ER Oxidative Stress Promotes Glutathione-Dependent Oxidation of Collagen-1A1 and Promotes Lung Fibroblast Activation.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in collagen 1A1 S-glutathionylation (COL1A1-SSG) in lung tissues from IPF subjects compared to control subjects in association with increases in ER oxidoreductin 1 (ERO1A) and enhanced oxidation of ER-localized peroxiredoxin 4 (PRDX4) reflecting an increased oxidative environment of the endoplasmic reticulum (ER). Human lung fibroblasts exposed to transforming growth factor beta 1 (TGFB1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 levels and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared to COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly, and increased expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling due to increased resistance to collagenase-mediated degradation and fibroblast activation.

Glutathione S-transferase: A versatile and dynamic enzyme.

The dynamic and versatile group of enzymes referred to as glutathione S-transferases (GSTs) play diverse roles in cellular detoxification, safeguarding hosts from oxidative damage, and performing various other functions. This review explores different classes of GST, existence of polymorphisms in GST, functions of GST and utilizations of GST inhibitors in treatment of human diseases. The study indicates that the cytosolic GSTs, mitochondrial GSTs, microsomal GSTs, and bacterial proteins that provide resistance to Fosfomycin are the major classes. Given a GST, variation in its expression and function among individuals is due to the presence of polymorphic alleles that encode it. Genetic polymorphism might result in the modification of GST activity, thereby increasing individuals' vulnerability to harmful chemical compounds. GSTs have been demonstrated to play a regulatory function in cellular signalling pathways through kinases, S-Glutathionylation, and in detoxification processes. Various applications of bacterial GSTs and their potential roles in plants were examined. Targeting GSTs, especially GSTP1-1, is considered a potential therapeutic strategy for treating cancer and diseases linked to abnormal cell proliferation. Their role in cancer cell growth, differentiation, and resistance to anticancer agents makes them promising targets for drug development, offering prospects for the future.

Thiol oxidative stress-dependent degradation of transglutaminase2 via protein S-glutathionylation sensitizes 5-fluorouracil therapy in 5-fluorouracil-resistant colorectal cancer cells.

5-Fluorouracil (5-Fu) is a first-line drug for colorectal cancer (CRC) therapy. However, the development of 5-Fu resistance limits its chemotherapeutic effectiveness and often leads to poor prognoses of CRC. Transglutaminase 2 (TGM2), a member of the transglutaminase family, is considered to be associated with chemoresistance through apoptotic prevention in various cancers including CRC. TGM2 was found to be overexpressed in two 5-Fu-resistant CRC cell lines and down-regulated by increased thiol oxidative stress induced by inhibition of glutathione reductase (GR). The present study aimed to explore the role of TGM2 in 5-Fu-resistant CRC and the mechanism of action by which the elevated thiol oxidative stress down-regulates TGM2 protein level. The results revealed that 5-Fu-resistance induced by overexpression of TGM2 in CRC cells was reversed through up-regulation of thiol oxidative stress. Knockdown of TGM2 increased the chemosensitivity of CRC cells to 5-Fu. Thiol oxidative stress potentially enhanced the therapeutic effect of 5-Fu in the resistant CRC cells by promotion of 5-Fu-induced apoptosis through down-regulation of TGM2. The elevated thiol oxidative stress increased the S-glutathionylation of TGM2 and led to proteasomal degradation of TGM2. Furthermore, Cys193 was identified as the S-glutathionylation site in TGM2, and its mutation resulted in thiol oxidative stress-mediated CRC cell apoptotic resistance. TGM2-induced EMT was also suppressed by the elevated thiol oxidative stress. A xenograft tumor model confirmed the effect of thiol oxidative stress in the reversal of 5-Fu resistance in CRC cells in vivo. TGM2 protein expression level was found to be significantly higher in human CRC specimens than in non-cancerous colorectal tissues. Taken together, the present data suggest an important role of TGM2 in 5-Fu resistance in CRC cells. Up-regulation of thiol oxidative stress could be a potential therapeutic approach for treating 5-Fu-resistant CRC and TGM2 may serve as a potential therapeutic target of thiol oxidative stress.

Glutathione-Dependent Pathways in Cancer Cells.

The most abundant tripeptide-glutathione (GSH)-and the major GSH-related enzymes-glutathione peroxidases (GPxs) and glutathione S-transferases (GSTs)-are highly significant in the regulation of tumor cell viability, initiation of tumor development, its progression, and drug resistance. The high level of GSH synthesis in different cancer types depends not only on the increasing expression of the key enzymes of the γ-glutamyl cycle but also on the changes in transport velocity of its precursor amino acids. The ability of GPxs to reduce hydroperoxides is used for cellular viability, and each member of the GPx family has a different mechanism of action and site for maintaining redox balance. GSTs not only catalyze the conjugation of GSH to electrophilic substances and the reduction of organic hydroperoxides but also take part in the regulation of cellular signaling pathways. By catalyzing the S-glutathionylation of key target proteins, GSTs are involved in the regulation of major cellular processes, including metabolism (e.g., glycolysis and the PPP), signal transduction, transcription regulation, and the development of resistance to anticancer drugs. In this review, recent findings in GSH synthesis, the roles and functions of GPxs, and GST isoforms in cancer development are discussed, along with the search for GST and GPx inhibitors for cancer treatment.

New insights into posttranslational modifications of proteins during bull sperm capacitation.

BACKGROUND: Due to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation. METHODS: Cryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach). RESULTS: Zona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated. CONCLUSIONS: Our research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.

Involvement of HIF1 stabilization and VEGF signaling modulated by Grx-1 in murine model of bronchopulmonary dysplasia.

Hypoxia inducible factor (HIF)-1α could be stabilized by Grx1 deletion, which is implicated critical in the pathogenesis of bronchopulmonary dysplasia (BPD). Until now, the stabilization of HIF-1α by glutathionylation to regulate the pulmonary microcirculation in BPD is not well addressed. In this study, we investigated whether the HIF-1α stabilization modulated by Grx1 ablation could ameliorate the pathological changes in the mouse model of BPD, including angiogenesis and alveolar formation. We found that depletion of Grx1 increased levels of GSH-protein adducts, which was associated with the improvement in the numbers of alveoli, the capillary density in the pulmonary microcirculation and the survival rate in the littermates with hyperoxic exposure. Grx1 ablation could promote HIF-1α glutathionylation by increasing GSH adducts to stabilize HIF-1α and to induce VEGF-A production in the lung tissue. The above phenotype of capillary density and VEGF-A production was removed by the pharmacological administration of YC-1, the HIF-1α inhibitor, suggesting the HIF-1α dependent manner for pulmonary microcirculatory perfusion. These data indicate that HIF-1α stabilization plays an critical role in modification pulmonary microcirculatory perfusion, which is associated with the pathological damage under hyperoxic conditions, suggesting that targeting with HIF-1α stabilization should be a potential clinical and therapeutic strategy for BPD treatment.

S-Glutathionylation and S-Nitrosylation as Modulators of Redox-Dependent Processes in Cancer Cell.

Development of oxidative/nitrosative stress associated with the activation of oncogenic pathways results from the increase in the generation of reactive oxygen and nitrogen species (ROS/RNS) in tumor cells, where they can have a dual effect. At high concentrations, ROS/RNS cause cell death and limit tumor growth at certain phases of its development, while their low amounts promote oxidative/nitrosative modifications of key redox-dependent residues in regulatory proteins. The reversibility of such modifications as S-glutathionylation and S-nitrosylation that proceed through the electrophilic attack of ROS/RNS on nucleophilic Cys residues ensures the redox-dependent switch in the activity of signaling proteins, as well as the ability of these compounds to control cell proliferation and programmed cell death. The content of S-glutathionylated and S-nitrosylated proteins is controlled by the balance between S-glutathionylation/deglutathionylation and S-nitrosylation/denitrosylation, respectively, and depends on the cellular redox status. The extent of S-glutathionylation and S-nitrosylation of protein targets and their ratio largely determine the status and direction of signaling pathways in cancer cells. The review discusses the features of S-glutathionylation and S-nitrosylation reactions and systems that control them in cancer cells, as well as their relationship with redox-dependent processes and tumor growth.

Robust AMBER Force Field Parameters for Glutathionylated Cysteines.

S-glutathionylation is an oxidative post-translational modification, which is involved in the regulation of many cell signaling pathways. Increasing amounts of studies show that it is crucial in cell homeostasis and deregulated in several pathologies. However, the effect of S-glutathionylation on proteins' structure and activity is poorly understood, and a drastic lack of structural information at the atomic scale remains. Studies based on the use of molecular dynamics simulations, which can provide important information about modification-induced modulation of proteins' structure and function, are also sparse, and there is no benchmarked force field parameters for this modified cysteine. In this contribution, we provide robust AMBER parameters for S-glutathionylation, which we tested extensively against experimental data through a total of 33 µs molecular dynamics simulations. We show that our parameter set efficiently describes the global and local structural properties of S-glutathionylated proteins. These data provide the community with an important tool to foster new investigations into the effect of S-glutathionylation on protein dynamics and function, in a common effort to unravel the structural mechanisms underlying its critical role in cellular processes.

Thiol-based Oxidative Posttranslational Modifications (OxiPTMs) of Plant Proteins.

The thiol group of cysteine (Cys) residues, often present in the active center of the protein, is of particular importance to protein function, which is significantly determined by the redox state of a protein's environment. Our knowledge of different thiol-based oxidative posttranslational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last 10 years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation and S-acylation. The role of each oxiPTM depends on the redox cellular state, which in turn depends on cellular homeostasis under either optimal or stressful conditions. Under such conditions, the metabolism of molecules such as glutathione, NADPH (reduced nicotinamide adenine dinucleotide phosphate), nitric oxide, hydrogen sulfide and hydrogen peroxide can be altered, exacerbated and, consequently, outside the cell's control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.

Measurement of S-glutathionylated proteins by HPLC.

S-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks -SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models.

NF-κB inactivation attenuates the M1 macrophage polarization in experimental necrotizing enterocolitis by glutaredoxin-1 deficiency.

The pathogenesis of necrotizing enterocolitis (NEC) is severe inflammatory injury in preterm infants, which resulted from macrophage polarization. Nuclear factor-κB (NF-κB) is implicated to be involved in macrophage polarization. We here evaluated the essential role of NF-κB in macrophage polarization in NEC in human samples from neonates with NEC and the mouse experimental NEC model. Enhanced intestinal macrophage (IM) infiltration was presented in human neonates with NEC, the majority of which were M1 macrophages. Meanwhile, NF-κB was activated in the IMs in human NEC samples. NF-κB inhibition by BAY promoted the M1 to M2 macrophage polarization. Furthermore, glutaredoxin-1 (Grx1) deficiency promoted M2 polarization via NF-κB inactivation from the lipopolysaccharide-induced proinflammatory macrophages. The IMs isolated from Grx1- / - mice presented with decreases in total numbers and less macrophage differentiation. Grx1- / - derived IM were effective in the increased survival in experimental NEC through inflammation blocking. Our study provides evidence that NF-κB inactivation by Grx1 depletion contributed to the alleviation of NEC via inhibiting M1 macrophage polarization. The modulation to alternative macrophages in the intestines may provide a promising benefits for NEC treatment.

Glutathionylation primes soluble glyceraldehyde-3-phosphate dehydrogenase for late collapse into insoluble aggregates.

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

Dysregulation of Pyruvate Kinase M2 Promotes Inflammation in a Mouse Model of Obese Allergic Asthma.

Obesity is a risk factor for the development of asthma and represents a difficult-to-treat disease phenotype. Aerobic glycolysis is emerging as a key feature of asthma, and changes in glucose metabolism are linked to leukocyte activation and adaptation to oxidative stress. Dysregulation of PKM2 (pyruvate kinase M2), the enzyme that catalyzes the last step of glycolysis, contributes to house dust mite (HDM)-induced airway inflammation and remodeling in lean mice. It remains unclear whether glycolytic reprogramming and dysregulation of PKM2 also contribute to obese asthma. The goal of the present study was to elucidate the functional role of PKM2 in a murine model of obese allergic asthma. We evaluated the small molecule activator of PKM2, TEPP46, and assessed the role of PKM2 using conditional ablation of the Pkm2 allele from airway epithelial cells. In obese C57BL/6NJ mice, parameters indicative of glycolytic reprogramming remained unchanged in the absence of stimulation with HDM. Obese mice that were subjected to HDM showed evidence of glycolytic reprogramming, and treatment with TEPP46 diminished airway inflammation, whereas parameters of airway remodeling were unaffected. Epithelial ablation of Pkm2 decreased central airway resistance in both lean and obese allergic mice in addition to decreasing inflammatory cytokines in the lung tissue. Lastly, we highlight a novel role for PKM2 in the regulation of glutathione-dependent protein oxidation in the lung tissue of obese allergic mice via a putative IFN-γ-glutaredoxin1 pathway. Overall, targeting metabolism and protein oxidation may be a novel treatment strategy for obese allergic asthma.

Enhanced epithelial sodium channel activity in neonatal Scnn1b mouse lung attenuates high oxygen-induced lung injury.

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen ([Formula: see text])-induced lung injury. Exposure to high [Formula: see text] from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG (400 µM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 µM) or amiloride (1 µM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10 µM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high [Formula: see text] exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.

Plasticity in plastid redox networks: evolution of glutathione-dependent redox cascades and glutathionylation sites.

BACKGROUND: Flexibility of plant metabolism is supported by redox regulation of enzymes via posttranslational modification of cysteine residues, especially in plastids. Here, the redox states of cysteine residues are partly coupled to the thioredoxin system and partly to the glutathione pool for reduction. Moreover, several plastid enzymes involved in reactive oxygen species (ROS) scavenging and damage repair draw electrons from glutathione. In addition, cysteine residues can be post-translationally modified by forming a mixed disulfide with glutathione (S-glutathionylation), which protects thiol groups from further oxidation and can influence protein activity. However, the evolution of the plastid glutathione-dependent redox network in land plants and the conservation of cysteine residues undergoing S-glutathionylation is largely unclear. RESULTS: We analysed the genomes of nine representative model species from streptophyte algae to angiosperms and found that the antioxidant enzymes and redox proteins belonging to the plastid glutathione-dependent redox network are largely conserved, except for lambda- and the closely related iota-glutathione S-transferases. Focussing on glutathione-dependent redox modifications, we screened the literature for target thiols of S-glutathionylation, and found that 151 plastid proteins have been identified as glutathionylation targets, while the exact cysteine residue is only known for 17% (26 proteins), with one or multiple sites per protein, resulting in 37 known S-glutathionylation sites for plastids. However, 38% (14) of the known sites were completely conserved in model species from green algae to flowering plants, with 22% (8) on non-catalytic cysteines. Variable conservation of the remaining sites indicates independent gains and losses of cysteines at the same position during land plant evolution. CONCLUSIONS: We conclude that the glutathione-dependent redox network in plastids is highly conserved in streptophytes with some variability in scavenging and damage repair enzymes. Our analysis of cysteine conservation suggests that S-glutathionylation in plastids plays an important and yet under-investigated role in redox regulation and stress response.

Role of protein S-Glutathionylation in cancer progression and development of resistance to anti-cancer drugs.

The survival, functioning and proliferation of mammalian cells are highly dependent on the cellular response and adaptation to changes in their redox environment. Cancer cells often live in an altered redox environment due to aberrant neo-vasculature, metabolic reprogramming and dysregulated proliferation. Thus, redox adaptations are critical for their survival. Glutathione plays an essential role in maintaining redox homeostasis inside the cells by binding to redox-sensitive cysteine residues in proteins by a process called S-glutathionylation. S-Glutathionylation not only protects the labile cysteine residues from oxidation, but also serves as a sensor of redox status, and acts as a signal for stimulation of downstream processes and adaptive responses to ensure redox equilibrium. The present review aims to provide an updated overview of the role of the unique redox adaptations during carcinogenesis and cancer progression, focusing on their dependence on S-glutathionylation of specific redox-sensitive proteins involved in a wide range of processes including signalling, transcription, structural maintenance, mitochondrial functions, apoptosis and protein recycling. We also provide insights into the role of S-glutathionylation in the development of resistance to chemotherapy. Finally, we provide a strong rationale for the development of redox targeting drugs for treatment of refractory/resistant cancers.

A Phase 1 dose-escalation study of disulfiram and copper gluconate in patients with advanced solid tumors involving the liver using S-glutathionylation as a biomarker.

BACKGROUND: Disulfiram and metals inactivate key oncoproteins resulting in anti-neoplastic activity. The goal of this study was to determine the maximum tolerated dose of copper when administered with disulfiram in patients with advanced solid tumors and liver involvement. METHODS: Disulfiram 250 mg was administered daily in 28-day cycles. Four doses of copper gluconate were tested (2, 4, 6, and 8 mg of elemental copper) in a standard 3 + 3 dose escalation design. Patients were evaluated for dose limiting toxicities and response. Protein S-glutathionylation was evaluated as a pharmacodynamic marker. RESULTS: Twenty-one patients were enrolled and 16 patients were evaluable for dose limiting toxicities. Among the 21 patients, there was a median of 4 lines of prior chemotherapy. Five Grade 3 toxicities were observed (anorexia, elevated aspartate aminotransferase or AST, elevated alkaline phosphatase, fever, and fatigue). Response data was available for 15 patients. Four patients had stable disease with the longest duration of disease control being 116 days. The median duration of treatment for evaluable patients was 55 days (range 28-124). Reasons for discontinuation included functional decline, disease progression, and disease-associated death. Increased S-glutathionylation of serum proteins was observed with treatment. CONCLUSION: Disulfiram 250 mg daily with copper gluconate (8 mg of elemental copper) was well-tolerated in patients with solid tumors involving the liver and was not associated with dose limiting toxicities. While temporary disease stabilization was noted in some patients, no objective responses were observed. Treatment was associated with an increase in S-glutathionylation suggesting that this combination could exert a suppressive effect on cellular growth and protein function. TRIAL REGISTRATION: NCT00742911 , first posted 28/08/2008.

Protein S-glutathionylation stimulate adipogenesis by stabilizing C/EBPß in 3T3L1 cells.

Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) ß. Protein S-glutathionylation decreased the interaction of C/EBPß and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier E3 ligase that facilitates C/EBPß degradation. Experiments with truncated mutant C/EBPß demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPß. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPß. The C201S, C296S double-mutant C/EBPß prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPß, stabilizing and increasing C/EBPß protein levels.

Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.

Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling.

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2-C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.