured under the same conditions. In addition, in vitro infection of host cells with the wild type, the FeoB2-deficient mutant, and the same-site revertant revealed that the mutant had a significantly decreased capability for intracellular survival in the host cells compared to the wild-type strain. Our results demonstrate that feoB2 encodes a major manganese transporter required for protection of the bacterium from oxidative stress generated by atmospheric oxygen and H(2)O(2). Furthermore, we show that FeoB2 and acquisition of manganese are required for intracellular survival of P. gingivalis in host cells.

Artificial insemination (AI) is a cornerstone technology for genetic improvement in livestock; however, the fertility outcomes of cryopreserved semen often remain inconsistent, particularly in tropical production systems where heat stress and oxidative damage compromise sperm function. Conventional semen extenders are largely developed through empirical approaches and may not adequately reflect the molecular characteristics of locally adapted cattle breeds. Proteomic profiling offers an opportunity to identify endogenous proteins involved in sperm function and resilience, thereby enabling the rational design of functionally targeted semen extenders. This study aimed to characterize the proteomic profiles of bull spermatozoa and seminal plasma and to identify functional proteins associated with semen quality traits to inform the development of biologically informed extender formulations for tropical cattle. Semen samples were collected from three sexually mature Bali bulls maintained under standardized management conditions. Three ejaculates were obtained from each bull, resulting in nine ejaculates for evaluation. Semen quality parameters, including motility, viability, abnormality, acrosome integrity, and membrane integrity, were assessed using conventional microscopic techniques and computer-assisted sperm analysis. For proteomic analysis, spermatozoa and seminal plasma fractions were separated by centrifugation and subjected to protein extraction, enzymatic digestion, and high-resolution liquid chromatography-tandem mass spectrometry. Identified proteins were analyzed using bioinformatics tools for functional annotation, Gene Ontology classification, and protein-protein interaction analysis to determine their biological roles and potential relevance to semen preservation. Fresh semen exhibited high motility (86.28% ± 3.26%), membrane integrity (86.35% ± 2.88%), and acrosome integrity (79.65% ± 6.93%), indicating overall favorable semen quality. Proteomic analysis identified 371 proteins, including 101 unique to spermatozoa and 270 shared between spermatozoa and seminal plasma. Functional annotation revealed that sperm proteins were predominantly associated with energy metabolism, cytoskeletal organization, and spermatogenesis, whereas seminal plasma proteins were enriched in antioxidant activity, immune response, and proteolytic processes. Key proteins involved in mitochondrial function, antioxidant defense, acrosomal activity, and structural integrity were identified and associated with semen quality parameters, including motility, viability, and membrane stability. Interaction network analysis further demonstrated coordinated relationships among mitochondrial enzymes, structural proteins, and fertilization-related molecules. The integration of semen quality assessment with proteomic profiling provides molecular insights into the biochemical environment supporting sperm function in Bali bulls. The identified proteins highlight critical pathways associated with energy production, oxidative stress protection, structural stability, and fertilization competence. These findings provide a conceptual framework for translating proteomic information into targeted additives for semen extenders, thereby supporting the development of biology-informed cryopreservation strategies tailored to tropical cattle production systems.

The chloroplast is a major sink for copper (Cu), which plays a crucial role in photosynthesis and oxidative stress protection in this organelle. The molecular mechanisms controlling Cu homeostasis in the chloroplast in rice remain unclear. Here, we identify OsHMA7, a P1B-type ATPase, as a chloroplast envelope-localized Cu transporter necessary for transporting Cu into the chloroplast. Temporal expression analysis revealed that OsHMA7 is expressed in both roots and leaves, with diurnal rhythmicity in the latter. Subcellular fractionation and confocal microscopy confirmed its exclusive localization to the leaf chloroplast envelope membrane and likely in plastids of roots. Heterologous expression in yeast demonstrated the Cu-transport activity of OsHMA7. Knockout of OsHMA7 by CRISPR/Cas9 significantly decreased the concentration of Cu in chloroplasts, resulting in large decreases in the abundance of the Cu-containing proteins plastocyanin and Cu/Zn-superoxide dismutase 2. Consequently, photosynthetic electron transfer efficiency and photosynthesis rate were reduced and the production of reactive oxygen species was increased. oshma7 mutants displayed severe growth impairments, with a 76-78% decrease in the tiller number and an 81-96% reduction in grain yield. Our findings establish OsHMA7 as an important Cu transporter in chloroplasts in rice, directly linking Cu homeostasis to photosynthetic efficiency and crop productivity.