Antioxidants

Plant growth and development are highly regulated processes and are majorly controlled by various environmental factors, whose extreme exposures lead to chronic stress conditions promoting reactive oxygen species (ROS) and carbonyl species (RCS) production. ROS and RCS extensively damage cellular biomolecules and organelles, affecting plants viability and development. Emerging reports highlight that the multi-stress responding DJ-1 superfamily proteins are critical in attenuating cytotoxic effects associated with abiotic stress. The current report, validated in yeast and plant models, shows that AtDJ-1C and AtDJ-1E are robust antioxidants that scavenge ROS and improve survival under oxidative stress. Although they lack conventional glyoxalases and do not attenuate the glycation of proteins, AtDJ-1C and AtDJ-1E preserve the GSH pool and regulate redox homeostasis. Moreover, transcriptome profiling indicates that levels of AtDJ-1C and AtDJ-1E are rapidly established to counter heat and oxidative stress conditions. Notably, the knockdown of AtDJ-1C and AtDJ-1E promotes detrimental alterations such as reduced chlorophyll retention, impaired root morphogenesis, and induced sensitivity to heat stress due to ROS elevation. Contrastingly, overexpression of AtDJ-1C and AtDJ-1E improved plant height and rosette formation under physiological conditions. In conclusion, our study unravels the pivotal functions of Arabidopsis thaliana DJ-1C and DJ-1E in governing plant health and survival under heat and oxidative stress conditions.

KatG and Msrs are important enzymes associated with ROS homeostasis and bacterial survival under oxidative stress. Consistent to this notion, mutant strains in these enzymes showed hypersensitivity to oxidants and accumulates elevated levels of ROS. In current study we observed that a pan msr deletion ({Delta}5msr mutant) strain of S. Typhimurium accumulates significantly higher levels of ROS. However, unexpectedly, as compared to S. Typhimurium, the {Delta}5msr mutant strain exhibits more than 2000 folds resistance to H2O2. Transcriptional and mass spectrometry analyses reveal the upregulation of KatG in {Delta}5msr mutant strain. Further, {Delta}5msr mutant strain exhibits [~]6 folds higher KatG activity. Supplementation of {Delta}5msr mutant culture with reduced glutathione resulted in ROS neutralization, decreased KatG activity and abrogation of H2O2 resistance. However, {Delta}5msr mutant strain showed negligible KatE and KatN activities. The findings of current study suggest that the Salmonella have evolved the mechanism to upregulate one antioxidant gene in absence of others to mitigate oxidative stress.

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain, and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 uM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection. Cellular protection was protective via the antioxidant proteins catalase and GGCS.

Attempts to improve the ascorbate (AsA) content of plants are still dealing with the limited understanding of why exists a wide variability of this powerful anti-oxidant molecule in different plant sources, species and environmental situations. In plant mitochondria, the last step of AsA synthesis is catalyzed by the enzyme L-galactone-1,4-lactone dehydrogenase (L-GalLDH). By using GalLDH-RNAi silencing plant lines, biochemical and proteomic approaches, we here discovered that, in addition to accumulate this antioxidant, mitochondria synthesize AsA to down-regulate the respiratory activity and the cellular energy provision. The work reveals that the AsA synthesis pathway within mitochondria is a branched electron transfer process that channels electrons towards the alternative oxidase, interfering with conventional electron transport. It was unexpectedly found that significant hydrogen peroxide is generated during AsA synthesis, which affects the AsA level. The induced AsA synthesis shows proteomic alterations of mitochondrial and extra-mitochondrial proteins related to oxidative and energetic metabolism. The most identified proteins were known components of plant responses to high light acclimation, programmed cell death, oxidative stress, senescence, cell expansion, iron and phosphorus starvation, different abiotic stress/pathogen attack responses and others. We propose that changing the electron flux associated with AsA synthesis might be part of a new mechanism by which the L-GalLDH enzyme would adapt plant mitochondria to fluctuating energy demands and redox status occurring under different physiological contexts.

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear superoxide dismutase with osmoprotective and antioxidative functions. However, its role in oxidative stress tolerance is not well understood. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. The findings demonstrated that the antioxidative function of FSD1 depends on the availability of Cu2+ in growth media. Prolonged MV exposure led to a decreased accumulation rate of superoxide, higher levels of hydrogen peroxide production, and higher protein carbonylation in the fsd1 mutants and transgenic plants lacking a plastidial pool of FSD1, compared to the wild type. MV led to a rapid increase in FSD1 activity, followed by a decrease. Chloroplastic localization of FSD1 is necessary for these changes. Proteomic analysis showed that the sensitivity of the fsd1 mutants coincided with decreased abundance of ferredoxin and light PSII harvesting complex proteins, with altered levels of signaling proteins. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

Geographic atrophy or late stage dry age-related macular degeneration (AMD) is characterized by drusen deposition and progressive retinal pigment epithelium (RPE) degeneration, leading to irreversible vision loss. The formation of drusen leads to dyshomeostasis, oxidative stress and irreversible damage to RPE. In this study, we used an in vitro model of oxidized-low density lipoproteins (ox-LDL) induced human RPE damage/death model to investigate the mechanism whereby a sterically hindered phenol antioxidant compound, PMC (2,2,5,7,8-pentamethyl-6-chromanol) protects RPE against ox-LDL-induced damage. We show that PMC exerts its protective effect by preventing the upregulation of stress-responsive heme oxygenase-1 (HMOX1/HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) at mRNA and protein levels. This effect was due to PMCs blockade of ROS generation, which in turn blocked nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, ultimately preventing the upregulation of antioxidant response elements (ARE), including HMOX1 and NQO1. A key role for HO-1 was demonstrated when the protective effect of PMC was inhibited by the knockdown of HMOX1. Additionally, treatment of PMC under different experimental conditions and time points revealed that the continuous presence of PMC is required for optimal protection against ox-LDL-induced cytotoxicity, defining the cellular pharmacokinetics of the molecule. Our data demonstrate the involvement of a key antioxidant pathway through which PMC mitigates oxidative stress induced by ox-LDL and provides a potential therapeutic strategy to suppress RPE degeneration/damage during AMD progression.

Cellular energy metabolism varies depending on tissue and cell type, as well as the availability of energy substrates and energy demands. We recently investigated the variations in cellular metabolism and antioxidant responses in primary bovine vascular endothelial cells (BAECs) under different energetic substrate conditions in vitro, specifically glucose or galactose. In this context, pharmacological agents may affect cells differently depending on their energy metabolism status. In this study, we aimed to characterize the effects of diphenyl diselenide ((PhSe)2), a redox-active molecule known for its prominent cardiovascular effects, on redox-bioenergetic cellular pathways under glycolytic or oxidative conditions in BAECs. Under glucose conditions, (PhSe)2 positively impacted mitochondrial oxidative capacity, as assessed by respirometry, and was associated with changes in mitochondrial cellular dynamics. However, these changes were not observed in cells cultured with galactose. Although (PhSe)2 induced the nuclear translocation of the redox sensitive nuclear factor erythroid 2-related factor 2 (Nrf2) in both glucose and galactose media, Nrf2 remained in the nuclei of cells cultured in galactose for a longer duration. Additionally, activation of another redox sensitive transcription factor, forkhead O3 (FOXO3a) was only detected in galactose media. Notably, (PhSe)2 induced the expression of genes controlling mitochondrial antioxidant capacity and glutathione synthesis and recycling in glucose media, whereas its effects in galactose media were primarily focused on glutathione homeostasis. In conclusion, our findings underscore the critical influence of cellular metabolic status on the antioxidant capacity of redox-active molecules such as (PhSe)2.

Hydrogen cyanamide (HC) is known to stimulate the production of reactive oxygen species (ROS) and also alter growth through modification of the cell cycle. However, the mechanisms by which HC alters cell proliferation and redox homeostasis are largely unknown. This study used roGFP2 expressing Arabidopsis seedlings to measure the oxidation states of the nuclei and cytosol in response to HC treatment. The Cytrap dual cell cycle phase marker system and flow cytometry were used to study associated changes in cell proliferation. HC (1.5mM) reversibly inhibited root growth during a 24h treatment. Higher concentrations were not reversible. HC did not synchronize the cell cycle. In contrast to hydroxyurea, HC caused a gradual accumulation of cells in the G2/M phase and decline of G1/S phase cells 16 to 24h post-treatment. This was accompanied by increased oxidation of both the nuclei and cytosol. Taken together, HC impairs proliferation of embryonic root meristem cells in a reversible manner through restriction of G2/M transition accompanied by increased oxidative poise.

Hydrogen sulfide (H2S) is a gaseous molecule historically regarded as toxic. Nevertheless, increasing evidence has brought to light important physiological roles in both animals and plants. In plants, H2S is involved in environmental and developmental responses, such as stomatal closure and seed germination, and in tolerance mechanisms to different stress conditions like salinity, drought and waterlogging. In this study, we report a function of H2S as a modulator of hypoxic responses in Arabidopsis thaliana. A combination of biochemical and genetic evidence demonstrates that H2S inhibits the activity of Plant Cysteine Oxidases, the molecular sensors of oxygen, through protein persulfidation to modulate hypoxia-associated responses. Furthermore, we show that H2S physiology contributes to responses to low oxygen, as disturbing H2S production impaired activation of hypoxia-responsive genes and submergence tolerance. Overall, this work introduces H2S as signalling modulator in plant hypoxic responses and adds a regulatory layer to the plant oxygen-sensing mechanism.

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.

Autophagy is one of the main degradative pathways used by eukaryotic organisms to eliminate useless or damaged intracellular material in order to maintain cellular homeostasis under stress conditions. Mounting evidence indicates a strong interplay between the generation of ROS and the activation of autophagy. Although a tight redox regulation of autophagy has been shown in several organisms including microalgae, the molecular mechanisms underlying this control remain poorly understood. In this study, we have performed an in-depth in vitro and in vivo redox characterization of ATG3, an E2-activating enzyme involved in ATG8 lipidation and autophagosome formation, from two evolutionary distant unicellular model organisms: the green microalga Chlamydomonas reinhardtii and the budding yeast Saccharomyces cerevisiae. Our results indicated that ATG3 activity from both organisms is subjected to redox regulation since these proteins require reducing equivalents to transfer ATG8 to the phospholipid phosphatidylethanolamine. We established the catalytic Cys of ATG3 as redox target in algal and yeast proteins, and showed that the oxidoreductase thioredoxin efficiently reduces ATG3. Moreover, in vivo studies revealed that the redox state of ATG3 from Chlamydomonas reinhardtii undergoes profound changes in the absence of photoprotective carotenoids, a stress condition that activates autophagy in algae.

Among the three active aldehyde oxidases in Arabidopsis thaliana leaves (AAO1-3), AAO3, which catalyzes the oxidation of abscisic-aldehyde to abscisic-acid, was shown recently to function as a reactive aldehyde detoxifier. Notably, aao2KO mutants exhibited less senescence symptoms and lower aldehyde accumulation, such as acrolein, benzaldehyde, and HNE than in wild-type leaves exposed to UV-C or Rose-Bengal. The effect of the absence of AAO2 expression on aldehyde detoxification by AAO3 and/or AAO1 was studied by comparing the response of wild-type plants to the response of aao1Single mutant, aao2KO mutants and single mutants of aao3Ss. Notably, aao3Ss exhibited similar aldehyde accumulation and chlorophyll content to aao2KO treated with UV-C or Rose-Bengal. In contrast, wild-type and aao1S exhibited higher aldehyde accumulation that resulted in lower remaining chlorophyll than in aao2KO leaves, indicating that the absence of active AAO2 enhanced AAO3 detoxification activity in aao2KO mutants. In support of this notion, employing abscisic-aldehyde as a specific substrate marker for AAO3 activity revealed enhanced AAO3 activity in aao2KO and aao3Ss leaves compared to wild-type treated with UV-C or Rose Bengal. The similar abscisic acid level accumulated in leaves of unstressed or stressed genotypes indicates that aldehyde detoxification by AAO3 is the cause for better stress resistance in aao2KO mutants. Employing the sulfuration process (known to activate aldehyde oxidases) in wild-type, aao2KO, and molybdenum-cofactor sulfurase (aba3-1) mutant plants revealed that the active AAO2 in WT employs sulfuration processes essential for AAO3 activity level, resulting in the lower AAO3 activity in WT than AAO3 activity in aao2KO.

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis genome codes for 53 GSTs, divided into seven subclasses, however understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5 and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-class GSTUs. Specifically, TGA2 controls the expression of several GSTUs under UV-B light and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutively expressed GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared to WT plants. GSTU7 was found to act as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus protecting cells from oxidative stress.

BackgroundDoxorubicin (DOX) remains a cornerstone in the treatment of various cancers. However, its clinical utilization is significantly hampered by dose-dependent cardiotoxicity. The generation of reactive oxygen species (ROS) constitutes the central component of the pathogenesis of DOX-induced cardiotoxicity. Activating transcription factor 4 (ATF4) has been demonstrated to exert a cardioprotective effect and augment cardiac antioxidative capacity in settings of heart failure. However, the role of ATF4 in DOX-induced cardiomyopathy (DIC) remains unknown. MethodsTo explore the role of ATF4 in DOX-induced cardiomyopathy, cardiac-specific ATF4 conditional heterozygous mice and AAV9 mediated ATF4 overexpression mouse models were utilized. Cardiac function was assessed by echocardiography. The upstream regulator and downstream mediator of ATF4 were evaluated using RNA-seq analysis and further verified using ChIP assay and luciferase reporter assay. ResultsWe found a substantial decrease in ATF4 expression levels in the heart of DIC mice. ATF4+/- mice exhibited a higher degree of susceptibility to DOX-induced cardiotoxicity in comparison with ATF4flox/flox mice, as evidenced by the manifestation of more severe cardiac dysfunction and a significantly earlier mortality rate. In contrast, cardiacc-specific overexpression ATF4 by AAV9 confers robust cardioprotection against DOX-induced cardiomyopathy. Mechanistically, we identified the upstream regulator of ATF4 as KLF16, which was significantly suppressed during DOX treatment. Further, the decrease of ATF4 led to a reduction in cystathionine {gamma}-lyase (CSE) transcription and hydrogen sulfide (H2S) production in the context of DOX-induced cardiotoxicity. ChIP and luciferase reporter assays revealed that ATF4 functioned as the transcription factor of the CSE gene, which is a key enzyme in the synthesis of H2S to counteract oxidative stress. Consistently, ROS scavengers or H2S donors was shown to mitigate the consequences of ATF4 deficiency. In contrast, the ectopic expression of ATF4 mitigated oxidative stress and apoptosis in DOX-induced cardiotoxicity, both in vivo and in vitro. ConclusionsOur study revealed a novel function of ATF4 in counteracting oxidative stress in DOX cardiotoxicity by promoting the transcription of CSE. ATF4 may represent a promising therapeutic target for the treatment of DOX-induced cardiomyopathy.

HIGHLIGHTSO_LID-PUFA diet prevents muscle atrophy in STZ-induced diabetic mice. C_LIO_LID-PUFA diet prevents muscle weakness depending on increased calcium release in STZ-induced diabetic mice. C_LIO_LID-PUFA diet may show a trend to decrease blood glucose in STZ-induced diabetic mice. C_LIO_LID-PUFA diet does not alter ferroptosis-related protein profiles including ACSL4, LPCAT3, ALOX12, and Gpx4. C_LI Oxidative stress and reactive oxygen species (ROS) have been linked to muscle atrophy and weakness. Diabetes increases the oxidative status of lipoproteins in nearly all tissues, including muscle tissues, but the role of lipid ROS on diabetes-induced muscle atrophy is not fully understood. Deuterium reinforced polyunsaturated fatty acids (D-PUFA) are more resistant to ROS-initiated chain reaction of lipid peroxidation than regular hydrogenated PUFA (H-PUFA). In this study, we tested the hypothesis that D-PUFA would protect muscle atrophy induced by diabetes driven by an accumulation of lipid hydroperoxides (LOOH). C57BL/6J mice were dosed with H-PUFA or D-PUFA for four weeks through dietary supplementation and then injected with streptozotocin (STZ) to induce insulin-deficient diabetes. After two weeks, muscles tissues were analyzed for individual muscle mass, force generating capacity and cross-sectional area. Skeletal muscle fibers from diabetic mice exhibited increased total ROS and LOOH. This was abolished by the D-PUFA supplementation regardless of accumulated iron. D-PUFA were found to be protective against muscle atrophy and weakness from STZ-induced diabetes. Prevention of muscle atrophy and weakness by D-PUFA might be independent of ACSL4/LPCAT3/15-LOX pathway. These findings provide novel insights into the role of LOOH in the mechanistic link between oxidative stress and diabetic myopathy and suggest a novel therapeutic approach to diabetes-associated muscle weakness.

The glyoxalase system is a ubiquitously expressed enzyme system with narrow substrate specificity and is responsible for the detoxification of harmful methylglyoxal (MG), a spontaneous by-product of energy metabolism. Glyoxalase 1 (Glo1) is the first and therefore rate limiting enzyme of this protective system. In this study we were able to show that a phosphorylation of threonine-107 in the Glo1 protein, mediated by Ca2+/Calmodulin-dependent Kinase II delta (CamKII{delta}), is associated with elevated catalytic efficiency of Glo1. In fact, Michaelis-Menten kinetics of Glo1 mutants revealed that a permanent phosphorylation of Glo1 was associated with increased Vmax (1.23 {micro}mol/min/mg) and decreased Km (0.19 mM HTA), whereas the non-phosphorylatable Glo1 showed significantly lower Vmax (0.66 {micro}mol/min/mg) and increased Km (0.31 mM HTA). This was also confirmed with human recombinant Glo1 (Vmax (Glo1phos) = 999 {micro}mol/min/mg; Km (Glo1phos) = 0.09 mM HTA vs. Vmax (Glo1red) = 497 {micro}mol/min/mg; Km (Glo1red) = 0.12 mM HTA). Additionally, proteasomal degradation of non-phosphorylated Glo1 via ubiquitination occurred more rapidly as compared to native Glo1. The absence of the responsible kinase CamKII{delta} was associated with poor MG detoxification capacity and decreased protein content of Glo1 in a murine CamKII{delta} knock-out model. Furthermore, this regulatory mechanism is also related to an altered Glo1 status in cancer, diabetes and during aging. In summary, phosphorylation of threonine-107 in the Glo1 protein by CamKII{delta} is a quick and precise mechanism regulating Glo1 activity.

Maternal obesity is a risk factor for increased fetal adiposity. The underlying mechanisms remain unclear, however, emerging evidence suggests that mesenchymal stem cells (MSCs), which are the precursors of adipocytes, from neonates of mothers with obesity exhibit enhanced adipogenic differentiation potential. We hypothesise that the MSCs of neonates from mothers with obesity have different stemness potential and redox state compared to the MSCs from mothers with normal weight. MSCs were isolated from neonates of women with obesity (BMI>30 kg/m{superscript 2}, OB-MSCs) and women with normal weight (BMI <25 kg/m{superscript 2}, NW-MSCs). OB-MSCs showed reduced stemness potential, as seen by a lower OCT3/4 expression and lower clonogenic capacity, than NW-MSCs (p<0.05). In addition, OB-MSCs showed higher levels of mitochondrial superoxide (O2*-), together with lower antioxidant SOD2 gene expression, compared to NW-MSCs (p<0.05). Conversely, OB-MSCs had higher levels of glutathione (GSH) compared to NW-MSCs (p<0.05). Upon exposure to H2O2 (250 M), OB-MSCs displayed attenuated antioxidant response, with lower SOD1, SOD2 and GPX1 gene expression as compared to NW-MSCs (p<0.05). Upon exposure to higher oxidative stress (H2O2, 400 M), total ROS levels were lower in OB-MSCs than in NW-MSCs. In contrast, when challenged for mitochondrial ROS, OB-MSCs showed higher levels of mitochondrial superoxide production as compared to NW-MSCs (p<0.05). Our results indicate that OB-MSCs have lower stemness potential, elevated mitochondrial O2*- and a different basal and oxidative stress-induced redox profile compared to NW-MSCs. These changes in OB-MSCs could predispose them to an increase adipogeneic commitment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/648714v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1638346org.highwire.dtl.DTLVardef@3f9caaorg.highwire.dtl.DTLVardef@46925eorg.highwire.dtl.DTLVardef@1336dfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chronic obstructive pulmonary disease (COPD), whose main risk factor is cigarette smoking (CS), is one of the most common diseases globally. Many COPD patients also develop pulmonary hypertension (PH), a severe complication that leads to premature death. Evidence suggests reactive oxygen species (ROS) involvement in COPD and PH, especially regarding pulmonary artery smooth muscle cells (PASMC) dysfunction. However, the effects of CS on the pulmonary vasculature are not completely understood. Herein we provide evidence on the effects of CS extract (CSE) exposure on PASMC regarding ROS production, antioxidant response and its consequences on vascular tone dysregulation. Our results indicate that CSE exposure promotes mitochondrial fission, mitochondrial membrane depolarization and increased mitochondrial superoxide levels. However, the increase in superoxide did not parallel a counterbalancing antioxidant response in human pulmonary artery (PA) cells. Interestingly, the mitochondrial superoxide chelator mitoTEMPO reduced mitochondrial fission and membrane potential depolarization caused by CSE. As we have previously shown, CSE reduces PA vasoconstriction and vasodilation. In this respect, mitoTEMPO prevented the impaired nitric oxide-mediated vasodilation, while vasoconstriction remained reduced. Finally, we observed a CSE-driven downregulation of the Cyb5R3 enzyme, which prevents soluble guanylyl cyclase oxidation in PASMC. This might explain the CSE-mediated decrease in PA vasodilation. These results provide evidence that there might be a connection between mitochondrial ROS and altered vasodilation responses in PH secondary to COPD, and strongly support the potential of antioxidant strategies specifically targeting mitochondria as a new therapy for these diseases. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Plant thioredoxins (TRXs) form a complex family involved in numerous metabolic and signalling pathways, such as the regulation of photosynthetic metabolism in relation with light conditions. The atypical CDSP32, chloroplastic drought-induced stress protein of 32 kDa, TRX includes two TRX-fold domains, one of which has an atypical redox-active HCGPC motif, and has been initially reported to participate in responses to oxidative stress as an electron donor to peroxiredoxins and methionine sulfoxide reductases. Here, we further characterized potato lines modified for CDSP32 expression to clarify the physiological roles of the TRX. Upon high salt treatments, modified lines displayed changes in the abundance and redox status of CDSP32 antioxidant partners, and exhibited sensitivity to NaHCO3, but not to NaCl. In non-stressed plants overexpressing CDSP32, a lower abundance of photosystem II PsbO and D1 subunits and ATP-synthase {gamma} subunit was noticed. The CDSP32 co-suppressed line showed altered chlorophyll a fluorescence induction and modified regulation of the plastidial ATP-synthase activity during dark/light and light/dark transitions, revealing the involvement of CDSP32 in the control of the photosynthetic machinery. In agreement with the previously reported interaction in planta between CDSP32 and the ATP-synthase {gamma} subunit, our data show that CDSP32 participates in the regulation of the transthylakoid membrane potential. Consistently, modeling of protein complex 3-D structure indicates that the CDSP32 TRX constitutes a suitable partner of ATP-synthase {gamma} subunit. We discuss the roles of CDSP32 in chloroplast redox homeostasis through the regulation of both photosynthetic activity and enzymatic antioxidant network.