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Guanine (Gua), among purines, is a preferred oxidation/nitration target because of its low one-electron redox potential. The reactive oxygen/nitrogen species peroxynitrite (ONOO

This study explored the metabolic adaptions to grazing conditions of two Holstein genetic strains (GS; North American, NAH; New Zealand, NZH) in two feeding strategies (FS; restricted, P30, vs. maximised, PMAX, grazing). Four groups (NAH-P30, NZH-P30, NAH-PMAX and NZH-PMAX; n = 10 cows each) were compared between -45 and 180 days in milk (DIM). NZH cows had lower (p = 0.02) fat and protein corrected milk (FPCM) yield and a tendency for lower (p = 0.09) body condition score concomitantly with a trend (p 0.07) for higher average plasma insulin and lower (p = 0.01) 3-methylhistidine (3MH) at -45 DIM than NAH. Plasma glucose tended to be affected by the triple interaction GS × FS × DIM (p = 0.06) as it was similar between NAH-P30 and NZH-P30, but higher (p ≤ 0.02) for NZH-PMAX than NAH-PMAX except at 21 DIM. The physiological imbalance index was affected by the GS × FS interaction (p 0.01) as it was lower (p 0.01) only for NZH-PMAX versus NAH-PMAX. NZH cows had higher (p = 0.01) plasma thiobarbituric acid reactive substances at -45 DIM and tended to have higher protein carbonyls (p = 0.10) and superoxide dismutase (SOD) activity (p = 0.06) on average, and had higher (p 0.01) α-tocopherol during mid-lactation than NAH Regarding the FS, FPCM was similar (p = 0.12) among them, but PMAX cows had higher (p 0.01) plasma non-esterified fatty acids and 3MH, and lower insulin (p 0.01) than P30 at 100 DIM. PMAX cows showed higher average SOD activity (p = 0.01) and plasma α-tocopherol at 100 and 180 DIM (p 0.01). Under grazing, NZH cows can have a better energy status and lower muscle mobilisation but a higher redox reactivity. Maximising grazing can worsen energy status and muscle mobilisation while improving antioxidant response with no effect on FPCM.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.

Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides. Fil: Benchoam, Dayana. Universidad de la República; Uruguay Fil: Semelak, Jonathan Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Cuevasanta, Ernesto. Universidad de la República; Uruguay Fil: Mastrogiovanni, Mauricio. Universidad de la Republica; Uruguay Fil: Grassano, Juan S.. Universidad de Buenos Aires; Argentina Fil: Ferrer-Sueta, Gerardo. Universidad de la Republica; Uruguay Fil: Zeida Camacho, Ari Fernando. Universidad de la Republica; Uruguay Fil: Trujillo, Madia. Universidad de la Republica; Uruguay Fil: Möller, Matías N.. Universidad de la Republica; Uruguay Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Alvarez, Beatriz. Universidad de la Republica; Uruguay

Under physiological and pathophysiological conditions, lipid nitration occurs generating nitro-fatty acids (NFA) with pleiotropic activities as modulation of inflammatory cell responses. Foam cell formation and atherosclerotic lesion development have been extensively related to low-density lipoprotein (LDL) oxidation. Considering our manuscript "Fatty acid nitration in human low-density lipoprotein" (https://doi.org/10.1016/j.abb.2019.108190), herein we report the oxidation versus nitration of human LDL protein and lipid fractions. Data is shown on LDL fatty acid nitration, in particular, formation and quantitation of nitro-conjugated linoleic acid (NO2-cLA) under mild nitration conditions. In parallel to NO2-cLA formation, depletion of endogenous antioxidants, protein tyrosine nitration, and carbonyl formation is observed. Overall, our data propose the formation of a potential anti-atherogenic form of LDL carrying NFA.

The reactions of proteins with biologically relevant oxidants have been widely studied, although most of the work has been performed in diluted homogenous solutions conditions that differ from those in intracellular environments. Cellular compartments represent highly crowded milieu in which high concentrations of biomolecules are present, unspecific intermolecular interactions are promoted, and physicochemical properties of constituents are modified. In this work, we propose that the high concentration at which proteins are present inside cells favours radical oxidative reactions between polypeptides which propagate in an oxygen-dependent process similar to membrane lipid peroxidation. The results presented herein show that highly concentrated solutions of bovine serum albumin (BSA) exposed to peroxynitrite, or metmyoglobin/H2O2, initiate the formation and propagation of protein peroxyl radicals, as evidenced by oxygen consumption, fluorescence spectroscopy, chemiluminescence, and electron paramagnetic resonance studies. Moreover, peroxyl radicals are capable of converting nitrite to nitrogen dioxide, which can oxidise amino acid residues to further assist radical-mediated protein oxidation. In addition, we also show that nitrone spin traps stop these propagation reactions in proteins, in line with the previously reported antioxidant role of these compounds in vivo. In summary, our results suggest that in crowded environments such as cellular compartments radical chain reactions propagate protein oxidative damage, highlighting a previously under recognised mechanism of cellular nitroxidative stress.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive liver fat deposition in the absence of significant alcohol intake. Since extra virgin olive oil (EVOO) reduces fat accumulation, we analyzed the involvement of nitro-fatty acids (NO(2)-FA) on the beneficial effects of EVOO consumption on NAFLD. Nitro-fatty acids formation was observed during digestion in mice supplemented with EVOO and nitrite. Mice fed with a high-fat diet (HF) presented lower plasma NO(2)-FA levels than normal chow, and circulating concentrations recovered when the HF diet was supplemented with 10% EVOO plus nitrite. Under NO(2)-FA formation conditions, liver hemoxygenase-1 expression significantly increased while decreased body weight and fat liver accumulation. Mitochondrial dysfunction plays a central role in the pathogenesis of NAFLD while NO(2)-FA has been shown to protect from mitochondrial oxidative damage. Accordingly, an improvement of respiratory indexes was observed when mice were supplemented with both EVOO plus nitrite. Liver mitochondrial complexes II and V activities were greater in mice with EVOO supplementation and further improved in the presence of nitrite. Overall, our results strongly suggest a positive correlation between NO(2)-OA formation from EVOO and the observed improvement of mitochondrial function in NAFLD. The formation of NO(2)-FA can account for the health benefits associated with EVOO consumption.

Background and aim Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. Results In the presence of Tempol (5–120 μM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC50 of 22.9 ± 10.8 μM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a′active site. More specifically, Cys400 was modified by Tempol. Conclusion We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.

Background Nitroarachidonic acid (NO 2 AA) exhibits pleiotropic anti-inflammatory actions in a variety of cell types. We have recently shown that NO 2 AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing the formation of the active complex. Recent work indicates the participation of protein disulfide isomerase (PDI) activity in NOX2 activation. Cysteine (Cys) residues at PDI active sites could be targets for NO 2 AA- nitroalkylation regulating PDI activity which could explain our previous observation. Methods PDI reductase and chaperone activities were assessed using the insulin and GFP renaturation methods in the presence or absence of NO 2 AA. To determine the covalent reaction with PDI as well as the site of reaction, the PEG-switch assay and LC–MS/MS studies were performed. Results and conclusions We determined that both activities of PDI were inhibited by NO 2 AA in a dose- and time- dependent manner and independent from release of nitric oxide. Since nitroalkenes are potent electrophiles and PDI has critical Cys residues for its activity, then formation of a covalent adduct between NO 2 AA and PDI is feasible. To this end we demonstrated the reversible covalent modification of PDI by NO 2 AA. Trypsinization of modified PDI confirmed that the Cys residues present in the active site a ′ of PDI were key targets accounting for nitroalkene modification. General significance PDI may contribute to NOX2 activation. As such, inhibition of PDI by NO 2 AA might be involved in preventing NOX2 activation. Future work will be directed to determine if the covalent modifications observed play a role in the reported NO 2 AA inhibition of NOX2 activity.

Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400 on CGHC redox motif of PDI a’ domain, a site for PDI prothrombotic activity. SummaryBackground Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI‘s actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

Hydrogen sulfide (H(2)S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H(2)S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H(2)S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE–SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE–SOH), which we chose here as a model to study the interactions between H(2)S and peroxiredoxins (Prx). MtAhpE–SOH reacted with H(2)S, forming a persulfide (MtAhpE–SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 10(3) m(−1) s(−1) (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H(2)S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE–SSH reacted 43 times faster than did MtAhpE–SH with the unspecific electrophile 4,4′-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE–SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE–SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pK(a) of the leaving thiol MtAhpE–SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.

Virgin olive oil (VOO) represents the main source of unsaturated lipids in the Mediterranean diet associated with low mortality. Health benefits of VOO rely on its composition, mainly fatty acids and minor components such as polyphenols. In addition, VOO contains nitro-fatty acids (NO2-FA), novel signaling mediators exhibiting pleiotropic anti-inflammatory responses. Previous work from our group reported the presence of nitro-oleic acid (NO2-OA), nitro-linoleic acid (NO2-LA) and nitro-conjugated linoleic acid (NO2-cLA) in extra virgin olive oil under gastric conditions. Herein, we analyzed the fatty acid profile, phenol, pigment and NO2-FA formation in two contrasting Uruguayan olive cultivars, Arbequina and Coratina at two ripening conditions. We demonstrate that VOO fatty acid nitration is dependent on olive cultivar as well as fruit ripening. Under gastric nitration conditions, the presence of polyphenols in Arbequina VOO promoted fatty acid nitration. In contrast, the absence of polyphenols favor lipid oxidation, decreasing fatty acid nitration. In Coratina, where the content of polyphenolic compounds is higher than in Arbequina, their absence did not affect the formation of NO2-FA. Coratina contains other bioactive constituents such as pigments that could play an important role in protection of VOO from lipid oxidation. Overall, we postulate that unsaponifiable constituents of VOO, e.g. polyphenols and pigments, contribute to the formation of NO2-FA in gastric conditions, thus potentiating their health beneficial

RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA.

Lipid nitration occurs during physiological and pathophysiological conditions, generating a variety of biomolecules capable to modulate inflammatory cell responses. Low-density lipoprotein (LDL) oxidation has been extensively related to atherosclerotic lesion development while oxidative modifications confer the particle pro-atherogenic features. Herein, we reviewed the oxidation versus nitration of human LDL protein and lipid fractions. We propose that unsaturated fatty acids present in LDL can be nitrated under mild nitration conditions, suggesting an anti-atherogenic role for LDL carrying nitro-fatty acids (NFA).

Early lactation is an energy-demanding period for dairy cows which may lead to negative energy balance, threatening animal health and consequently productivity. Herein we studied hepatic mitochondrial function in Holstein-Friesian multiparous dairy cows during lactation, under two different feeding strategies. During the first 180 days postpartum the cows were fed a total mixed ration (70% forage: 30% concentrate) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Mendicago sativa plus supplementation (grazing group, G1). From 180 to 250 days postpartum, all cows grazed Festuca arundinacea and were supplemented with total mixed ration. Mitochondrial function was assessed measuring oxygen consumption rate in liver biopsies and revealed that maximum respiratory rate decreased significantly in grazing cows during early lactation, yet was unchanged in non-grazing cows during the lactation curve. While no differences could be found in mitochondrial content or oxidative stress markers, a significant increase in protein lysine acetylation was found in grazing cows during early lactation but not in cows from the non-grazing group. Mitochondrial acetylation positively correlated with liver triglycerides and β-hydroxybutyrate plasma levels, well-known markers of negative energy balance, while a negative correlation was found with the maximum respiratory rate and sirtuin 3 levels. To our knowledge this is the first report of mitochondrial function in liver biopsies of dairy cows during lactation. On the whole our results indicate that mitochondrial function is impaired during early lactation in grazing cows and that acetylation may account for changes in mitochondrial function in this period. Additionally, our results suggest that feeding total mixed ration during early lactation may be an efficient protective strategy.

Syzygium cumini (L.) Skeels has been reported to exert anti-inflammatory and cardiometabolic activities due to its high content of polyphenols. We characterized the chemical composition and assessed the antidiabetic effects of a novel polyphenol-rich extract (PESc) obtained from S. cumini leaf. Rats were injected with alloxan (150 mg/kg, ip, ALX group) and followed up for 7 days. Some were orally treated with PESc (50 mg/kg/day) for 7 days before and after diabetes induction (ALX-PP) or only for 7 days after alloxan injection (ALX-P). ALX-P and ALX-PP decreased fasting glycemia in 37 and 43%, respectively, as compared to ALX. Triglycerides and total cholesterol serum levels were also significantly reduced in comparison to ALX. PESc presented high polyphenol concentration (71.78 ± 8.57 GAE/100 g), with flavonoid content of 8.21 ± 0.42 QE/100 g. Upon HPLC-MS/MS and MS/MS studies, five main polyphenols—gallic acid, quercetin, myricetin, and its derivatives—were identified. Myricetin was predominant (192.70 ± 16.50 μg/mg PESc), followed by measurable amounts of gallic acid (11.15 ± 0.90 μg/mg PESc) and quercetin (4.72 ± 0.06 μg/mg PESc). Kinetic assessment of total antioxidant capacity revealed PESc high potency, since maximum response was reached within 5 min reaction time in a concentration-dependent manner. Specific antioxidant activity of PESc was assessed against both DPPH• and ABTS•+, showing strong activity (IC50: 3.88 ± 1.09 and 5.98 ± 1.19 μg/mL, resp.). PESc also inhibited lipoxygenase activity (IC50: 27.63 ± 8.47), confirming its antioxidant activity also on biologically relevant radicals. Finally, PESc induced insulin secretion by directly stimulating INS-1E β cells in the absence of any cytotoxic effect. Overall, our results support that PESc is a potent antioxidant phytocomplex with potential pharmacological use as a preventive antidiabetic natural product.

Trypanosoma cruzi, the causative agent of Chagas disease, affects 8–10 million people in Latin America. Parasite antioxidant systems are essential for parasite survival and infectivity in the vertebrate host. Herein, we characterized the enzymic properties, subcellular localization, and contribution to parasite virulence of a T. cruzi hybrid type A member of class I heme peroxidases. The enzyme reacts fast with hydrogen peroxide and utilizes both ferrocytochrome c and ascorbate as reducing substrates [T. cruzi ascorbate peroxidase (TcAPx)-cytochrome c peroxidase (CcP)]. A unique subcellular distribution of TcAPx-CcP in the infective stages suggests a role during parasite–host interactions. Infection of macrophages and cardiomyocytes, as well as in mice, confirmed the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1·s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1·s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium. Fil: Hugo, Martín. Universidad de la República; Uruguay Fil: Martínez, Alejandra. Universidad de la República; Uruguay Fil: Trujillo, Madia. Universidad de la República; Uruguay Fil: Estrada, Damián. Universidad de la República; Uruguay Fil: Mastrogiovanni, Mauricio. Universidad de la República; Uruguay Fil: Linares, Edlaine. Universidade de Sao Paulo; Brasil Fil: Augusto, Ohara. Universidade de Sao Paulo; Brasil Fil: Issoglio, Federico Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Heijnen, Harry F. G.. University Medical Center; Países Bajos Fil: Piacenza, Lucía. Universidad de la República; Uruguay Fil: Radi, Rafael. Universidad de la República; Uruguay

Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol.In the presence of Tempol (5-120 μM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an ICWe have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by excessive fat deposition in hepatocytes in the absence of significant alcohol intake. We decided to evaluate the effects on NAFLD of extra virgin olive oil (EVOO) consumption and the role that nitro-fatty acids (NO2-FA) present or formed during digestion in EVOO may have. We postulate that NO2-FA, due to its well-characterized pleiotropic anti-inflammatory properties, could spare mitochondria form the deleterious effects of high fat diet (HFD), contributing to the health benefits associated with EVOO consumption. Accordingly, we analyzed liver mitochondrial function in mice fed with HFD and supplemented with 10% EVOO (w/w) in the absence or presence of nitrite. We have been demonstrated that gastric conditions i.e EVOO plus nitrite at acidic pH favor NO2-FA formation (PLOS One 2014). HFD mice supplemented with EVOO plus nitrite exhibited lower increase in body weight than HFD controls or even animals under normal diet, in addition to a decreased accumulation of fat liver. Liver mitochondrial function was studied by high resolution respirometry. The HFD mice group supplemented with EVOO plus nitrite showed the best mitochondrial respiratory control ratio (RCR). When looking for electron transport respiratory chain complexes activities, complexes II and V were significantly improved by EVOO supplementation and even better in the presence of nitrite. As part of the antioxidant/anti-inflammatory actions of NO2-FA, liver hemoxygenase-1 (HO-1) expression increased in the EVOO/nitrite condition. Plasma NO2-FA levels were lower in the condition of HFD when compared to normal diet while increased in the HFD plus EVOO/nitrite condition suggesting an association between NO2-FA formation by EVOO and the observed improvement of mitochondrial function.

Hydrogen sulfide (H2S) has been proposed to be a signaling agent. However, its mechanisms of action are usually unknown. H2S can modify cysteine residues to persulfides (RSSH), poorly characterized species proposed to mediate the signaling effects of H2S. The successful intracellular pathogen Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis disease, is able to produce H2S and to import it from the host, although the effects of H2S on Mt physiology and virulence are still unknown. In this work, we utilized the one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mt (MtAhpE-S-) as a model to study the formation and reactions of protein persulfides. The peroxidatic cysteine in MtAhpE is oxidized by different peroxides to a stable sulfenic acid (MtAhpE-SOH), which reacts with H2S to form a persulfide derivative (MtAhpE-SS-). The formation of MtAhpE-SS- was evidenced by treatment with iodoacetamide and detection of the alkyl derivative (MtAhpE-SS-R) by mass spectrometry. The kinetics of persulfide formation reaction was determined (103 M-1s-1 at pH 7.4, 25oC) by two independent competition assays. In addition, MtAhpE catalyzed the reduction of H2O2 by H2S indicating that H2S was able to complete the catalytic cycle of MtAhpE. However, H2S is not expected to be the preferred reducing substrate in vivo, since it would not compete with mycothiol/mycoredoxin-1. Reactivity of MtAhpE-SS- was compared with that of MtAhpE-S-: MtAhpE-SS- reacted faster in non-specific nucleophilic reactions (such as with the disulfide 4,4’-dithiodipyridine) but lost the specific fast reactivity with hydroperoxides (H2O2 and peroxynitrite). Computational simulations indicated that persulfidation of peroxidatic cysteine leads to a disruption of the hydrogen bond network at the active site, which is required for fast hydroperoxide reduction. Overall, the data indicate that persulfuration is a posttranslational modification that can alter the reactivities of cysteine residues in different directions depending on their specific roles and protein microenvironments.

We sought to explore the fate of the fatty acid synthesis pathway in human fibroblasts exposed to DNA damaging agents capable of inducing senescence, a state of irreversible growth arrest. Induction of premature senescence by doxorubicin or hydrogen peroxide led to a decrease in protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1), the enzyme that catalyzes the rate-limiting step in fatty-acid biosynthesis. ACC1 decay accompanied the activation of the DNA damage response (DDR), and resulted in decreased lipid synthesis. A reduction in protein and mRNA levels of ACC1 and in lipid synthesis was also observed in human primary fibroblasts that underwent replicative senescence. We also explored the consequences of inhibiting fatty acid synthesis in proliferating non-transformed cells. Using shRNA technology, we knocked down ACC1 in human fibroblasts. Interestingly, this metabolic perturbation was sufficient to arrest proliferation and trigger the appearance of several markers of the DDR and increase senescence associated β-galactosidase activity. Reactive oxygen species and p38 mitogen activated protein kinase phosphorylation participated in the induction of senescence. Similar results were obtained upon silencing of fatty acid synthase (FAS) expression. Together our results point towards a tight coordination of fatty acid synthesis and cell proliferation in human fibroblasts.

Arachidonic acid (AA) represents a precursor of potent signaling molecules, i.e., prostaglandins and thromboxanes through enzymatic and non-enzymatic oxidative pathways. Arachidonic acid can be nitrated by reactive nitrogen species leading to the formation of nitro-arachidonic acid (NO2-AA). In fact, nitration of AA by inflammatory stimulus could divert the “normal metabolic pathway” of AA to gain novel responses. In this prospective article, we describe the mechanisms of NO2-AA formation in human cells as well as its key chemical and biological properties. This includes the ability of NO2-AA to mediate unique redox signaling anti-inflammatory actions along with its therapeutic potential.

Quillaja brasiliensis (Quillajaceae) is a saponin producing species native from southern Brazil and Uruguay. Its saponins are remarkably similar to those of Q. saponaria , which provides most of the saponins used as immunoadjuvants in vaccines. The immunostimulating capacities of aqueous extract (AE) and purified saponin fraction (QB-90) obtained from leaves of Q. brasiliensis were favorably comparable to those of a commercial saponin-based adjuvant preparation (Quil-A ® ) in experimental vaccines against bovine herpesvirus type 1 and 5, poliovirus and bovine viral diarrhea virus in mice model. Herein, the immunogenicity and protection efficacy of rabies vaccines adjuvanted with Q. brasiliensis AE and its saponin fractions were compared with vaccines adjuvanted with either commercial Quil-A or Alum. Mice were vaccinated with one or two doses (on days 0 and 14) of one of the different vaccines and serum levels of total IgG, IgG1 and IgG2a were quantified over time. A challenge experiment with a lethal dose of rabies virus was carried out with the formulations. Viral RNA detection in the brain of mice was performed by qPCR, and RNA copy-numbers were quantified using a standard curve of in vitro transcribed RNA. All Q. brasiliensis saponin-adjuvanted vaccines significantly enhanced levels of specific IgG isotypes when compared with the no adjuvant group ( P ≤ 0.05). Overall, one or two doses of saponin-based vaccine were efficient to protect against the lethal rabies exposure. Both AE and saponin fractions from Q. brasiliensis leaves proved potent immunological adjuvants in vaccines against a lethal challenge with a major livestock pathogen, hence confirming their value as competitive or complementary sustainable alternatives to saponins of Q. saponaria .

Nitroarachidonic acid (NO 2 AA) is a nitroalkene exhibiting pleiotropic anti-inflammatory actions in many cell types. We have recently shown that NO 2 AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing formation of the active complex in the membrane, without affecting the phosphorylation of cytosolic subunits. Recent work indicates the participation of Protein disulfide isomerase (PDI) activity on NOX2 active complex formation. Thus, the nitroalkene could affect PDI activity which might help to explain our previous observations on NOX2 inhibition. We determined that both the reductase and chaperone activities of PDI were inhibited by NO 2 AA in a dose and time- dependent manner, being a reversible effect. The mechanism of inhibition did not involve the release of nitric oxide ( ● NO). Since nitroalkenes are potent electrophiles and PDI has critical cysteine residues for its activity, the formation of a reversible covalent adduct between NO 2 AA and PDI may occur. We demonstrated the reversible covalent modification of PDI by NO 2 AA using the modified PEG switch assay as well as LC-MS/MS. Trypsinization of modified PDI confirmed that Cys residues present in the active site of PDI were key targets accounting for nitroalkene modification. Future work will be directed to determine if these modifications participates in the reported NO 2 AA modulation of NOX2.

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the CNS, it is mainly located in the cytosol of astrocytes, playing an important role in detoxification of ammonia and excess glutamate. Alterations in GS activity and oxidative posttranslational modifications have been found in vivo. Herein, we studied the effect of oxidants on recombinant hGS function and structure with a combination of experimental and computational studies. Peroxynitrite caused a dose-dependent inactivation of hGS and 3-nitrotyrosine (NT) formation. In addition, higher molecular weight aggregates were formed likely due to 3,3´-dityrosine crosslinks. pH studies suggested that 3,3´-dityrosine formation had a larger impact on GS activity than nitration. MS analysis identified some critical residues modified, namely Tyr 185 and 269. H 2 O 2 -dependent thiol oxidation did not impact on GS activity. Exposure of human astrocytes to SIN-1 led to GS nitration and decreased enzyme activity. Molecular dynamic studies in GS (Issoglio et al. Biochemistry 2016) are being performed in nitrated forms of the enzyme to define with an atomic level of detail how tyrosine oxidative modifications affect GS structure and activity.

The lack of current treatments for Amyotrophic Lateral Sclerosis (ALS) highlight the absence of a comprehensive understanding of the biological mechanisms that occur during the process of neurodegeneration. A consistent neuropathological feature of ALS is the extensive inflammation around motor neurons and axonal degeneration, evidenced by the accumulation of reactive astrocytes and activated microglia. Final products of inflammatory processes may be detected in blood and may act as a screening tool to identify treatment response. Herein, we focus on a) detecting products derived from arachidonic acid (AA) metabolization by the lipoxygenase (LOX) or prostaglandin endoperoxide H synthase pathways in blood from G93ASOD mice and b) evaluate the pharmacological modulation of the progress of the disease by electrophilic nitro-fatty acids (NFA), in particular nitro oleic acid (OA-NO2), because its well-documented unique anti-inflammatory properties. Results showed a different behavior of LOX-derived products in G93ASOD mice: an increase in 12-HETE levels was observed when clinical symptoms appeared whereas no changes were detected in age-matched wt mice. In addition, 5-HETE levels decreased faster in G93ASOD mice than in controls. This is in agreement with reports of 12/15-LOX formation in the central nervous system, suggesting that 12-HETE may be involved in brain oxidative damage. No changes in prostaglandins levels were observed in the plasma of transgenic mice while wt levels were greater at day 120. In addition, analysis of plasma samples from both wt and G93ASOD mice showed quantitative differences in lipid-derived products, which correlated with disease onset and progression. Administration of OA-NO2 16 mg/kg, s/c three times a week, to G93ASOD female mice, significantly improved grip strength and reduced weight loss compared to vehicle treated animals. Improved grip strength data were associated to increased heme oxygenase 1 (HO-1) expression. In fact, HO-1 co-localized with reactive astrocytes in the spinal cord of symptomatic mice. Furthermore, significant levels of OA-NO2 were detected in brain and spinal cord from OA-NO2-treated mice. We conclude that LOX-derived oxidation products correlate with disease progression and demonstrate that NFA can cross the brain barrier and be effective to reach the central nervous system to induce neuroprotective mechanisms. In this way, changes in the levels of AA-derived products were observed as well as different expression and activity of LOX in both brain and spinal cord from G93ASOD animals treated with OA-NO2. Overall, we are proposing key mediators of AA-derived pathways as novel footprints of ALS onset and progression with the aim to determine molecules associated with ALS and their underlying biology.