Glutathionylation Research Articles

Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function

BackgroundThe interplay between oxidative stress and other signaling pathways in the contractile machinery regulation during cardiac stress and its consequences on cardiac function remains poorly understood. We evaluated the effect of the crosstalk between β-adrenergic and redox signaling on post-translational modifications of sarcomeric regulatory proteins, Myosin Binding Protein-C (MyBP-C) and Troponin I (TnI). Methods and resultsWe mimicked in vitro high level of physiological cardiac stress by forcing rat hearts to produce high levels of oxidized glutathione. This led to MyBP-C S-glutathionylation associated with lower protein kinase A (PKA) dependent phosphorylations of MyBP-C and TnI, increased myofilament Ca2+ sensitivity, and decreased systolic and diastolic properties of the isolated perfused heart. Moderate physiological cardiac stress achieved in vivo with a single 35 min exercise (Low stress induced by exercise, LSE) increased TnI and cMyBP-C phosphorylations and improved cardiac function in vivo (echocardiography) and ex-vivo (isolated perfused heart). High stress induced by exercise (HSE) altered strongly oxidative stress markers and phosphorylations were unchanged despite increased PKA activity. HSE led to in vivo intrinsic cardiac dysfunction associated with myofilament Ca2+ sensitivity defects. To limit protein S-glutathionylation after HSE, we treated rats with N-acetylcysteine (NAC). NAC restored the ability of PKA to modulate myofilament Ca2+ sensitivity and prevented cardiac dysfunction observed in HSE animals. ConclusionUnder cardiac stress, adrenergic and oxidative signaling pathways work in concert to alter myofilament properties and are key regulators of cardiac function.

Glutathionylation: a regulatory role of glutathione in physiological processes.

Glutathione (γ-glutamyl-cysteinyl-glycine) is an intracellular thiol molecule and a potent antioxidant that participates in the toxic metabolism phase II biotransformation of xenobiotics. It can bind to a variety of proteins in a process known as glutathionylation. Protein glutathionylation is now recognised as one of important posttranslational regulatory mechanisms in cell and tissue physiology. Direct and indirect regulatory roles in physiological processes include glutathionylation of major transcriptional factors, eicosanoids, cytokines, and nitric oxide (NO). This review looks into these regulatory mechanisms through examples of glutathione regulation in apoptosis, vascularisation, metabolic processes, mitochondrial integrity, immune system, and neural physiology. The focus is on the physiological roles of glutathione beyond biotransformational metabolism.

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities.

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100–500 μM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80–90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Hydrogen sulfide increases glutathione biosynthesis, and glucose uptake and utilisation in C2C12 mouse myotubes

Diabetic patients have lower blood concentrations of hydrogen sulfide (H2S), L-cysteine (LC), and glutathione (GSH). Using C2C12 mouse myotubes as a model, this study investigates the hypothesis that the beneficial effects of LC supplementation are mediated by upregulation of the H2S status under diabetic conditions. Results show that exogenous administration of sodium hydrosulfide (NaHS, 10 or 20 µM; 6 hours), a H2S donor, significantly (p < .05) upregulates the gene expression of cystathionine-γ-lyase (CSE), LC transporter (Slc7a11/xCT), and the genes involved in GSH biosynthesis. Additionally, it reduces homocysteine (HCys), reactive oxygen species (ROS) production, and enhances cellular LC, H2S, and glucose uptake and utilisation in myoblasts. The use of CSE siRNA to induce deficient endogenous H2S production causes an increase in H2O2, ROS, HCys levels, and downregulation of GSH biosynthesis pathway enzymes. In additional, CSE knockdown downregulates glucose transporter type 4 (GLUT4) and gene expression of its key transcription factors, and reduces glucose uptake in C2C12 myotubes. CSE knockdown cells showed specific increases in the protein S-glutathionylation of LC transporter and GLUT4 along with increased total protein S-glutathionylation. Taken together, evidence from this study provides molecular insights into the importance of the CSE/H2S system in maintaining the cellular glutathione and glucose homeostasis in C2C12 myotubes.

Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

Cystathionine β-synthase-deficient homocystinuria (HCU) is a poorly understood, life-threatening inborn error of sulfur metabolism. Analysis of hepatic glutathione (GSH) metabolism in a mouse model of HCU demonstrated significant depletion of cysteine, GSH, and GSH disulfide independent of the block in trans-sulfuration compared with wild-type controls. HCU induced the expression of the catalytic and regulatory subunits of γ-glutamyl ligase, GSH synthase (GS), γ-glutamyl transpeptidase 1, 5-oxoprolinase (OPLAH), and the GSH-dependent methylglyoxal detoxification enzyme, glyoxalase-1. Multiple components of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant-response regulatory axis were induced without any detectable activation of Nrf2. Metabolomic analysis revealed the accumulation of multiple γ-glutamyl amino acids and that plasma ophthalmate levels could serve as a noninvasive marker for hepatic redox stress. Neither cysteine, nor betaine treatment was able to reverse the observed enzyme inductions. Taurine treatment normalized the expression levels of γ-glutamyl ligase C/M, GS, OPLAH, and glyoxalase-1, and reversed HCU-induced deficits in protein glutathionylation by acting to double GSH levels relative to controls. Collectively, our data indicate that the perturbation of the γ-glutamyl cycle could contribute to multiple sequelae in HCU and that taurine has significant therapeutic potential for both HCU and other diseases for which GSH depletion is a critical pathogenic factor.-Maclean, K. N., Jiang, H., Aivazidis, S., Kim, E., Shearn, C. T., Harris, P. S., Petersen, D. R., Allen, R. H., Stabler, S. P., Roede, J. R. Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

Proteomic analysis of human glutathione transferase omega (hGSTO1) stable transfection in a 6-hydroxydopamine-induced neuronal cells.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. The disease is associated with dopaminergic neuron losses in the substantia nigra area of the brain and the formation of cytoplasmic inclusion bodies. Human glutathione transferase omega 1 (hGSTO1) appears to have a role in modulating stress response. The study was aimed to elucidate differentially expressed proteins caused by oxidative stress induced by 6-hydroxydopamine (6-OHDA). Human neuronal cells SH-SY5Y overexpressing hGSTO1 were used to investigate protein glutathionylation and the modulation of cellular protein expression. Therefore SH-SY5Y/hGSTO1 and SH-SY5Y/control lysate proteins were separated by 2D-gel electrophoresis compared with untreated conditions in both standard and non-reducing conditions. In standard conditions, the analysis of protein profiles demonstrated 25 differentially expressed spots and 10 spots were chosen for further protein identification by LC-MS analysis. Several proteins were later identified as vimentin, galectin-1, high mobility group protein B2, clathrin, tropomyosin, heterogenous nuclear ribonucleoprotein and peroxiredoxin-2. Search Tool for Interactions of Chemicals (STITCH) analysis suggested that oxidative stress induced by 6-OHDA involved carbohydrate metabolism in SH-SY5Y via a lactose metabolic pathway. Our results raise the possibility that hGSTO1 modulates the functions of many proteins that play a role in the degenerative cell response of a Parkinson's model.

Ascorbyl stearate and ionizing radiation potentiate apoptosis through intracellular thiols and oxidative stress in murine T lymphoma cells

Ascorbyl stearate (Asc-s) is a derivative of ascorbic acid with better anti-tumour efficacy compared to its parent compound ascorbic acid. In this study, we have examined radio-sensitizing effect of Asc-s in murine T cell lymphoma (EL4) cells at 4 Gy. Asc-s and radiation treatment reduced cell proliferation, induced apoptosis in a dose dependent manner by arresting the cells at S/G2-M phase of cell cycle. It also decreased the frequency of cancer stem cells per se, with significantly higher decrease in combination with radiation treatment./Further, Asc-s and radiation treatment increased the level of reactive oxygen species (ROS), drop in mitochondrial membrane potential (MMP) and increased caspase-3 activity resulting in apoptosis of EL4 cells. Further it also significantly decreased GSH/GSSG ratio due to binding of Asc-s with thiols. The increase in oxidative stress induced by Asc-s and radiation treatment was abrogated by thiol antioxidants in EL4 cells. Interestingly, this redox modulation triggered significant increase in protein glutathionylation in a time dependent manner. Asc-s treatment resulted in glutathionylation of IKK, p50-NF-kB and mutated p53, thereby inhibiting cancer progression during oxidative stress. Asc-s quenches GSH ensuing Asc-s + GSH adduct thereby further modulating GSH/GSSG ratio as evident from HPLC and docking studies. The anti-tumour effect of Asc-s along with radiation was studied by injecting EL4 cells in synegenicC57/BL6 male mice. Intraperitoneal injection of Asc-s followed by radiation exposure at 4 Gy to the tumour bearing mice resulted in radio-sensitization which is evident from significant regression of tumour as evident from tumour burden index. The survival study supports the data that Asc-s pre-treatment enhances radio-sensitization in murine lymphoma. Our data, suggest that Asc-s and ionizing radiation induced cell cycle arrest and apoptosis by perturbing redox balance through irreversible complexes of thiols with Asc-s, disturbed mitochondrial membrane permeability and activation of caspase-3 in EL4 cells.

Characterization of the impact of glutaredoxin-2 (GRX2) deficiency on superoxide/hydrogen peroxide release from cardiac and liver mitochondria

Mitochondria are critical sources of hydrogen peroxide (H2O2), an important secondary messenger in mammalian cells. Recent work has shown that O2•-/H2O2 emission from individual sites of production in mitochondria is regulated by protein S-glutathionylation. Here, we conducted the first examination of O2•-/H2O2 release rates from cardiac and liver mitochondria isolated from mice deficient for glutaredoxin-2 (GRX2), a matrix-associated thiol oxidoreductase that facilitates the S-glutathionylation and deglutathionylation of proteins. Liver mitochondria isolated from mice heterozygous (GRX2+/-) and homozygous (GRX2-/-) for glutaredoxin-2 displayed a significant decrease in O2•-/H2O2 release when oxidizing pyruvate or 2-oxoglutarate. The genetic deletion of the Grx2 gene was associated with increased protein expression of pyruvate dehydrogenase (PDH) but not 2-oxoglutarate dehydrogenase (OGDH). By contrast, O2•-/H2O2 production was augmented in cardiac mitochondria from GRX2+/- and GRX2-/- mice metabolizing pyruvate or 2-oxoglutarate which was associated with decreased PDH and OGDH protein levels. ROS production was augmented in liver and cardiac mitochondria metabolizing succinate. Inhibitor studies revealed that OGDH and Complex III served as high capacity ROS release sites in liver mitochondria. By contrast, Complex I and Complex III were found to be the chief O2•-/H2O2 emitters in cardiac mitochondria. These findings identify an essential role for GRX2 in regulating O2•-/H2O2 release from mitochondria in liver and cardiac tissue. Our results demonstrate that the GRX2-mediated regulation of O2•-/H2O2 release through the S-glutathionylation of mitochondrial proteins may play an integral role in controlling cellular ROS signaling.

Allosteric Control of a Plant Receptor Kinase through S-Glutathionylation

Growing evidence supports the importance of protein S-glutathionylation as a regulatory post-translational modification with functional consequences for proteins. Discoveries of redox-state-dependent protein kinase S-glutathionylation have fueled discussion of redox-sensitive signaling. Following previously published experimental evidence for S-glutathionylation induced deactivation of the Arabidopsis thaliana kinase BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1), we investigated the consequences of S-glutathionylation on the equilibrium conformational ensemble of BAK1 using all-atom molecular dynamics simulations. We found that glutathionylation of C408 allosterically destabilizes the active-like state of BAK1 and stabilizes an inactive conformation known to recur in protein kinases. Glutathionylation of C408 also has structural consequences throughout the BAK1 kinase domain, whereas glutathionylation of C353 in the N-lobe and C374 near the ATP-binding site have few notable effects on BAK1 compared with the unmodified protein. Our results suggest an allosteric mechanism for inhibition of BAK1 by C408 S-glutathionylation, and more generally, support the notion of protein kinase S-glutathionylation as a means of redox signaling in plant cells.

Abstract 21314: Glutaredoxin-1: An Endothelial Protector in Hyperlipidemia

Background: Hypercholesterolemia (HC) dominates the risk factors for atherosclerosis. Sustained oxidative stress is a central mechanism driving endothelial dysfunction (ED) in HC. But the underlying redox mechanism remains unclear. Protein S-glutathionylation, a reversible oxidative modification of cysteine residues is tightly controlled by a specific de-glutathionylation enzyme: glutaredoxin-1 (Glrx-1), which thereby plays a critical role in redox regulation of cellular function. Methods and Results: The protein levels of Glrx1 in aorta and lung tissues are inversely correlated with circulating cholesterol in WT and ApoE -/- mice. Overexpression of Glrx1 in ApoE -/- mice attenuates HC-induced ED as measured by flow-mediated dilation of femoral artery using a new vascular imaging method with optical coherence tomography technique, and impaired eNOS function as evidenced by increased eNOS monomer to dimer ratio and suppressed phosphorylation of vasodilator-stimulated phosphoprotein in the aorta. In HAECs oxidized LDL promotes downregulation of Glrx1 without altering its mRNA level. The consequent elevated PrS-SG inactivates Akt/eNOS and stimulates degradation of Rac1, a key regulator of eNOS. All of these deteriorate effects of oxLDL are prevented by adenoviral overexpression of Glrx1. Conclusions: HC-induced downregulation of Glrx1 promotes ED by redox inactivation of eNOS and its upstream regulators: Akt1 and Rac1.

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.

Immunological and Oxidative Responses Caused by Cellulose Nanocrystal (CNCs) Derivatives Designed for Biomedical Applications, in Human and Mouse Cells

Cellulose nanocrystals (CNCs) are utilized for a wide variety of important applications from nanofillers to potential drug delivery. CNCs have a fiber-like structure that can be modified with different moieties to change the physicochemical characteristics and proper suit to biomedical applications. However, these modifications also can lead to undesirable effects, such as robust immune response. Previously we demonstrated that cationic CNCs evoke immunological responses through NLRP3 inflammasome/IL-1β inflammatory pathway, and this effect is associated, at least in part, with changes in mitochondrial ROS. In this work we expend our studies by analyzing biological responses of series of newly modified CNCs designed for different biomedical purposes. We modified CNCs with β-cyclodextrin (CNCs-β-CD), with N-(2-aminoethylmethacrylamide) (CNCs-AEMA1), and with N-isopropylacrylamide (CNCs-NIPAAm), designed for delivery of lipophilic, anionic and thermo-responsive drugs, respectively. We also synthesized CNCs-grafted with the monomer 2-methacryloyloxyethylphosphorylcholine (MPC), which can mimic the phospholipid polar groups within cell membranes, making it suitable for coating cardiovascular stents. We evaluated the immunological response of these nanomaterials by assessing NLRP3 inflammasome signaling pathway (ELISA and western blotting) and oxidative stress by analyzing mitochondrial ROS and protein S-glutathionylation (PSSG) in mouse (J774A.1) and human (THP-1) myeloid cells as well in human peripheral mononuclear blood cells (PMBCs). In summary, we found that the both CNCs-β-CD and CNCs-MPC had a tendency to decrease IL-1β secretion in LPS-stimulated cell lines and also in non-stimulated PMBCs. Mitochondrial ROS was elevated with all nanomaterial in non-stimulated J774A.1 cells. LPS-stimulated J774A.1 cells treated with CNCs-MPC mitochondrial ROS and PSSG were both decreased. Overall our results indicated that different modification on the surface of CNCs indeed affect their biomedical properties and also can cause undesirable effects. Therefore, further and more in depth interdisciplinary studies are necessary to fully understand and explore the potential biomedical applicability of CNCs.

145 - GSTP1-catalyzed PKM2 S-Glutathionylation Regulates Glycolysis in Non-small Cell Lung Cancer and is Attenuated with a Clinically Relevant inhibitor of Glutathione-S-transferase P

Rationale Lung cancer is the most lethal form of any of the common cancers, resulting in over 160,000 deaths in the US every year. Unfortunately, the 5-year survival rate has remained almost unchanged over the past 3 decades. Glutathione S-transferase P1 (GSTP1) is expressed significantly higher in lung tumor tissue from NSCLC compared to matched non-tumorous lung tissue. Originally this was attributed to its role in phase II drug metabolism. However, GSTP1 was recently described as a catalyst of protein S-glutathionylation (PSSG), a reversible cysteine oxidation and post-translational modification pointing towards a different role of GSTP in lung cancer. The goal of this study is to determine whether GSTP1 expression and subsequent enhanced PSSG are important in tumorigenesis. Methods In a tissue micro array of 106 human NSCLC specimens together with adjacent healthy tissue GSTP1 expression and overall PSSG was evaluated. The most common “driver” mutation in NSCLC occurs in the KRAS gene. To study NSCLC in mice we established an inducible KRASG12D-driven lung adenocarcinoma model that enables lung-epithelial specific expression of mutant KRAS. Alternatively, primary Mouse Tracheal Epithelial (MTE) Cells derived from KRASG12D mice were exposed to adeno-Cre to activate mutant KRAS. Results Assessment of 106 NSCLC TMAs revealed that 47% were completely negative and 41% were highly positive for GSTP1, and the lung cancers expressing GSTP1 had high PSSG reactivity. GSTP1 expression and PSSG were associated with a worse prognosis. In a mouse model of NSCLC, activation of mutant KRAS resulted in an increase of GSTP1 protein and PSSG in the lung. Further, lactate content and glycolysis regulators were significantly upregulated in lungs following KRASG12D activation. Following pharmaceutical inhibition of GSTP with TLK117, a phase II tested compound, KRAS-induced increases in PSSG and lactate were strongly decreased in lungs compared to vehicle- treated animals. KRASG12D activation in primary lung epithelial cells by adeno-cre resulted in a strong increase of GSTP1 protein and a striking increase in overall protein S-glutathionylation. More specifically Pyruvate kinase M2 (PKM2), an important enzyme in cancer metabolism, was highly S-glutathionylated in KRASG12D activated mouse lung and in MTE cells and increase in lactate production and expression of glycolysis enzymes was observed. Pharmacological GSTP1 inhibition with TLK117 decreased PKM2-and overall PSSG, glycolysis and tumor migration. Conclusion Together, these data reveal a potential pivotal new role of GSTP1 in regulating lung adenocarcinoma metabolism by promoting glycolysis in part through oxidative inactivation (S-glutathionylation) of PKM2.

Fatiguing Contractions Induce Acute Redox Signaling in Mouse Muscle

Protein S-glutathionylation (P-SSG) is a reversible redox modification known to alter the activity of transcription factors, kinases, and contractile proteins as well as protect critical cysteine residues from irreversible oxidation. Exercise elicits an acute oxidative stress that is reported to result in beneficial adaptive responses, a process known as redox signaling. We hypothesized that redox signaling will occur through P-SSG of regulatory cysteines following fatiguing muscle contractions. To test this hypothesis, we performed acute fatiguing contractions using an in vivo stimulation protocol on anesthetized CB6F1 (BALB/cBy x C57BL/6) mice. Muscle force production was monitored by a force transducer throughout the 15 minutes of fatiguing stimulations. The right (stimulated) and left (unstimulated) gastrocnemius were collected 60 minutes after the last stimulation and processed for redox proteomics using resin-assisted thiol enrichment and selective reduction. Redox proteomics revealed over 1,500 P-SSG modifications that were significantly increased by fatiguing contractions. Mitochondrial proteins were among the most sensitive to glutathionylation. Ingenuity Pathway Analysis revealed mitochondrial dysfunction, calcium signaling, cytoskeleton signaling, and oxidative stress response in the top ten affected pathways. Critical cysteines on beta-actin, troponin I, ryanodine receptor 1, SERCA1, ATP synthase subunit alpha and NADH dehydrogenase flavoprotein 1 were among those significantly glutathionylated that have previously been reported to alter protein and muscle function. These modifications of redox sensitive proteins implicate rapid regulation of metabolism, excitation/contraction coupling, kinase activity, and contractile protein function, and protection from irreversible oxidation. Together, these data suggest a direct role of ROS in acute exercise signaling and map the redox sensitive proteome in mouse muscle. This study lays the groundwork for future investigation into the redox signaling of altered exercise adaptation associated with chronic conditions, such as sarcopenia, the age-related loss of muscle mass and function.

Glutaredoxin 1 Deficiency Causes Late-Age Onset Metabolic Disease in Female C57BL/6 Mice, Associated with Monocyte and Macrophage Dysfunction

Glutaredoxin 1 (Grx1) reduces mixed disulfides between glutathione and protein thiols thereby controlling thiol-redox regulated cellular functions, including intracellular signaling, migration, angiogenesis, autophagy and apoptosis. Grx1 protects monocytes and macrophages from metabolic-stress-induced “priming’ and dysfunction. Here we report that female but not male Grx1-/- mice on a C57BL/6 background fed a normal chow diet become obese and hyperglycemic by 6 and 8 months of age respectively. At 18 months female Grx1-/- mice developed spontaneous atherosclerosis and late-age onset associated metabolic disease, characterized by mild hyperlipidemia, hyperglycemia and obesity. Atherosclerotic lesions in 18 month female Grx1-/- mice, measured by en-face analysis, were localized to both the ascending arch and abdominal aortic regions. In aged female Grx1-/- mice, monocyte priming was also measured in vivo using the Matrigel plug assay and ex vivo in multi-well Boyden chambers using peritoneal macrophages isolated from female Grx1-/- mice and age-matched WT mice. Furthermore, aged Grx1-/- female mice possessed increased numbers of circulating Ly6Chi inflammatory blood monocytes compared to male Grx1-/- and WT mice. Macrophages isolated from female Grx1-/- mice also displayed increased protein-S-glutathionylation, increased expression of pro-inflammatory “M1”-associated genes, increased susceptibility to ketocholesterol-induced apoptosis, and impaired autophagy. Together, these data underscore the critical role of protein-S-glutathionylation in monocyte macrophage dysfunction and the development of atherosclerosis and obesity and identify a protective role for Grx1 in these processes. Monocytic Grx1 and metabolic stress-induced protein-S glutathionylation may therefore represent a new mechanistic link between metabolic disorders and chronic inflammatory diseases.

Stress-Induced Protein S-Glutathionylation and S-Trypanothionylation in African Trypanosomes-A Quantitative Redox Proteome and Thiol Analysis.

Aims: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation.Results: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.Innovation: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides.Conclusion: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517–533.

GLUTATHIONE AND GLUTAREDOXIN IN ROSCOVITINE-MEDIATED INHIBITION OF BREAST CANCER CELL PROLIFERATION

Background: Breast tumors are number one cause of cancer morbidity and mortality among women around the world, and Russia is not an exception. Many proteins that control proliferation of immortalized cells are redox-regulated, which is essential for modulating cellular proliferative activity, especially during tumor growth. Studying the role of glutaredoxin and glutathione in cell cycle phase distribution will allow not only to identify the molecular targets regulating cell proliferation, but also to develop methods of diagnosis and targeted therapy of socially sensitive diseases, including breast cancer, in the future.Aims: To evaluate the role of glutathione and glutaredoxin in the molecular mechanisms regulating MCF-7 breast cancer cell proliferation under the effects of roscovitine, a cyclin-dependent protein kinase inhibitor.Materials and methods: The MCF-7 cell line (human breast adenocarcinoma) was used in the study. The cell culture was incubated in the presence and absence of roscovitine in the final concentration of 20 µmol for 18 h. The production of reactive oxygen species, the distribution of cells between cell cycle phases and the amount of Annexin V positive cells were determined using flow cytometry. The concentrations of total, reduced and oxidized glutathione, protein SH groups and protein-bound glutathione were measured by spectrophotometry. The levels of glutaredoxin, cyclin E and cyclin-dependent protein kinases were estimated by Western blotting with monoclonal antibodies.Results: The effects of roscovitine in the MCF-7 cells resulted in cell cycle arrest in G2/М phases with the decreased levels of cyclin E and cyclin-dependent protein kinase 2. It was accompanied by activation of programmed cell death. In tumor cells incubated in the presence of roscovitine, oxidative stress was triggered, which was accompanied by the elevated generation of reactive oxygen species, the decrease in the concentration of reduced glutathione, and the rise in the level of glutaredoxin. It contributed to the increase in protein glutathionylation against the backdrop of the decreased SH group concentration.Conclusions: Breast cancer cell proliferation under the effects of roscovitine is reduced following not only the decrease in the cyclin level and cyclin-dependent protein kinase activity, but also the shift in the intracellular oxidant/antioxidant ratio. Roscovitine-induced oxidative stress in the MCF-7 cells contributed to protein glutathionylation with the changes in the protein structure and functions. It results in impaired cell cycle progression, indicating a possibility to regulate cellular proliferation through modulating functional properties of redox-dependent proteins using the glutathione/glutaredoxin system.

A mechanistic insight into curcumin modulation of the IL-1β secretion and NLRP3 S-glutathionylation induced by needle-like cationic cellulose nanocrystals in myeloid cells

Recently we have demonstrated that needle-like cationic cellulose nanocrystals (CNC-AEMA2) evoke immunological responses through NLRP3 inflammasome/IL-1β inflammatory pathway. In this study we demonstrated that curcumin, a naturally occurring polyphenolic compound isolated from Curcuma longa (Zingiberaceae), was able to suppress, at least in part, this immunological response, as observed by diminished IL-1β secretion in CNC-AEMA2-stimulated macrophages primed with LPS. Curcumin is a well-known antioxidant and anti-inflammatory natural compound and in addition to acting as “scavenger” of reactive oxygen species (ROS), it can also upregulates antioxidant enzymes. However, the mechanisms by which this natural compound exerts its protective activity is still under investigation. We hypothesize that curcumin may also affect S-glutathionylation of key proteins involved in the NLRP3 inflammasome/IL-1β pathway, and therefore impact their protein-protein interactions. The goal of this study was to investigate the effects of curcumin on the S-glutathionylation of NLRP3 induced by CNC-AEMA2 in LPS-primed mouse macrophages (J774A.1), as well as interactions among proteins of the NLRP3 inflammasome complex. Our main finding indicates that the addition of curcumin concomitantly with LPS caused the greatest decrease in NLRP3 S-glutathionylation and a respective increase in caspase-1 S-glutathionylation, which appears to favor protein-protein interactions in the NLRP3 complex. Taking together, our results suggest that, at least in part, the anti-inflammatory activity of curcumin is associated with changes in S-glutathionylation of key NLRP3 inflammasome components, and perhaps resulting in sustained complex assembly and suppression of IL-1β secretion.

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid, a glutathione reductase inhibitor, induces G2/M cell cycle arrest through generation of thiol oxidative stress in human esophageal cancer cells.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant cancer with poor response to both of chemotherapy and radiotherapy. 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid (2-AAPA), an irreversible inhibitor of glutathione reductase (GR), is able to induce intracellular oxidative stress, and has shown anticancer activity in many cancer cell lines. In this study, we investigated the effects of 2-AAPA on the cell proliferation, cell cycle and apoptosis and aimed to explore its mechanism of action in human esophageal cancer TE-13 cells. It was found that 2-AAPA inhibited growth of ESCC cells in a dose-dependent manner and it did not deplete reduced glutathione (GSH), but significantly increased the oxidized form glutathione (GSSG), resulting in decreased GSH/GSSG ratio. In consequence, significant reactive oxygen species (ROS) production was observed. The flow cytometric analysis revealed that 2-AAPA inhibited growth of esophageal cancer cells through arresting cell cycle in G2/M phase, but apoptosis-independent mechanism. The G2/M arrest was partially contributed by down-regulation of protein expression of Cdc-25c and up-regulation of phosphorylated Cdc-2 (Tyr15), Cyclin B1 (Ser147) and p53. Meanwhile, 2-AAPA-induced thiol oxidative stress led to increased protein S-glutathionylation, which resulted in α-tubulin S-glutathionylation-dependent depolymerization of microtubule in the TE-13 cells. In conclusion, we identified that 2-AAPA as an effective thiol oxidative stress inducer and proliferation of TE-13 cells were suppressed by G2/M phase cell cycle arrest, mainly, through α-tubulin S-glutathionylation-mediated microtubule depolymerization. Our results may introduce new target and approach for esophageal cancer therapy through generation of GR-mediated thiol oxidative stress.