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Danyelle M. Townsend - One of the best experts on this subject based on the ideXlab platform.
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Altered redox regulation and S-Glutathionylation of BiP contribute to bortezomib resistance in multiple myeloma
Free radical biology & medicine, 2020Co-Authors: Jie Zhang, Lauren E. Ball, Kenneth D. Tew, Wei Chen, John Culpepper, Haiming Jiang, Shikhar Mehrotra, Anna Blumental-perry, Danyelle M. TownsendAbstract:Multiple myeloma (MM) cells have high rates of secretion of proteins rich in disulfide bonds and depend upon compartmentalized redox balance for accurate protein folding. The proteasome inhibitor bortezomib (Btz) is a successful frontline treatment for the disease, but its long-term efficacy is restricted by the acquisition of resistance. We found that MM cell lines resistant to Btz maintain high levels of oxidative stress and are cross resistant to endoplasmic reticulum (ER) stress-inducing agents thapsigargin (ThG), and tunicamycin (TuM). Moreover, cells expressing high/wild type levels of glutathione S-transferase P (GSTP) are more resistant than Gstp1/p2 knockout cells. In agreement, basal levels of S-glutathionylated proteins and redox regulation enzymes, including GSTP are elevated at mRNA and protein levels in resistant cells. GSTP mediated S-Glutathionylation (SSG) regulates the activities of a number of redox active ER proteins. Here we demonstrated that the post-translational modification determines the balance between foldase and ATPase activities of the binding immunoglobulin protein (BiP), with Cys41-SSG important for ATPase, and Cys420-SSG for foldase. BiP expression and S-Glutathionylation are increased in clinical specimens of bone marrow from MM patients compared to non-cancerous samples. Preventing S-Glutathionylation in MM cells with a GSTP specific inhibitor restored BiP activities and reversed resistance to Btz. Therefore, S-Glutathionylation of BiP confers pro-survival advantages and represents a novel mechanism of drug resistance in MM cells. We conclude that altered GSTP expression leads to S-Glutathionylation of BiP, and contributes to acquired resistance to Btz in MM.
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Post-translational S-Glutathionylation of cofilin increases actin cycling during cocaine seeking
PloS one, 2019Co-Authors: Anna Kruyer, Lauren E. Ball, Danyelle M. Townsend, Peter W. Kalivas, Joachim D. UysAbstract:Neuronal defense against oxidative damage is mediated primarily by the glutathione redox system. Traditionally considered a mechanism to protect proteins from irreversible oxidation, mounting evidence supports a role for protein S-Glutathionylation in cell signaling in response to changes in intracellular redox status. Here we determined the specific sites on the actin binding protein cofilin that undergo S-Glutathionylation. In addition, we show that S-Glutathionylation of cofilin reduces its capacity to depolymerize F-actin. We further describe an assay to determine the S-Glutathionylation of target proteins in brain tissue from behaving rodents. Using this technique, we show that cofilin in the rat nucleus accumbens undergoes S-Glutathionylation during 15-minutes of cued cocaine seeking in the absence of cocaine. Our findings demonstrate that cofilin S-Glutathionylation is increased in response to cocaine-associated cues and that increased cofilin S-Glutathionylation reduces cofilin-dependent depolymerization of F-actin. Thus, S-Glutathionylation of cofilin may serve to regulate actin cycling in response to drug-conditioned cues.
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An evolving understanding of the S-Glutathionylation cycle in pathways of redox regulation.
Free radical biology & medicine, 2018Co-Authors: Jie Zhang, Danyelle M. Townsend, Shweta Singh, Kenneth D. TewAbstract:By nature of the reversibility of the addition of glutathione to low pKa cysteine residues, the post-translational modification of S-Glutathionylation sanctions a cycle that can create a conduit for cell signaling events linked with cellular exposure to oxidative or nitrosative stress. The modification can also avert proteolysis by protection from over-oxidation of those clusters of target proteins that are substrates. Altered functions are associated with S-Glutathionylation of proteins within the mitochondria and endoplasmic reticulum compartments, and these impact energy production and protein folding pathways. The existence of human polymorphisms of enzymes involved in the cycle (particularly glutathione S-transferase P) create a scenario for inter-individual variance in response to oxidative stress and a number of human diseases with associated aberrant S-Glutathionylation have now been identified.
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S-Glutathionylation of estrogen receptor α affects dendritic cell function.
The Journal of biological chemistry, 2018Co-Authors: Jie Zhang, Lauren E. Ball, Yefim Manevich, Kenneth D. Tew, Yvonne M. W. Janssen-heininger, Wei Chen, Shikhar Mehrotra, Danyelle M. TownsendAbstract:Glutathione S-transferase Pi (GSTP) is a thiolase that catalyzes the addition of glutathione (GSH) to receptive cysteines in target proteins, producing an S-glutathionylated residue. Accordingly, previous studies have reported that S-Glutathionylation is constitutively decreased in cells from mice lacking GSTP (Gstp1/p2−/−). Here, we found that bone marrow–derived dendritic cells (BMDDCs) from Gstp1/p2−/− mice have proliferation rates that are greater than those in their WT counterparts (Gstp1/p2+/+). Moreover, Gstp1/p2−/− BMDDCs had increased reactive oxygen species (ROS) levels and decreased GSH:glutathione disulfide (GSSG) ratios. Estrogen receptor α (ERα) is linked to myeloproliferation and differentiation, and we observed that its steady-state levels are elevated in Gstp1/p2−/− BMDDCs, indicating a link between GSTP and ERα activities. BMDDCs differentiated by granulocyte–macrophage colony-stimulating factor had elevated ERα levels, which were more pronounced in Gstp1/p2−/− than WT mice. When stimulated with lipopolysaccharide for maturation, Gstp1/p2−/− BMDDCs exhibited augmented endocytosis, maturation rate, cytokine secretion, and T-cell activation; heightened glucose uptake and glycolysis; increased Akt signaling (in the mTOR pathway); and decreased AMPK-mediated phosphorylation of proteins. Of note, GSTP formed a complex with ERα, stimulating ERα S-Glutathionylation at cysteines 221, 245, 417, and 447; altering ERα's binding affinity for estradiol; and reducing overall binding potential (receptor density and affinity) 3-fold. Moreover, in Gstp1/p2−/− BMDDCs, ERα S-Glutathionylation was constitutively decreased. Taken together, these findings suggest that GSTP-mediated S-Glutathionylation of ERα controls BMDDC differentiation and affects metabolic function in dendritic cells.
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S-Glutathionylation of buccal cell proteins as biomarkers of exposure to hydrogen peroxide.
BBA clinical, 2014Co-Authors: Christina L. Grek, Danyelle M. Townsend, Leticia Reyes, Kenneth D. TewAbstract:Abstract Background Exogenous or endogenous hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species (ROS) that can lead to oxidation of cellular nucleophiles, particularly cysteines in proteins. Commercial mouthwashes containing H 2 O 2 provide the opportunity to determine clinically whether changes in S -glutathionylation of susceptible proteins in buccal mucosa cells can be used as biomarkers of ROS exposure. Methods Using an exploratory clinical protocol, 18 disease-free volunteers rinsed with a mouthwash containing 1.5% H 2 O 2 (442 mM) over four consecutive days. Exfoliated buccal cell samples were collected prior and post-treatment and proteomics were used to identify S -glutathionylated proteins. Results Four consecutive daily treatments with the H 2 O 2 -containing mouthwash induced significant dose and time-dependent increases in S -glutathionylation of buccal cell proteins, stable for at least 30 min following treatments. Elevated levels of S -glutathionylation were maintained with subsequent daily exposure. Increased S -glutathionylation preceded and correlated with transcriptional activation of ROS sensitive genes, such as ATF3, and with the presence of 8-hydroxy deoxyguanosine. Data from a human buccal cell line TR146 were consistent with the trial results. We identified twelve proteins that were S -glutathionylated following H 2 O 2 exposure. Conclusions Buccal cells can predict exposure to ROS through increased levels of S -glutathionylation of proteins. These post-translationally modified proteins serve as biomarkers for the effects of H 2 O 2 in the oral cavity and in the future, may be adaptable as extrapolated pharmacodynamic biomarkers for assessing the impact of other systemic drugs that cause ROS and/or impact redox homeostasis. General significance S -glutathionylation of buccal cell proteins can be used as a quantitative measure of exposure to ROS.
Kenneth D. Tew - One of the best experts on this subject based on the ideXlab platform.
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Altered redox regulation and S-Glutathionylation of BiP contribute to bortezomib resistance in multiple myeloma
Free radical biology & medicine, 2020Co-Authors: Jie Zhang, Lauren E. Ball, Kenneth D. Tew, Wei Chen, John Culpepper, Haiming Jiang, Shikhar Mehrotra, Anna Blumental-perry, Danyelle M. TownsendAbstract:Multiple myeloma (MM) cells have high rates of secretion of proteins rich in disulfide bonds and depend upon compartmentalized redox balance for accurate protein folding. The proteasome inhibitor bortezomib (Btz) is a successful frontline treatment for the disease, but its long-term efficacy is restricted by the acquisition of resistance. We found that MM cell lines resistant to Btz maintain high levels of oxidative stress and are cross resistant to endoplasmic reticulum (ER) stress-inducing agents thapsigargin (ThG), and tunicamycin (TuM). Moreover, cells expressing high/wild type levels of glutathione S-transferase P (GSTP) are more resistant than Gstp1/p2 knockout cells. In agreement, basal levels of S-glutathionylated proteins and redox regulation enzymes, including GSTP are elevated at mRNA and protein levels in resistant cells. GSTP mediated S-Glutathionylation (SSG) regulates the activities of a number of redox active ER proteins. Here we demonstrated that the post-translational modification determines the balance between foldase and ATPase activities of the binding immunoglobulin protein (BiP), with Cys41-SSG important for ATPase, and Cys420-SSG for foldase. BiP expression and S-Glutathionylation are increased in clinical specimens of bone marrow from MM patients compared to non-cancerous samples. Preventing S-Glutathionylation in MM cells with a GSTP specific inhibitor restored BiP activities and reversed resistance to Btz. Therefore, S-Glutathionylation of BiP confers pro-survival advantages and represents a novel mechanism of drug resistance in MM cells. We conclude that altered GSTP expression leads to S-Glutathionylation of BiP, and contributes to acquired resistance to Btz in MM.
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An evolving understanding of the S-Glutathionylation cycle in pathways of redox regulation.
Free radical biology & medicine, 2018Co-Authors: Jie Zhang, Danyelle M. Townsend, Shweta Singh, Kenneth D. TewAbstract:By nature of the reversibility of the addition of glutathione to low pKa cysteine residues, the post-translational modification of S-Glutathionylation sanctions a cycle that can create a conduit for cell signaling events linked with cellular exposure to oxidative or nitrosative stress. The modification can also avert proteolysis by protection from over-oxidation of those clusters of target proteins that are substrates. Altered functions are associated with S-Glutathionylation of proteins within the mitochondria and endoplasmic reticulum compartments, and these impact energy production and protein folding pathways. The existence of human polymorphisms of enzymes involved in the cycle (particularly glutathione S-transferase P) create a scenario for inter-individual variance in response to oxidative stress and a number of human diseases with associated aberrant S-Glutathionylation have now been identified.
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S-Glutathionylation of estrogen receptor α affects dendritic cell function.
The Journal of biological chemistry, 2018Co-Authors: Jie Zhang, Lauren E. Ball, Yefim Manevich, Kenneth D. Tew, Yvonne M. W. Janssen-heininger, Wei Chen, Shikhar Mehrotra, Danyelle M. TownsendAbstract:Glutathione S-transferase Pi (GSTP) is a thiolase that catalyzes the addition of glutathione (GSH) to receptive cysteines in target proteins, producing an S-glutathionylated residue. Accordingly, previous studies have reported that S-Glutathionylation is constitutively decreased in cells from mice lacking GSTP (Gstp1/p2−/−). Here, we found that bone marrow–derived dendritic cells (BMDDCs) from Gstp1/p2−/− mice have proliferation rates that are greater than those in their WT counterparts (Gstp1/p2+/+). Moreover, Gstp1/p2−/− BMDDCs had increased reactive oxygen species (ROS) levels and decreased GSH:glutathione disulfide (GSSG) ratios. Estrogen receptor α (ERα) is linked to myeloproliferation and differentiation, and we observed that its steady-state levels are elevated in Gstp1/p2−/− BMDDCs, indicating a link between GSTP and ERα activities. BMDDCs differentiated by granulocyte–macrophage colony-stimulating factor had elevated ERα levels, which were more pronounced in Gstp1/p2−/− than WT mice. When stimulated with lipopolysaccharide for maturation, Gstp1/p2−/− BMDDCs exhibited augmented endocytosis, maturation rate, cytokine secretion, and T-cell activation; heightened glucose uptake and glycolysis; increased Akt signaling (in the mTOR pathway); and decreased AMPK-mediated phosphorylation of proteins. Of note, GSTP formed a complex with ERα, stimulating ERα S-Glutathionylation at cysteines 221, 245, 417, and 447; altering ERα's binding affinity for estradiol; and reducing overall binding potential (receptor density and affinity) 3-fold. Moreover, in Gstp1/p2−/− BMDDCs, ERα S-Glutathionylation was constitutively decreased. Taken together, these findings suggest that GSTP-mediated S-Glutathionylation of ERα controls BMDDC differentiation and affects metabolic function in dendritic cells.
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S-Glutathionylation of buccal cell proteins as biomarkers of exposure to hydrogen peroxide.
BBA clinical, 2014Co-Authors: Christina L. Grek, Danyelle M. Townsend, Leticia Reyes, Kenneth D. TewAbstract:Abstract Background Exogenous or endogenous hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species (ROS) that can lead to oxidation of cellular nucleophiles, particularly cysteines in proteins. Commercial mouthwashes containing H 2 O 2 provide the opportunity to determine clinically whether changes in S -glutathionylation of susceptible proteins in buccal mucosa cells can be used as biomarkers of ROS exposure. Methods Using an exploratory clinical protocol, 18 disease-free volunteers rinsed with a mouthwash containing 1.5% H 2 O 2 (442 mM) over four consecutive days. Exfoliated buccal cell samples were collected prior and post-treatment and proteomics were used to identify S -glutathionylated proteins. Results Four consecutive daily treatments with the H 2 O 2 -containing mouthwash induced significant dose and time-dependent increases in S -glutathionylation of buccal cell proteins, stable for at least 30 min following treatments. Elevated levels of S -glutathionylation were maintained with subsequent daily exposure. Increased S -glutathionylation preceded and correlated with transcriptional activation of ROS sensitive genes, such as ATF3, and with the presence of 8-hydroxy deoxyguanosine. Data from a human buccal cell line TR146 were consistent with the trial results. We identified twelve proteins that were S -glutathionylated following H 2 O 2 exposure. Conclusions Buccal cells can predict exposure to ROS through increased levels of S -glutathionylation of proteins. These post-translationally modified proteins serve as biomarkers for the effects of H 2 O 2 in the oral cavity and in the future, may be adaptable as extrapolated pharmacodynamic biomarkers for assessing the impact of other systemic drugs that cause ROS and/or impact redox homeostasis. General significance S -glutathionylation of buccal cell proteins can be used as a quantitative measure of exposure to ROS.
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Abstract 3546: S-Glutathionylation of buccal mucosal cell proteins as biomarkers of oxidative stress.
Clinical Research, 2013Co-Authors: Christina L. Grek, Danyelle M. Townsend, Yefim Manevich, Kenneth D. TewAbstract:Many anticancer drugs produce reactive oxygen species (ROS) that directly or indirectly lead to toxicities. As a conduit to drug development we have identified S-glutathionylated (post-translational addition of glutathione to susceptible cysteine residues) serine proteinase inhibitors (serpin9s A1 and A3) as plausible biomarkers for drug exposure in both rodent and human blood. Commercial mouthwashes often contain redox-altering agents such as hydrogen peroxide (H2O2) and/or ethanol. Oxidative damage in buccal mucosa cells is associated with etiology of oral cancers and we hypothesized that these cells may also serve to provide a surrogate tissue source for biomarker analysis to measure S-glutathionylated proteins. Using an approved clinical protocol for exfoliated buccal cell samples, we have identified a number of S-glutathionylated proteins, including actin, in samples from volunteers following standard treatments with a commercial mouthwash containing H2O2 (1.5%). Their identities are similar to those discovered using in vitro experiments with the human buccal cell line TR146. Our current data show a time and dose-dependent increase in S-Glutathionylation of buccal cell sample profiles in normal volunteers following four days of exposure dosing to H2O2 containing mouthwash. Increases in protein S-Glutathionylation occurred immediately following exposure and remained for 30min following treatments. Higher levels of S-Glutathionylation were maintained with each subsequent daily exposure. These increased levels of S-Glutathionylation preceded the transcriptional activation of ROS sensitive genes, incuding ATF3. The identification of biomarkers that evaluate the effects of oxidative stress in the oral cavity may define at-risk populations for oral cancer and may be useful as direct biomarkers for the impact of drugs on general redox status. Citation Format: Christina L. Grek, Yefim Manevich, Danyelle M. Townsend, Kenneth D. Tew. S-Glutathionylation of buccal mucosal cell proteins as biomarkers of oxidative stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3546. doi:10.1158/1538-7445.AM2013-3546
Chun Jiang - One of the best experts on this subject based on the ideXlab platform.
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S-Glutathionylation of ion channels: insights into the regulation of channel functions, thiol modification crosstalk, and mechanosensing.
Antioxidants & redox signaling, 2013Co-Authors: Yang Yang, Xin Jin, Chun JiangAbstract:Ion channels control membrane potential, cellular excitability, and Ca(++) signaling, all of which play essential roles in cellular functions. The regulation of ion channels enables cells to respond to changing environments, and post-translational modification (PTM) is one major regulation mechanism. Many PTMs (e.g., S-Glutathionylation, S-nitrosylation, S-palmitoylation, S-sulfhydration, etc.) targeting the thiol group of cysteine residues have emerged to be essential for ion channels regulation under physiological and pathological conditions. Under oxidative stress, S-Glutathionylation could be a critical PTM that regulates many molecules. In this review, we discuss S-Glutathionylation-mediated structural and functional changes of ion channels. Criteria for testing S-Glutathionylation, methods and reagents used in ion channel S-Glutathionylation studies, and thiol modification crosstalk, are also covered. Mechanotransduction, and S-Glutathionylation of the mechanosensitive KATP channel, are discussed. Further investigation of the ion channel S-Glutathionylation, especially the physiological significance of S-Glutathionylation and thiol modification crosstalk, could lead to a better understanding of the thiol modifications in general and the ramifications of such modifications on cellular functions and related diseases.
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S-Glutathionylation underscores the modulation of the heteromeric Kir4.1-Kir5.1 channel in oxidative stress.
The Journal of physiology, 2012Co-Authors: Xin Jin, Yang Yang, Xianfeng Chen, Shuang Zhang, Zhenda Shi, Xiaoli Zhang, Chun JiangAbstract:The Kir4.1 channel is expressed in the brainstem, retina and kidney where it acts on K(+) transportation and pH-dependent membrane potential regulation. Its heteromerization with Kir5.1 leads to K(+) currents with distinct properties such as single-channel conductance, rectification, pH sensitivity and phosphorylation modulation. Here we show that Kir5.1 also enables S-Glutathionylation to the heteromeric channel. Expressed in HEK cells, an exposure to the oxidant H(2)O(2) or diamide produced concentration-dependent inhibitions of the whole-cell Kir4.1-Kir5.1 currents. In inside-out patches, currents were inhibited strongly by a combination of diamide/GSH or H(2)O(2)/GSH but not by either alone. The currents were also suppressed by GSSG and the thiol oxidants pyridine disulfides (PDSs), suggesting S-Glutathionylation. In contrast, none of the exposures had significant effects on the homomeric Kir4.1 channel. Cys158 in the TM2 helix of Kir5.1 was critical for the S-Glutathionylation, which was accessible to intracellular but not extracellular oxidants. Site-directed mutagenesis of this residue (C158A or C158T) abolished the Kir4.1-Kir5.1 current modulation by oxidants, and eliminated almost completely the biochemical interaction of Kir5.1 with GSH. In tandem Kir4.1-Kir5.1 constructs, the channel with a single Cys158 was inhibited to the same degree as the wild-type channel, suggesting that one glutathione moiety is sufficient to block the channel. Consistent with the location of Cys158, GSSG inhibited the channel only when the channel was open, indicating that the channel inhibition was state dependent. The finding that the heteromeric Kir4.1-Kir5.1 channel but not the homomeric Kir4.1 is subject to the S-Glutathionylation thus suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress.
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Molecular Basis and Structural Insight of Vascular KATP Channel Gating by S-Glutathionylation
The Journal of biological chemistry, 2011Co-Authors: Yang Yang, Weiwei Shi, Xianfeng Chen, Ningren Cui, Anuhya S. Konduru, Yun Shi, Timothy C. Trower, Shuang Zhang, Chun JiangAbstract:The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-Glutathionylation. Here we show evidence for the molecular basis of the S-Glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-Glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-Glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.
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Oxidative stress inhibits vascular KATP channels by S-Glutathionylation
The Journal of biological chemistry, 2010Co-Authors: Yang Yang, Weiwei Shi, Ningren Cui, Chun JiangAbstract:The KATP channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-Glutathionylation was a modulation mechanism underlying oxidant-mediated vascular KATP channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H2O2) impaired the KATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H2O2 or diamide caused a strong inhibition of the vascular KATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-Glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H2O2. These results suggest that S-Glutathionylation is an important mechanism for the vascular KATP channel modulation in oxidative stress.
Ryan J. Mailloux - One of the best experts on this subject based on the ideXlab platform.
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Protein S-Glutathionylation and the regulation of cellular functions
Oxidative Stress, 2020Co-Authors: Ryan J. Mailloux, Robert Gill, Adrian YoungAbstract:Abstract Protein S-Glutathionylation is a ubiquitous and reversible posttranslation modification that involves the addition and removal of GSH to and from a protein cysteine thiol. These reactions are catalyzed by glutaredoxins (GRX), thiol oxidoreductases that S-glutathionylate and deglutathionylate target proteins in response to fluctuations in the redox state of cellular glutathione pools. The modification of proteins with GSH modulates a number cell functions in response to environmental and physiological cues including energy metabolism and sensing, apoptosis, calcium handling, signaling, and protein folding. Protein S-Glutathionylation also serves as a superimposed cosignal that works in tandem with other pathways to regulate cell functions. Here, we discuss the various ways that protein S-Glutathionylation serves as one of the main devices for the regulation of various cell proteins during oxidative eustress signaling.
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Protein S-Glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria.
Chemico-biological interactions, 2018Co-Authors: Adrian Young, Robert Gill, Ryan J. MaillouxAbstract:Abstract Protein S-Glutathionylation reactions are a ubiquitous oxidative modification required to control protein function in response to changes in redox buffering capacity. These reactions are rapid and reversible and are, for the most part, enzymatically mediated by glutaredoxins (GRX) and glutathione S-transferases (GST). Protein S-Glutathionylation has been found to control a range of cell functions in response to different physiological cues. Although these reactions occur throughout the cell, mitochondrial proteins seem to be highly susceptible to reversible S-Glutathionylation, a feature attributed to the unique physical properties of this organelle. Indeed, mitochondria contain a number of S-Glutathionylation targets which includes proteins involved in energy metabolism, solute transport, reactive oxygen species (ROS) production, proton leaks, apoptosis, antioxidant defense, and mitochondrial fission and fusion. Moreover, it has been found that conjugation and removal of glutathione from proteins in mitochondria fulfills a number of important physiological roles and defects in these reactions can have some dire pathological consequences. Here, we provide an updated overview on mitochondrial protein S-Glutathionylation reactions and their importance in cell functions and physiology.
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Protein S-Glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.
PloS one, 2018Co-Authors: Robert Gill, Marisa O’brien, Adrian Young, Danielle Gardiner, Ryan J. MaillouxAbstract: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.
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Protein S-Glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex
Free radical biology & medicine, 2017Co-Authors: Marisa O’brien, Danielle Gardiner, Julia Chalker, Liam Slade, Ryan J. MaillouxAbstract:Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O2•-/ H2O2 emission from pyruvate dehydrogenase (Pdh) is altered by S-Glutathionylation. Glutathione disulfide (GSSG) amplified O2•-/ H2O2 production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-Glutathionylation. S-Glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD+ lowering O2•-/ H2O2 production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-Glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O2•-/ H2O2 production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-Glutathionylation.
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Induction of mitochondrial reactive oxygen species production by GSH mediated S-Glutathionylation of 2-oxoglutarate dehydrogenase.
Redox biology, 2016Co-Authors: Ryan J. Mailloux, D. Craig Ayre, Sherri L. ChristianAbstract:2-Oxoglutarate dehydrogenase (Ogdh) is an important mitochondria redox sensor that can undergo S-Glutathionylation following an increase in H2O2 levels. Although S-Glutathionylation is required to protect Ogdh from irreversible oxidation while simultaneously modulating its activity it remains unknown if glutathione can also modulate reactive oxygen species (ROS) production by the complex. We report that reduced (GSH) and oxidized (GSSG) glutathione control O2(∙-)/H2O2 formation by Ogdh through protein S-Glutathionylation reactions. GSSG (1mM) induced a modest decrease in Ogdh activity which was associated with a significant decrease in O2(∙-)/H2O2 formation. GSH had the opposite effect, amplifying O2(∙-)/H2O2 formation by Ogdh. Incubation of purified Ogdh in 2.5mM GSH led to significant increase in O2(∙-)/H2O2 formation which also lowered NADH production. Inclusion of enzymatically active glutaredoxin-2 (Grx2) in reaction mixtures reversed the GSH-mediated amplification of O2(∙-)/H2O2 formation. Similarly pre-incubation of permeabilized liver mitochondria from mouse depleted of GSH showed an approximately ~3.5-fold increase in Ogdh-mediated O2(∙-)/H2O2 production that was matched by a significant decrease in NADH formation which could be reversed by Grx2. Taken together, our results demonstrate GSH and GSSG modulate ROS production by Ogdh through S-Glutathionylation of different subunits. This is also the first demonstration that GSH can work in the opposite direction in mitochondria-amplifying ROS formation instead of quenching it. We propose that this regulatory mechanism is required to modulate ROS emission from Ogdh in response to variations in glutathione redox buffering capacity.
M S Shahul Hameed - One of the best experts on this subject based on the ideXlab platform.
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S-Glutathionylation OF CYSTEINE-217 ALLOSTERICALLY INHIBITS TRIOSE-PHOSPHATE ISOMERASE: A MOLECULAR DYNAMICS STUDY
Journal of Proteins & Proteomics, 2016Co-Authors: M S Shahul HameedAbstract:Reversible S-Glutathionylation is an important post-translational modification of proteins involved in redox signaling under normal physiological conditions and plays a protective role during oxidative / nitrosative stress by preventing irreversible oxidation of cysteine residues in proteins. Several enzymes of the glycolytic pathway have been shown to be targets of S-Glutathionylation, wherein S-Glutathionylation results in inhibition of the pathway. In this study the effects of S-Glutathionylation of Triose-phosphate Isomerase are reported. The effect of S-Glutathionylation on the structure and dynamics of TIM were examined through molecular dynamics simulations. MD simulation study has provided interesting insights into a novel mechanism of allosteric regulation of this enzyme by S-Glutathionylation of Cysteine-217 in Helix G. The simulations predict that S-Glutathionylation of Cys-217 leads to a complete loss of active site loop structure and causes alterations in its dynamics. This leads to premature substrate dissociation from active site leading to enzyme inhibition. Keywords : Protein S-Glutathionylation; metabolic regulation; triose-phosphate isomerase; Molecular Dynamics simulations; allosteric inhibition.
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ALLOSTERIC INHIBITION OF TRIOSE-PHOSPHATE ISOMERASE BY S- GLUTATHIONYLATION
Journal of Proteins & Proteomics, 2015Co-Authors: M S Shahul HameedAbstract:S-Glutathionylation regulates several cellular processes by modulating protein function. Many enzymes of the glycolytic pathway have been shown to be targets of S-Glutathionylation, wherein S-Glutathionylation results in inhibition of the pathway. In this study the effects of S-Glutathionylation of triose-phosphate isomerase are reported. The kinetics and sites of S-Glutathionylation of triose-phosphate isomerase were identified using high resolution mass spectrometric analysis and their consequences on enzyme activity were examined by in vitro biochemical assays. Combined analysis of data from biochemical assays and mass spectrometry have provided interesting insights into a possible novel mechanism of regulation of this enzyme by S-Glutathionylation of cysteine-217 present in helix G of triose-phosphate isomerase.
