Search Results (23)

Hydrogen peroxide (H₂O₂) plays a crucial role in cell signaling in response to physiological and environmental perturbations. H₂O₂ can oxidize typical 2-Cys peroxiredoxin (PRX) first into a sulfenic acid, which resolves into a disulfide that can be reduced by thioredoxin (TRX)/TRX reductase (TR). At high levels, H₂O₂ can also hyperoxidize sulfenylated PRX into a sulfinic acid that can be reduced by sulfiredoxin (SRX). Therefore, PRX, TRX, TR, and SRX (abbreviated as PTRS system here) constitute the coupled sulfenylation and sulfinylation cycle (CSSC), where certain oxidized PRX and TRX forms also function as redox signaling intermediates. Earlier studies have revealed that the PTRS system is capable of rich signaling dynamics, including linearity, ultrasensitivity/switch-like response, nonmonotonicity, circadian oscillation, and possibly, bistability. However, the origins of ultrasensitivity, which is fundamentally required for redox signal amplification, have not been adequately characterized, and their roles in enabling complex nonlinear dynamics of the PTRS system remain to be determined. Through in-depth mathematical modeling analyses, here we revealed multiple sources of ultrasensitivity that are intrinsic to the CSSC, including zero-order kinetic cycles, multistep H₂O₂ signaling, and a mechanism arising from diminished H₂O₂ removal at high PRX hyperoxidation state. The CSSC, structurally a positive feedback loop, is capable of bistability under certain parameter conditions, which requires embedding multiple sources of ultrasensitivity identified. Forming a negative feedback loop with cytosolic SRX as previously observed in energetically active cells, the mitochondrial PTRS system (where PRX3 is expressed) can produce sustained circadian oscillations through supercritical Hopf bifurcations. In conclusion, our study provided novel quantitative insights into the dynamical complexity of the PTRS system and improved appreciation of intracellular redox signaling. Full article

A series of novel 2-alkythio-4-chloro-N-[imino-(heteroaryl)methyl]benzenesulfonamide derivatives, 8–24, were synthesized in the reaction of the N-(benzenesulfonyl)cyanamide potassium salts 1–7 with the appropriate mercaptoheterocycles. All the synthesized compounds were evaluated for their anticancer activity in HeLa, HCT-116 and MCF-7 cell lines. The most promising compounds, 11–13, molecular hybrids containing benzenesulfonamide and imidazole moieties, selectively showed a high cytotoxic effect in HeLa cancer cells (IC₅₀: 6–7 μM) and exhibited about three times less cytotoxicity against the non-tumor cell line HaCaT cells (IC₅₀: 18–20 μM). It was found that the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells. The compounds increased the early apoptotic population of cells, elevated the percentage of cells in the sub-G1 phase of the cell cycle and induced apoptosis through caspase activation in HeLa cells. For the most active compounds, susceptibility to undergo first-phase oxidation reactions in human liver microsomes was assessed. The results of the in vitro metabolic stability experiments indicated values of the factor t_½ for 11–13 in the range of 9.1–20.3 min and suggested the hypothetical oxidation of these compounds to sulfenic and subsequently sulfinic acids as metabolites. Full article

Thiol antioxidants play important roles in cell and body defense against oxidative stress. In body fluid, albumin is the richest source of thiol antioxidants. One recent study showed that the reductive modification of thiol residues in albumin potentiated its antioxidative activity. Given that whey protein (WP) contains albumin and other thiol-active proteins, this property of WP could be exploited to develop novel thiol antioxidants. The aim of this study was to address this possibility. WP was reductively modified with dithiothreitol (DTT). The modified protein exhibited significantly elevated free sulfhydryl groups (-SH) and thiol antioxidative activity. It detoxified H₂O₂ and prevented H₂O₂-initiated protein oxidation and cell death in a -SH group-dependent way in vitro. In addition, it reacted with GSH/GSSG and altered the GSH/GSSG ratio via thiol–disulfide exchange. In vivo, oral administration of the reductively modified WP prevented oxidative stress and renal damage in a mouse model of renal injury caused by ischemia reperfusion. It significantly improved renal function, oxidation, inflammation, and cell injury. These protective effects were not observed in the WP control and were lost after blocking the -SH groups with maleimide. Furthermore, albumin, one of the ingredients of WP, also exhibited similar protective effects when reductively modified. In conclusion, the reductive modification of thiol residues in WP transformed it into a potent thiol antioxidant that protected kidneys from ischemia reperfusion injury. Given that oxidative stress underlies many life-threatening diseases, the reductively modified dietary protein could be used for the prevention and treatment of many oxidative-stress-related conditions, such as cardiovascular diseases, cancer, and aging. Full article

Vitamin C (ascorbic acid; AA) and copper (Cu²⁺) are well used supplements with many health-promoting actions. However, when they are used in combination, the Fenton reaction occurs, leading to the formation of highly reactive hydroxyl radicals. Given that kidney is vulnerable to many toxicants including free radicals, we speculated that the in vivo administration of AA plus Cu²⁺ may cause oxidative kidney injury. The purpose of this study was to address this possibility. Mice were administered with AA and Cu²⁺, alone or in combination, via oral gavage once a day for various periods. Changes in the systemic oxidative status, as well renal structure and functions, were examined. The administration of AA plus Cu²⁺ elevated protein oxidation in serum, intestine, bladder, and kidney, as evidenced by the increased sulfenic acid formation and decreased level of free sulfhydryl groups (-SH). The systemic oxidative stress induced by AA plus Cu²⁺ was associated with a significant loss of renal function and structure, as indicated by the increased blood urea nitrogen (BUN), creatinine and urinary proteins, as well as glomerular and tubular cell injury. These effects of AA and Cu²⁺ were only observed when used in combination, and could be entirely prevented by thiol antioxidant NAC. Further analysis using cultured renal tubular epithelial cells revealed that AA plus Cu²⁺ caused cellular protein oxidation and cell death, which could be abolished by NAC and catalase. Moreover, coincubation of AA and Cu²⁺ led to H₂O₂ production. Collectively, our study revealed that a combined administration of AA and Cu²⁺ resulted in systemic oxidative stress and renal cell injury. As health-promoting supplements, AA and Cu²⁺ should not be used together. Full article

Oxidation of glyceraldehyde−3−phosphate dehydrogenase (GAPDH) by reactive oxygen species such as H₂O₂ activate pleiotropic signaling pathways is associated with pathophysiological cell fate decisions. Oxidized GAPDH binds chaperone proteins with translocation of the complex to the nucleus and mitochondria initiating autophagy and cellular apoptosis. In this study, we establish the mechanism by which H₂O₂−oxidized GAPDH subunits undergo a subunit conformational rearrangement. H₂O₂ oxidizes both the catalytic cysteine and a vicinal cysteine (four residues downstream) to their respective sulfenic acids. A ‘two−cysteine switch’ is activated, whereby the sulfenic acids irreversibly condense to an intrachain thiosulfinic ester resulting in a major metastable subunit conformational rearrangement. All four subunits of the homotetramer are uniformly and independently oxidized by H₂O₂, and the oxidized homotetramer is stabilized at low temperatures. Over time, subunits unfold forming disulfide−linked aggregates with the catalytic cysteine oxidized to a sulfinic acid, resulting from thiosulfinic ester hydrolysis via the highly reactive thiosulfonic ester intermediate. Molecular Dynamic Simulations provide additional mechanistic insights linking GAPDH subunit oxidation with generating a putative signaling conformer. The low−temperature stability of the H₂O₂−oxidized subunit conformer provides an operable framework to study mechanisms associated with gain−of−function activities of oxidized GAPDH to identify novel targets for the treatment of neurodegenerative diseases. Full article

This review focuses on the consequences of GAPDH S-nitrosylation at the catalytic cysteine residue. The widespread hypothesis according to which S-nitrosylation causes a change in GAPDH structure and its subsequent binding to the Siah1 protein is considered in detail. It is assumed that the GAPDH complex with Siah1 is transported to the nucleus by carrier proteins, interacts with nuclear proteins, and induces apoptosis. However, there are several conflicting and unproven elements in this hypothesis. In particular, there is no direct confirmation of the interaction between the tetrameric GAPDH and Siah1 caused by S-nitrosylation of GAPDH. The question remains as to whether the translocation of GAPDH into the nucleus is caused by S-nitrosylation or by some other modification of the catalytic cysteine residue. The hypothesis of the induction of apoptosis by oxidation of GAPDH is considered. This oxidation leads to a release of the coenzyme NAD⁺ from the active center of GAPDH, followed by the dissociation of the tetramer into subunits, which move to the nucleus due to passive transport and induce apoptosis. In conclusion, the main tasks are summarized, the solutions to which will make it possible to more definitively establish the role of nitric oxide in the induction of apoptosis. Full article

Hydrogen sulfide (H₂S), which is generated mainly by cystathionine γ-lyase (CSE) in the cardiovascular system, plays a pivotal role in a wide range of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is poorly understood. Herein, we show that oxidation induces the disulfide bond formation between Cys252 and Cys255 in the CXXC motif, thus stimulating the H₂S-producing activity of CSE. The activity of oxidized CSE is approximately 2.5 fold greater than that of the reduced enzyme. Molecular dynamics and molecular docking suggest that the disulfide bond formation induces the conformational change in the active site of CSE and consequently increases the affinity of the enzyme for the substrate L-cysteine. Mass spectrometry and mutagenesis studies further established that the residue Cys255 is crucial for oxidation sensing. Oxidative stress-mediated sulfenylation of Cys255 leads to a sulfenic acid intermediate that spontaneously forms an intramolecular disulfide bond with the vicinal thiol group of Cys252. Moreover, we demonstrate that exogenous hydrogen peroxide (H₂O₂) and endogenous H₂O₂ triggered by vascular endothelial growth factor (VEGF) promote cellular H₂S production through the enhancement of CSE activity under oxidative stress conditions. By contrast, incubation with H₂O₂ or VEGF did not significantly enhance cellular H₂S production in the presence of PEG-catalase, an enzymatic cell-permeable H₂O₂ scavenger with high H₂O₂ specificity. Taken together, we report a new posttranslational modification of CSE that provides a molecular mechanism for H₂O₂/H₂S crosstalk in cells under oxidative stress. Full article

Ovothiol A (OSH) is one of the strongest natural antioxidants. So far, its presence was found in tissues of marine invertebrates, algae and fish. Due to very low pKa value of the SH group, under physiological conditions, this compound is almost entirely present in chemically active thiolate form and reacts with ROS and radicals significantly faster than other natural thiols. In biological systems, OSH acts in tandem with glutathione GSH, with OSH neutralizing oxidants and GSH maintaining ovothiol in the reduced state. In the present work, we report the rate constants of OSH oxidation by H₂O₂ and of reduction of oxidized ovothiol OSSO by GSH and we estimate the Arrhenius parameters for these rate constants. The absorption spectra of reaction intermediates, adduct OSSG and sulfenic acid OSOH, were obtained. We also found that OSH effectively quenches the triplet state of kynurenic acid with an almost diffusion-controlled rate constant. This finding indicates that OSH may serve as a good photoprotector to inhibit the deleterious effect of solar UV irradiation; this assumption explains the high concentrations of OSH in the fish lens. The unique antioxidant and photoprotecting properties of OSH open promising perspectives for its use in the treatment of human diseases. Full article

Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y₁₂ receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL⁻¹ of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h⁻¹ H₂O₂ and 20% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS² spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays. Full article

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H₂O₂ acting as a second messenger. However, H₂O₂ actually reacts poorly with most cysteine thiols and it is not clear how H₂O₂ discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H₂O₂ scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H₂O₂-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses. Full article

The catalytic activity of peroxiredoxins (Prx) is determined by the conserved peroxidatic cysteine (Cys_P), which reacts with peroxides to form sulfenic acid (Cys-SOH). Under conditions of oxidative stress, Cys_P is oxidized to catalytically inactive sulfinic (Cys-SO₂) and sulfonic (Cys-SO₃) forms. The Cys-SO₂ form can be reduced in a reaction catalyzed by sulfiredoxin (Srx). To explore the physiological significance of peroxiredoxin overoxidation, we investigated daily variations in the oxidation state of 2-Cys peroxiredoxins in flies of different ages, or under conditions when the pro-oxidative load is high. We found no statistically significant changes in the 2-Cys Prxs monomer:dimer ratio, which indirectly reflects changes in the Prx catalytic activity. However, we found daily variations in Prx-SO_2/3 that were more pronounced in older flies as well as in flies lacking Srx. Unexpectedly, the srx mutant flies did not exhibit a diminished survivorship under normal or oxidative stress conditions. Moreover, the srx mutant was characterized by a higher physiological activity. In conclusion, catalytically inactive forms of Prx-SO_2/3 serve not only as a marker of cellular oxidative burden, but may also play a role in an adaptive response, leading to a positive effect on the physiology of Drosophila melanogaster. Full article

L-Cysteinate-intercalated CaAl-layered double hydroxide (LDH) was prepared by the co-precipitation method producing highly crystalline hydrocalumite phase with a well-pillared interlayer gallery. The obtained materials were characterized by X-ray diffractometry, IR as well as Raman spectroscopies. By performing interlamellar oxidation reactions with peracetic acid as oxidant, oxidation of cysteinate to cystinate in aqueous and cysteinate sulfenic acid in acetonic suspensions occurred. The oxidations could be performed under mild conditions, at room temperature, under neutral pH and in air. It has been shown that the transformation pathways are due to the presence of the layered structure, that is, the confined space of the LDH behaved as molecular reactor. Full article

In this communication we demonstrate that two natural isothiocyanates, sulforaphane (SFN) and erucin (ERN), inhibit autoxidation of lipids at 140 °C but not below 100 °C. This effect is due to thermal decomposition of ERN and SFN to sulfenic acids and methylsulfinyl radicals, species able to trap lipidperoxyl radicals. Our observations shed new light on thermal processing of vegetables containing these two isothiocyanates. Full article

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20–300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca²⁺-activated maximal (active) force (F_o) by about 50% and evoked an approximately 3-fold increase in Ca²⁺-independent (passive) force (F_passive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations. Full article

We analyzed antitumor effects of a series of curcumin analogues. Some of them were obtained by reaction of substitution involving the two phenolic OH groups of curcumin while the analogues with a substituent at C-4 was prepared following an original procedure that regards the condensation of benzenesulfenic acid onto the nucleophilic central carbon of the curcumin skeleton. We analyzed cytotoxic effects of such derivatives on two TNBC (triple negative breast cancer) cell lines, SUM 149 and MDA-MB-231, but only three of them showed an IC₅₀ in a lower micromolar range with respect to curcumin. We also focused on these three derivatives that in both cell lines exhibited a higher or at least equivalent pro-apoptotic effect than curcumin. The analysis of molecular mechanisms of action of the curcumin derivatives under study has highlighted that they decreased NF-κB transcriptional factor activity, and consequently the expression of some NF-κB targets. Our data confirmed once again that curcumin may represent a very good lead compound to design analogues with higher antitumor capacities and able to overcome drug resistance with respect to conventional ones, even in tumors difficult to treat as TNBC. Full article