Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.

Investigation of the prebiotic synthesis of amino acids and RNA bases from CO2 using FeS/H2S as a reducing agent.

An autotrophic theory of the origin of metabolism and life has been proposed in which carbon dioxide is reduced by ferrous sulfide and hydrogen sulfide by means of a reversed citric acid cycle, leading to the production of amino acids. Similar processes have been proposed for purine synthesis. Ferrous sulfide is a strong reducing agent in the presence of hydrogen sulfide and can produce hydrogen as well as reduce alkenes, alkynes, and thiols to saturated hydrocarbons and reduce ketones to thiols. However, the reduction of carbon dioxide has not been demonstrated. We show here that no amino acids, purines, or pyrimidines are produced from carbon dioxide with the ferrous sulfide and hydrogen sulfide system. Furthermore, this system does not produce amino acids from carboxylic acids by reductive amination and carboxylation. Thus, the proposed autotrophic theory, using carbon dioxide, ferrous sulfide, and hydrogen sulfide, lacks the robustness needed to be a geological process and is, therefore, unlikely to have played a role in the origin of metabolism or the origin of life.

Characterization of Arabidopsis thaliana cDNAs that render yeasts tolerant toward the thiol-oxidizing drug diamide.

Diamide oxidizes cellular thiols and induces oxidative stress. To isolate plant genes which may, when overexpressed, increase tolerance of plants toward oxidative damage, an in vivo diamide tolerance screening in yeasts was used. An Arabidopsis cDNA library in a yeast expression vector was used to transform a yeast strain with intact antioxidant defense. Cells from approximately 10(5) primary transformants were selected for resistance to diamide. Three Arabidopsis cDNAs which confer diamide tolerance were isolated. This drug tolerance was specific and no cross tolerance toward hydroperoxides was found. One cDNA (D3) encodes a polypeptide which has an amino-terminal J domain characteristic of a divergent family of DnaJ chaperones. Another (D18) encodes a putative dTDP-D-glucose 4,6-dehydratase. Surprisingly, the third cDNA (D22) encodes a plant homolog of gamma-glutamyltransferases. It would have been difficult to predict that the expression of those genes would lead to an improved survival under conditions of depletion of cellular thiols. Hence, we suggest that this cloning approach may be a useful contribution to the isolation of plant genes that can help to cope with oxidative stress.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Desenvolvimento de eletrodos de ouro modificados com camadas auto-arranjadas de tióis para formação in situ de nanopartículas de ouro

Eletrodos de ouro foram utilizados para preparação de eletrodos modificados com monocamadas auto arranjadas de tióis. A limpeza do substrato metálico é fundamental para que a arquitetura molecular superficial possa ser efetuada com boa estabilidade e reprodutibilidade, além de fornecer dados utilizados no cálculo de área ativa, necessários na normalização dos valores de recobrimento superficial obtidos nas determinações de dessorção do agente modificador interno, o ácido 3-mercaptopropiônico. Os eletrodos modificados consistiram no recobrimento de transdutores de ouro com ácido 3-mercaptopropiônico através da imersão do eletrodo de Au em solução 25 mmolL-1 deste composto e, em seguida, com moléculas de cisteína, através da imersão do eletrodo de Au/3-MPA em solução 0,1 molL-1 deste composto, originando um sensor do tipo Au/3-MPA/CSH. As moléculas de cisteína foram utilizadas como agente redutor para obtenção de nanopartículas de Au na superfície do eletrodo modificado, através da aplicação de 20 µL de solução de HAuCl4. Após a confirmação da ausência do par tiólico superficial responsável pela redução das nanopartículas, o eletrodo Au/3-MPA/CSH/AuNp foi utilizado na determinação de peróxido de hidrogênio em soluções de concentrações crescentes em tampão fosfato 0,1 molL-1 pH 7,2.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Synthesis of monocyclic and linear polystyrene using the reversible coupling/cleavage of thiol/disulfide groups

By carefully controlling the concentration of alpha,omega-thiol polystyrene in solution, we achieved formation of unique monocyclic polystyrene chains (i.e., polymer chains with only one disulfide linkage). The presence of cyclic polystyrene was confirmed by its lower than expected molecular weight due to a lower hydrodynamic volume and loss of thiol groups as detected by using Ellman's reagent. The alpha,omega-thiol polystyrene was synthesized by polymerizing styrene in the presence of a difunctional RAFT agent and subsequent conversion of the dithioester end groups to thiols via the addition of hexylamine. Oxidation gave either monocyclic polymer chains (i.e., with only one disulfide linkage) or linear multiblock polymers with many disulfide linkages depending on the concentration of polymer used with greater chance of cyclization in more dilute solutions. At high polymer concentrations, linear multiblock polymers were formed. To control the MWD of these linear multiblocks, monofunctional X-PSTY (X = PhCH2C(S)-S-) was added. It was found that the greatest ratio of X-PSTY to X-PSTY-X resulted in a low M-n and PDI. We have shown that we can control both the structure and MWD using this chemistry, but more importantly such disulfide linkages can be readily reduced back to the starting polystyrene with thiol end groups, which has potential use for a recyclable polymer material.

Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione.

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone 'Compound 1' caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1's MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols. © 2004 Elsevier B.V. All rights reserved.

A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro

Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.

Healthy ageing and depletion of intracellular glutathione influences T cell membrane thioredoxin-1 levels and cytokine secretion

Background: During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Intracellular thiols are maintained in their reduced state by a network of redox regulating peptides, proteins and enzymes such as glutathione, thioredoxins and thioredoxin reductase. Here we have investigated whether any relationship exists between age and secreted or cell surface thioredoxin-1, intracellular glutathione concentration and T cell surface thioredoxin 1 (Trx-1) and how this is related to interleukin (IL)-2 production.Results: Healthy older adults have reduced lymphocyte surface expression and lower circulating plasma Trx-1 concentrations. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show that cell surface Trx-1 is lowered, secretion of Trx-1 is decreased and the response to the lectin phytohaemagglutinin measured as IL-2 production is also affected. These effects are recapitulated by another glutathione depleting agent, diethylmaleate.Conclusion: Together these data suggest that a relationship exists between the intracellular redox compartment and Trx-1 proteins. Loss of lymphocyte surface Trx-1 may be a useful biomarker of healthy ageing. © 2013 Carilho Torrao et al.; licensee Chemistry Central Ltd.

Stabilising cysteinyl thiol oxidation and nitrosation for proteomic analysis

Oxidation and S-nitrosylation of cysteinyl thiols (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H), sulfonic acids (Cys-SO3H), disulphides and S-nitrosothiols are suggested as important post-translational modifications that can activate or deactivate the function of many proteins. Non-enzymatic post-translational modifications to cysteinyl thiols have been implicated in a wide variety of physiological and pathophysiological states but have been difficult to monitor in a physiological setting because of a lack of experimental tools. The purpose of this review is to bring together the approaches that have been developed for stably trapping cysteine either in its reduced or oxidised forms for enrichment and or subsequent mass spectrometric analysis. These tools are providing insight into potential targets for post-translational modifications to cysteine modification in vivo. This article is part of a Special Issue entitled: Special Issue: Posttranslational Protein modifications in biology and Medicine. © 2013.