Cellular Thiol Redox State Research Articles

Real-time thiol detection in iPSC-derived neuron cultures using SemKur-IM, a novel fluorescent dithio probe

Neurological disorders associated with inflammation and oxidative stress show reduced glutathione (GSH) levels in the human brain. Drug discovery efforts and pharmacological studies would benefit from tools (e.g. chemical probes) that detect changes to oxidative stress, from the perspective of physiologically-relevant reporters like cellular thiols, including GSH. To this end, we have developed a fluorescence visualization assay using iPSC-derived cortical glutamatergic neurons that were loaded with 25 μM of a novel thiol-detection fluorescent probe, SemKur-IM. This probe enables visualization of cellular thiol level changes in the neuronal somas and neurites, in response exposure to N-acetyl-cysteine (NAC). Cellular thiol redox state was observed to change, based on an increase in green fluorescence (485 nm excitation maximum; 525 nm emission maximum) due to changes in thiol levels, from 0 to 40 mM. Interestingly, prior to treatment with NAC, cells did not appear to have significant levels of reduced thiols. Our studies demonstrate the utility of SemKur-IM in the detection of thiol levels in live cells in response to chemical exposures, such as from drugs that return the cell to a healthier reduced state. An initial application to screening the effects of an Alzheimer's disease drug candidate, Posiphen, using fluorescence cell sorting is presented. Other potential applications include high throughput screening of central nervous system (CNS) drugs thought to work by affecting cellular redox state in neurons.

Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Deletion of Rap1 disrupts redox balance and impairs endothelium-dependent relaxations

AimsRepressor activator protein 1 (Rap1) is conventionally known as a static structural component of the telomere, but recent evidence indicates that it exerts functions within and outside the nucleus taking part in metabolic regulation and promoting inflammatory responses. The present study investigated whether or not Rap1 deletion affects oxidative stress and nitric oxide (NO) bioavailability in the vascular wall, thus modulating endothelial function. Methods and resultsVascular responsiveness was studied in wire myographs in aortae from Rap1 wildtype and knockout mice. Deletion of Rap1 impaired endothelium-dependent relaxations elicited by acetylcholine. Rap1 deficiency did not affect the activation of endothelial NO synthase or the sensitivity of vascular smooth muscle to NO donors. The blunted acetylcholine-mediated relaxations in Rap1 deficient aortae were restored with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, apocynin or VAS2870. Rap1 deletion lowered cellular thiol-redox status and diminished activities of thiol-redox enzymes, thioredoxin 1 and glutaredoxin 1. ConclusionsThe capacity of thioredoxin 1 and glutaredoxin 1 to reduce intra-protein disulfide bridges is weakened in Rap1 deficient mice, resulting in hyper-activation of NADPH oxidase and greater reactive oxygen species generation. The high oxidative stress in Rap1 deficient mice is implicated with greater oxidative breakdown of NO, explaining the blunted acetylcholine-mediated relaxations in this animal. These findings imply that Rap1 plays an unanticipated role in regulating the fate of NO (a pivotal determinant of vascular homeostasis) and thus identify a new physiological importance of the telomere-associated protein.

169 - Cardiac-Specific Constitutive Activation of Nrf2 Induces a Stable Reductive State in Mice

Redox homeostasis regulates key cellular signaling pathways in both physiology and pathology. The cell's antioxidant response provides a defense against oxidative stress and establishes a redox tone permissive for cell signaling. The molecular regulation of the well-known Keap1/Nrf2 system acts as sensor responding to changes in redox homeostasis and is poorly studied in the heart. Importantly, it is not yet known whether Nrf2 alone can serve as a master regulator of cellular redox homeostasis without compensation of the transcriptional regulation of antioxidant response element (ARE) genes through alternate mechanisms. Here, we addressed this question using cardiac-specific transgenic expression at two different levels of constitutively active nuclear erythroid related factor 2 (caNrf2) functioning independently of Keap1. The caNrf2 mice showed augmentation of glutathione (GSH), the key regulator of the cellular thiol redox state. The Trans-AM assay for Nrf2-binding to the antioxidant response element (ARE) showed a dose-dependent increase associated with upregulation of several major antioxidant genes and proteins. This was accompanied by a significant decrease in dihydroethidium staining and malondialdehyde (MDA) in the caNrf2-TG mice myocardium. Interestingly, caNrf2 gene-dosage dependent redox changes were noted resulting in generation of a multi-stage model of pro-reductive and reductive conditions in the myocardium of TG-low and TG-high mice, respectively. These data clearly show that Nrf2 levels alone are capable of serving as the master regulator of the ARE. These models provide an important platform to investigate the impact of the Nrf2 system independent of the need to regulate the activity of Keap1 and the consequent exposure to pro-oxidants or electrophiles, which have numerous off-target effects.

Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions.Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2′-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated.Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 μM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress.In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Constitutive activation of Nrf2 induces a stable reductive state in the mouse myocardium

Redox homeostasis regulates key cellular signaling pathways in both physiology and pathology. The cell's antioxidant response provides a defense against oxidative stress and establishes a redox tone permissive for cell signaling. The molecular regulation of the well-known Keap1/Nrf2 system acts as sensor responding to changes in redox homeostasis and is poorly studied in the heart. Importantly, it is not yet known whether Nrf2 alone can serve as a master regulator of cellular redox homeostasis without compensation of the transcriptional regulation of antioxidant response element (ARE) genes through alternate mechanisms. Here, we addressed this question using cardiac-specific transgenic expression at two different levels of constitutively active nuclear erythroid related factor 2 (caNrf2) functioning independently of Keap1. The caNrf2 mice showed augmentation of glutathione (GSH), the key regulator of the cellular thiol redox state. The Trans-AM assay for Nrf2-binding to the antioxidant response element (ARE) showed a dose-dependent increase associated with upregulation of several major antioxidant genes and proteins. This was accompanied by a significant decrease in dihydroethidium staining and malondialdehyde (MDA) in the caNrf2-TG mice myocardium. Interestingly, caNrf2 gene-dosage dependent redox changes were noted resulting in generation of a multi-stage model of pro-reductive and reductive conditions in the myocardium of TG-low and TG-high mice, respectively. These data clearly show that Nrf2 levels alone are capable of serving as the master regulator of the ARE. These models provide an important platform to investigate the impact of the Nrf2 system independent of the need to regulate the activity of Keap1 and the consequent exposure to pro-oxidants or electrophiles, which have numerous off-target effects.

Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.

Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

Stable Isotope Metabolic Labeling-Based Quantitative Thiol Redox Proteomic Analysis of Hydrogen Peroxide-treated Arabidopsis plant

Homeostasis of protein thiol redox plays an important role in numerous cellular processes and stress responses. The thiol moiety of cysteine residue is easily altered to various redox isoforms that play critical roles in regulation of both structure and function of proteins. In the present study, a quantitative thiol redox proteomic method, termed OxNSIL, has been developed, which integrates the chemical labeling by biotin-tagged alkylating reagents with the heavy nitrogen stable isotope-coded salt metabolic labeling and acquires both advantages of biotin-avidin enrichment and MS-based quantitation of cysteine residue-containing peptides. Both the reduced and the reversibly oxidized cysteine thiol moieties are finally identified in a site-specific manner and measured in a single experiment. Hydrogen peroxide, H2O2, was applied on the whole Arabidopsis plant to trigger thiol redox state alteration in order to obtain a proof-of-concept result for application of OxNSIL approach in plants. A total of 438 non-redundant biotin-tagged thiolalkylated peptides representing 391 different cysteine-containing proteins were eventually obtained, among which 17 redox-sensitive biotin tag-labeled peptides were significantly altered by H2O2. Some well-known ROS-related proteins, such as Ferredoxins 1/2, RuBisCO large subunit, RuBisCO activase, and Fructose-bisphosphate aldolase 6/8 were also detected as the oxidation- sensitive thiol redox proteins. These results substantiate the usefulness of OxNSIL approach in study of in planta ROS-induced cellular thiol redox state alterations.

Diminishing glutathione availability and age-associated decline in neuronal excitability

Oxidative stress is frequently implicated in diminished electrical excitability of aging neurons yet the foundations of this phenomenon are poorly understood. This study explored links between alterations in cellular thiol-redox state and age-associated decline in electrical excitability in identified neurons (right pedal dorsal 1 [RPeD1]) of the gastropod Lymnaea stagnalis. Intracellular thiol redox state was modulated with either dithiothreitol or membrane permeable ethyl ester of the antioxidant glutathione (et-GSH). Neuronal antioxidant demand was manipulated through induction of lipid peroxidation with 2,2′-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH). Glutathione synthesis was manipulated with buthionine sulfoximine (BSO). We show that; glutathione content of snail brains declines with age, whereas pyroglutamate content increases; treatment with AAPH and BSO alone aggravated the natural low excitability state of old RPeD1, but only the combination of AAPH + BSO affected electrical excitability of young RPeD1; et-GSH reversed this effect in young RPeD1; et-GSH and dithiothreitol treatment reversed age-associated low excitability of old RPeD1. Together, these data argue for a tight association between glutathione availability and the regulation of neuronal electrical excitability and indicate perturbation of cellular thiol-redox metabolism as a key factor in neuronal functional decline in this gastropod model of biological aging.

Identification of potential protein dithiol-disulfide substrates of mammalian Grx2

BackgroundGlutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. Methods and resultsHere, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. ConclusionsOur study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state. General significanceThe knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.

Effects of Redox State on the Efficient Uptake of Cell Permeable Peptide in Mammalian Cells

We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC). The FRET peptide construct was easily detected in cells cultured in 96-well plates using a plate-reader. Treatment of cells with various classes of reducing or oxidizing agents resulted in an increase or decrease in FAM fluorescence, respectively. Changes in FAM fluorescence correlated significantly with redox-sensitive green fluorescent protein ratios in cells treated with hydrogen peroxide but not NAC. Detection of relative changes in cellular redox state was enhanced by the fact that uptake of the cell-penetrating peptide occurred more quickly in relatively reduced compared with oxidized cells. We conclude that cell-penetrating peptides coupled via disulfide bonds to detectable cargo is a novel and specific approach for assessment of relative changes in cellular thiol redox state.

Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870)

Specific inhibitors of the production of reactive oxygen species (ROS) by the NADPH oxidases (Nox's) are potentially important therapeutic agents in the wide range of human diseases that are characterized by excessive ROS production. It has been proposed that VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3- triazolo[4,5-d]pyrimidine), identified as an inhibitor of Nox2 by small-molecule screening, may serve as an example of such an agent. Here we show that VAS2870 inhibits ROS production in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle, previously identified with Nox4, and thereby abrogates O2-coupled redox regulation of the ryanodine receptor–Ca2+ channel (RyR1). However, we also find that VAS2870 modifies directly identified cysteine thiols within RyR1. Mass spectrometric analysis of RyR1 exposed in situ to VAS2870 and of VAS2870-treated glutathione indicated that thiol modification is through alkylation by the benzyltriazolopyrimidine moiety of VAS2870. Thus, VAS2870 exerts significant off-target effects, and thiol alkylation by VAS2870 (and closely related Nox inhibitors) may in fact replicate some of the effects of ROS on cellular thiol redox status. In addition, we show that SR-localized Nox4 is inhibited by other thiol-alkylating agents, consistent with a causal role for cysteine modification in the inhibition of ROS production by VAS2870.

Abstract P270: Resin-Assisted Capture Reveals Unique Targets of Oxidation in Ischemic Preconditioning

Proper maintenance of cellular thiol redox status is critical in the regulation of many processes that dictate normal myocardial function. Cell death signaling, in particular, is especially susceptible to alterations in thiol redox state. Myocardial ischemic preconditioning (IPC) is a redox sensitive process that is thought to provide protection from ischemia-reperfusion (IR) injury, in part, via reversible oxidative signaling. Protein S -nitrosylation (SNO) is a reversible, thiol-based modification that we and others have shown to provide cardioprotection by modulating the activity of target proteins and by shielding cysteine residues from irreversible oxidation. Additional thiol-based modifications are also thought to play an important role in cardioprotection, and these include disulfide bridges and glutathiolation. Therefore, it is of interest to identify these redox sensitive targets that are unique to IPC. Langendorff perfused mouse hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR) and homogenized. Specific sites of oxidation were identified using oxidation-resin assisted capture (Ox-RAC) with mass spectrometry. Consistent with a role for oxidative signaling in cardioprotection, Ox-RAC analysis identified 56 proteins that were oxidized with IPC, but not IR or IPC-IR. These proteins are likely to be reversibly oxidized, as evidenced by the fact that they are oxidized with IPC, but are no longer oxidized following reperfusion (IPC-IR). These proteins may represent important targets in cardioprotective signaling and include F-actin-capping protein β, Egl nine homolog 1, and numerous members of the heat shock family (HSP60, HSP70, HSP90β). Heat shock proteins are important in the cellular stress response and have been shown to play an important role in cardioprotection. Pathway analysis also revealed that more than 30% (19 of 56) of the proteins uniquely oxidized following IPC were involved with cell death pathways. These proteins include Aminoacyl tRNA synthase complex-interacting multifunctional protein 1, Charged multivesicular body protein 5, and Lamin-A/C. These results indicate that the cardioprotective effects of IPC may occur, in part, through the reversible oxidation of key protein targets.

Expression of Glutathione Reductase Splice Variants in Human Tissues

Glutathione is an antioxidant that helps protect cells from reactive oxygen species, such as free radicals and peroxides (Meister 1988; Pompella et al. 2003). Synthesized from the amino acids cysteine, glutamic acid, and glycine, it contains a peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. In healthy cells and tissues, more than 90% of the glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG) (Pastore et al. 2003). Glutathione reductase is a homodimeric flavoprotein that maintains the cellular thiol redox state by catalyzing the reduction of GSSG to GSH, with NADPH as the reducing cofactor (Tutic et al. 1990). Glutathione reductase recycles GSSG to maintain high levels of reduced GSH by catalyzing the reaction GSSG ? NADPH ? H = 2 GSH ? NADP (Chan et al. 1999; Bohme et al. 2000). Glutathione reductase is a key enzyme of the glutathione-dependent antioxidant system, which protects cells against oxidative injury (Kanzok et al. 2001). The human glutathione reductase gene (GSR) maps to p21.1 of the short arm of chromosome 8, spans over 4.9 kb, and consists of 13 exons separated by intronic sequences (Tutic et al. 1990; Kelner and Montoya 2000). The gene’s complete cDNA sequence encodes a polypeptide of 522 amino acids. Mammalian glutathione reductase is localized in the cytosol and in the mitochondria. Mitochondrial GSR lacks the first exon and the corresponding N-terminal leader peptide sequence (Tamura et al. 1996). Many scientists have analyzed GSR mutations (Berkholz et al. 2008; Mavaddat et al. 2009); however, alternative splice variants (ASVs) of GSR have not been reported. Alternative splicing of the primary transcript is a

Treatment of human cancer cells with selenite or tellurite in combination with auranofin enhances cell death due to redox shift

Selenium is an essential trace element incorporated as selenocysteine in 25 human selenoproteins. Among them are thioredoxin reductases (TrxR) and glutathione peroxidases, all central proteins in the regulation of the cellular thiol redox state. In this paper the effects of selenite and tellurite treatment in human cancer cells are reported and compared. Our results show that both selenite and tellurite, at relatively low concentrations, are able to increase the expression of mitochondrial and cytosolic TrxR in cisplatin-sensitive (2008) and -resistant (C13⁎) phenotypes. We further investigated the cellular effects induced by selenite or tellurite in combination with the specific TrxR inhibitor auranofin. Selenite pretreatment induced a dramatic increase in auranofin cytotoxicity in both resistant and sensitive cells. Investigation of TrxR activity and expression levels as well as the cellular redox state demonstrated the involvement of TrxR inhibition and redox changes in selenite and auranofin combined action.

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD + and NADPH/NADP +. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].

The inhibition of glyceraldehyde-3-phosphate dehydrogenase by nitroxyl (HNO)

Nitroxyl (HNO) has received recent and significant interest due to its novel and potentially important pharmacology. However, the chemical/biochemical mechanism(s) responsible for its biological activity remain to be established. Some of the most important biological targets for HNO are thiols and thiol proteins. Consistent with this, it was recently reported that HNO inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with a catalytically important cysteine thiol at its active site. Interestingly, it was reported that intracellular GAPDH inhibition occurred without significantly altering the cellular thiol redox status of glutathione. Herein, the nature of this reaction specificity was examined. HNO is found to irreversibly inhibit GAPDH in a manner that can be protected against by one of its substrates, glyceraldehyde-3-phosphate (G-3-P). These results are consistent with the idea that HNO has the ability to react with and oxidize a variety of intracellular thiols and the ease or facility of cellular re-reduction of the thiol targets can determine the target specificity.

Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system

Reactive oxygen species (ROS) and the cellular thiol redox state are crucial mediators of multiple cell processes like growth, differentiation, and apoptosis. Excessive ROS production or oxidative stress is associated with several diseases, including cardiovascular disorders like ischemia-reperfusion. To prevent ROS-induced disorders, the heart is equipped with effective antioxidant systems. Key players in defense against oxidative stress are members of the thioredoxin-fold family of proteins. Of these, thioredoxins and glutaredoxins maintain a reduced intracellular redox state in mammalian cells by the reduction of protein thiols. The reversible oxidation of Cys-Gly-Pro-Cys or Cys-Pro(Ser)-Tyr-Cys active site cysteine residues is used in reversible electron transport. Thioredoxins and glutaredoxins belong to corresponding systems consisting of NADPH, thioredoxin reductase, and thioredoxin or NADPH, glutathione reductase, glutathione, and glutaredoxin, respectively. Thioredoxin as well as glutaredoxin activities appear to be very important for the progression and severity of several cardiovascular disorders. These proteins function not only as antioxidants, they inhibit or activate apoptotic signaling molecules like apoptosis signal-regulating kinase 1 and Ras or transcription factors like NF-kappaB. Thioredoxin activity is regulated by the endogenous inhibitor thioredoxin-binding protein 2 (TBP-2), indicating an important role of the balance between thioredoxin and TBP-2 levels in cardiovascular diseases. In this review, we will summarize cardioprotective effects of endogenous thioredoxin and glutaredoxin systems as well as the high potential in clinical applications of exogenously applied thioredoxin or glutaredoxin or the induction of endogenous thioredoxin and glutaredoxin systems.

Redox Regulation of 4-Hydroxy-2-nonenal-mediated Endothelial Barrier Dysfunction by Focal Adhesion, Adherens, and Tight Junction Proteins

4-Hydroxy-2-nonenal (4-HNE), one of the major biologically active aldehydes formed during inflammation and oxidative stress, has been implicated in a number of cardiovascular and pulmonary disorders. 4-HNE has been shown to increase vascular endothelial permeability; however, the underlying mechanisms are unclear. Hence, in the current study, we tested our hypothesis that 4-HNE-induced changes in cellular thiol redox status may contribute to modulation of cell signaling pathways that lead to endothelial barrier dysfunction. Exposure of bovine lung microvascular endothelial cells (BLMVECs) to 4-HNE induced reactive oxygen species generation, depleted intracellular glutathione, and altered cell-cell adhesion as measured by transendothelial electrical resistance. Pretreatment of BLM-VECs with thiol protectants, N-acetylcysteine and mercaptopropionyl glycine, attenuated 4-HNE-induced decrease in transendothelial electrical resistance, reactive oxygen species generation, Michael protein adduct formation, protein tyrosine phosphorylation, activation of ERK, JNK, and p38 MAPK, and actin cytoskeletal rearrangement. Treatment of BLMVECs with 4-HNE resulted in the redistribution of FAK, paxillin, VE-cadherin, beta-catenin, and ZO-1, and intercellular gap formation. Western blot analyses confirmed the formation of 4-HNE-derived Michael adducts with the focal adhesion and adherens junction proteins. Also, 4-HNE decreased tyrosine phosphorylation of FAK without affecting total cellular FAK contents, suggesting the modification of integrins, which are natural FAK receptors. 4-HNE caused a decrease in the surface integrin in a time-dependent manner without altering total alpha5 and beta3 integrins. These results, for the first time, revealed that 4-HNE in redox-dependent fashion affected endothelial cell permeability by modulating cell-cell adhesion through focal adhesion, adherens, and tight junction proteins as well as integrin signal transduction that may lead dramatic alteration in endothelial cell barrier dysfunction during heart infarction, brain stroke, and lung diseases.

MRP2-Mediated Reduced Glutathione Transport in Protection Against Oxidant Stress

Although the primary physiological role of multidrug resistance-associated protein 2 (MRP2) appears to be the export of a wide variety of organic molecules from the cell, including glutathione disulfide (GSSG) and glutathione S-conjugates, there is now strong evidence that MRP2 also is able to export reduced glutathione (GSH) from the cell. The ability to export both GSH and GSSG would allow MRP2 to contribute directly to the regulation of the cellular thiol-redox status and therefore to the protection against oxidative stress. In addition, because GSH export is intimately linked to the other biological functions of this tripeptide, MRP2 may contribute to the regulation of these other cellular functions. Evidence for a role of MRP2 in GSH export comes from several studies, including (a) studies in MRP2-deficient rats demonstrating that these animals are unable to transport GSH into bile; (b) in vivo and in vitro studies demonstrating that cellular GSH export rates correlate with MRP2 expression levels; (c) comparative studies with the yeast orthologue of MRP2, Ycflp, showing that the yeast orthologue functions as an ATP-driven GSH transporter; (d) direct measurement of ATP-dependent GSH transport in rat liver canalicular membrane vesicles; and (e) studies with MRP1, the functional orthologue of MRP2, demonstrating that MRP1 is able to transport GSH. Taken together these data indicate that GSH is a substrate for MRP2; however, neither the precise biochemical mechanism of transport nor the quantitative significance of this process is yet known.