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1.
Int J Mol Sci ; 25(20)2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39456676

RESUMO

Glyoxalase 2 (Glo2) is an enzyme of the glyoxalase system whose pathway parallels glycolysis and which aims to remove methylglyoxal (MGO). This study analyzed the possible additional roles of the Glo2 enzyme in breast cancer (MCF7) and non-cancer (HDF) cell lines, investigating its presence at the nuclear level and its potential involvement in cell proliferation and chemotherapy resistance. The results revealed that Glo2 is overexpressed in cancer cells, and its expression is higher during the proliferative (S and G2/M) phases of the cell cycle. The study also examined a post-translational modification (PTM) in which Glo2 could be involved, with S-glutathionylation revealing that Glo2 enhances this PTM in cancer cells both in the cytoplasm and nucleus. Inhibition of Glo2 by p-NCBG resulted in increased sensitivity to doxorubicin, a common chemotherapeutic agent. This suggests that Glo2 increases cancer cell resistance to chemotherapy, potentially through its role in regulating oxidative stress. These results highlight Glo2 as a potential therapeutic target to improve the efficacy of existing treatments.


Assuntos
Neoplasias da Mama , Proliferação de Células , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Lactoilglutationa Liase , Humanos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Feminino , Lactoilglutationa Liase/metabolismo , Lactoilglutationa Liase/genética , Células MCF-7 , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Estresse Oxidativo/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Tioléster Hidrolases
2.
Biomed Pharmacother ; 180: 117514, 2024 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-39362067

RESUMO

Alcohol use disorder (AUD) is the most prevalent substance use disorder but there is incomplete knowledge of the underlying molecular etiology. Here, we examined the cytosolic proteome from the nucleus accumbens core (NAcC) of ethanol drinking rhesus macaques to identify ethanol-sensitive signaling proteins. The targets were subsequently investigated using bioinformatics, genetic, and pharmacological manipulations in mouse models of ethanol drinking. Of the 1000+ cytosolic proteins identified in our screen, 50 proteins differed significantly between control and ethanol drinking macaques. Gene Ontology analysis of the differentially expressed proteins identified enrichment in pathways regulating metabolic processes and proteasome activity. Because the family of Glutathione S-transferases (GSTs) was enriched in these pathways, validation studies targeted GSTs using bioinformatics and genetically diverse mouse models. Gstp1 and Gstm2 were identified in Quantitative Trait Loci and published gene sets for ethanol-related phenotypes (e.g., ethanol preference, conditioned taste aversion, differential expression), and recombinant inbred strains that inherited the C57BL/6J allele at the Gstp2 interval consumed higher amounts of ethanol than those that inherited the DBA/2J allele. Genetic deletion of Gstp1/2 led to increased ethanol consumption without altering ethanol metabolism or sucrose preference. Administration of the pharmacologic activator of Gstp1/2, carnosic acid, decreased voluntary ethanol drinking. Proteomic analysis of the NAcC cytosolic of heavy drinking macaques that were validated in mouse models indicate a role for glutathione-mediated redox regulation in ethanol-related neurobiology and the potential of pharmacological interventions targeting this system to modify excessive ethanol drinking.

3.
Biochem Biophys Res Commun ; 734: 150774, 2024 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-39366175

RESUMO

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.


Assuntos
Glutationa Transferase , Humanos , Glutationa Transferase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/enzimologia , Neoplasias/metabolismo , Animais , Polimorfismo Genético , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química
4.
Int J Mol Sci ; 25(15)2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39125992

RESUMO

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.


Assuntos
Glutationa Transferase , Glutationa , Neoplasias , Transdução de Sinais , Humanos , Glutationa/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Glutationa Transferase/metabolismo , Animais , Glutationa Peroxidase/metabolismo
5.
Nutrients ; 16(16)2024 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-39203889

RESUMO

Protein glutathionylation is a reversible post-translational modification that involves the attachment of glutathione to cysteine residues. It plays a role in the regulation of several cellular processes and protection against oxidative damage. Glutathionylation (GS-ylation) modulates protein function, inhibits or enhances enzymatic activity, maintains redox homeostasis, and shields several proteins from irreversible oxidative stress. Aberrant GS-ylation patterns are thus implicated in various diseases, particularly those associated with oxidative stress and inflammation, such as cardiovascular diseases, neurodegenerative disorders, cancer, and many others. Research in the recent years has highlighted the potential to manipulate protein GS-ylation for therapeutic purposes with strategies that imply both its enhancement and inhibition according to different cases. Moreover, it has become increasingly evident that monitoring the GS-ylation status of selected proteins offers diagnostic potential in different diseases. In this review, we try to summarize recent research in the field with a focus on our current understanding of the molecular mechanisms related to aberrant protein GS-ylation.


Assuntos
Glutationa , Doenças Neurodegenerativas , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Humanos , Glutationa/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Oxirredução , Neoplasias/metabolismo , Inflamação/metabolismo
6.
Sci Total Environ ; 947: 174534, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-38986690

RESUMO

Arsenic, a toxicant widely distributed in the environment, is considered as a risk factor for liver fibrosis. At present, the underlying mechanism still needs to be explored. In the present study, we found that, for mice, chronic exposure to arsenic induced liver fibrosis, activated the NLRP3 inflammasome, and increased the levels of reactive oxygen species (ROS). After hepatocytes were co-cultured with hepatic stellate cells (HSCs), we observed the arsenic-activated NLRP3 inflammasome in hepatocytes, and the co-cultured HSCs were activated. Further, we found that, in livers of mice, arsenic disturbed GSH metabolism and promoted protein S-glutathionylation. A 3D molecular docking simulation suggested that NLRP3 binds with GSH, which was confirmed by immunoprecipitation experiments. N-acetylcysteine (NAC) increased the levels of GSH in hepatocytes, which suppressed the S-glutathionylation of NLRP3 and blocked arsenic-induced activation of the NLRP3 inflammasome. Mechanistically, an imbalance of the redox state induced by arsenic promotes the S-glutathionylation of NLRP3, which regulates activation of the NLRP3 inflammasome, leading into the activation of HSCs. Moreover, NAC increases the levels of GSH to block arsenic-induced S-glutathionylation of NLRP3, thereby blocking arsenic-induced liver fibrosis. Thus, via activating HSCs, the S-glutathionylation of NLRP3 in hepatocytes is involved in arsenic-induced liver fibrosis, and, for hepatocytes, NAC alleviates these effects by increasing the levels of GSH. These results reveal a new mechanism and provide a possible therapeutic target for the liver fibrosis induced by environmental factors.


Assuntos
Arsênio , Glutationa , Hepatócitos , Inflamassomos , Cirrose Hepática , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Cirrose Hepática/induzido quimicamente , Camundongos , Hepatócitos/efeitos dos fármacos , Animais , Inflamassomos/metabolismo , Glutationa/metabolismo , Arsênio/toxicidade , Acetilcisteína/farmacologia , Espécies Reativas de Oxigênio/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-39042020

RESUMO

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.

8.
Antioxidants (Basel) ; 13(4)2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38671848

RESUMO

Glutathione (GSH), a robust endogenous antioxidant, actively participates in the modulation of the redox status of cysteine residues in proteins. Previous studies have indicated that GSH can prevent ß-cell failure and prediabetes caused by chronic oscillating glucose (OsG) administration. However, the precise mechanism underlying the protective effect is not well understood. Our current research reveals that GSH is capable of reversing the reduction in Nrf2 levels, as well as downstream genes Grx1 and HO-1, in the islet ß-cells of rats induced by chronic OsG. In vitro experiments have further demonstrated that GSH can prevent ß-cell dedifferentiation, apoptosis, and impaired insulin secretion caused by OsG. Additionally, GSH facilitates the translocation of Nrf2 into the nucleus, resulting in an upregulation of Nrf2-targeted genes such as GCLC, Grx1, HO-1, and NQO1. Notably, when the Nrf2 inhibitor ML385 is employed, the effects of GSH on OsG-treated ß-cells are abrogated. Moreover, GSH enhances the S-glutathionylation of Keap1 at Cys273 and Cys288, but not Cys151, in OsG-treated ß-cells, leading to the dissociation of Nrf2 from Keap1 and facilitating Nrf2 nuclear translocation. In conclusion, the protective role of GSH against OsG-induced ß-cell failure can be partially attributed to its capacity to enhance Keap1 S-glutathionylation, thereby activating the Nrf2 signaling pathway. These findings provide novel insights into the prevention and treatment of ß-cell failure in the context of prediabetes/diabetes, highlighting the potential of GSH.

9.
Redox Biol ; 71: 103116, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38479222

RESUMO

Oxidative stress plays an important role in the pathogenesis of acute lung injury (ALI). As a typical post-translational modification triggered by oxidative stress, protein S-glutathionylation (PSSG) is regulated by redox signaling pathways and plays diverse roles in oxidative stress conditions. In this study, we found that GSTP downregulation exacerbated LPS-induced injury in human lung epithelial cells and in mice ALI models, confirming the protective effect of GSTP against ALI both in vitro and in vivo. Additionally, a positive correlation was observed between total PSSG level and GSTP expression level in cells and mice lung tissues. Further results demonstrated that GSTP inhibited KEAP1-NRF2 interaction by promoting PSSG process of KEAP1. By the integration of protein mass spectrometry, molecular docking, and site-mutation validation assays, we identified C434 in KEAP1 as the key PSSG site catalyzed by GSTP, which promoted the dissociation of KEAP1-NRF2 complex and activated the subsequent anti-oxidant genes. In vivo experiments with AAV-GSTP mice confirmed that GSTP inhibited LPS-induced lung inflammation by promoting PSSG of KEAP1 and activating the NRF2 downstream antioxidant pathways. Collectively, this study revealed the novel regulatory mechanism of GSTP in the anti-inflammatory function of lungs by modulating PSSG of KEAP1 and the subsequent KEAP1/NRF2 pathway. Targeting at manipulation of GSTP level or activity might be a promising therapeutic strategy for oxidative stress-induced ALI progression.


Assuntos
Lesão Pulmonar Aguda , Fator 2 Relacionado a NF-E2 , Animais , Humanos , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/tratamento farmacológico , Antioxidantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Simulação de Acoplamento Molecular , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo
10.
Mol Ther Methods Clin Dev ; 32(1): 101214, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38496303

RESUMO

Inducible nitric oxide synthase (iNOS), regulated by nuclear factor kappa B (NF-κB), is crucial for intestinal inflammation and barrier injury in the progression of necrotizing enterocolitis (NEC). The NF-κB pathway is inhibited by S-glutathionylation of inhibitory κB kinase ß (IKKß), which can be restored by glutaredoxin-1 (Grx1). Thus, we aim to explore the role of Grx1 in experimental NEC. Wild-type (WT) and Grx1-knockout (Grx1-/-) mice were treated with an NEC-inducing regimen. Primary intestinal epithelial cells (IECs) were subjected to LPS treatment. The production of iNOS, NO, and inflammation injuries were assessed. NF-κB and involved signaling pathways were also explored. The severity of NEC was attenuated in Grx1-/- mice. Grx1 ablation promoted IKKß glutathionylation, NF-κB inactivation, and decreased iNOS, NO, and O2·- production in NEC mice. Furthermore, Grx1 ablation restrained proinflammatory cytokines and cell apoptosis, ameliorated intestinal barrier damage, and promoted proliferation in NEC mice. Grx1 ablation protected NEC through iNOS and NO inhibition, which related to S-glutathionylation of IKKß to inhibit NF-κB signaling. Grx1-related signaling pathways provide a new therapeutic target for NEC.

11.
Redox Biol ; 69: 103015, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38183796

RESUMO

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.


Assuntos
Glutarredoxinas , Peróxido de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Oxirredução , Glutationa/metabolismo , Estresse Oxidativo , Cloroplastos/metabolismo , Dissulfetos/química
12.
J Leukoc Biol ; 115(2): 322-333, 2024 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-37726110

RESUMO

Scavenger receptor A (SRA) is preferentially expressed in macrophages and implicated as a multifunctional pattern recognition receptor for innate immunity. Hepatic macrophages play a primary role in the pathogenesis of alcoholic liver disease. Herein, we observed that SRA expression was significantly increased in the liver tissues of mice with alcohol-related liver injury. SRA-deficient (SRA-/-) mice developed more severe alcohol-induced liver disease than wild-type mice. Enhanced liver inflammation existed in alcohol-challenged SRA-/- mice and was associated with increased Notch activation in hepatic macrophages compared with wild-type control animals. Mechanistically, SRA directly bound with Notch1 and suppressed its S-glutathionylation, thereby inhibiting Notch pathway activation. Further, we determined that the SRA interacted with thioredoxin-1 (Trx-1), a redox-active protein. SRA inhibited Trx-1 dimerization and facilitated the interaction of Trx-1 with Notch1. Application of a Trx-1-specific inhibitory agent during macrophage stimulation abolished SRA-mediated regulation of the Notch pathway and its downstream targets. In summary, our study revealed that SRA plays a critical role in macrophage inflammatory response by targeting Notch1 for its glutathionylation. SRA-mediated negative regulation of Notch activation might serve as a novel therapeutic strategy for alcohol-induced liver injury.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Camundongos , Animais , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Receptores Depuradores Classe A/metabolismo , Macrófagos/metabolismo , Receptores Depuradores/metabolismo , Fígado/metabolismo , Fatores Imunológicos , Etanol/toxicidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout
13.
Plants (Basel) ; 12(21)2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37960101

RESUMO

12-oxo-phytodienoic acid (OPDA) is a primary precursor of jasmonates, able to trigger autonomous signaling cascades that activate and fine-tune plant defense responses, as well as growth and development. However, its mechanism of actions remains largely elusive. Here we describe a dual-function messenger of OPDA signaling, reduced glutathione (GSH), that cross-regulates photosynthesis machinery and stress protection/adaptation in concert, optimizing plant plasticity and survival potential. Under stress conditions, the rapid induction of OPDA production stimulates GSH accumulation in the chloroplasts, and in turn leads to protein S-glutathionylation in modulating the structure and function of redox-sensitive enzymes such as 2-cysteine (Cys) peroxiredoxin A (2CPA), a recycler in the water-water cycle. GSH exchanges thiol-disulfides with the resolving CysR175, while donating an electron (e-, H+) to the peroxidatic CysP53, of 2CPA, which revives its reductase activity and fosters peroxide detoxification in photosynthesis. The electron flow protects photosynthetic processes (decreased total non-photochemical quenching, NPQ(T)) and maintains its efficiency (increased photosystem II quantum yield, ΦII). On the other hand, GSH also prompts retrograde signaling from the chloroplasts to the nucleus in adjusting OPDA-responsive gene expressions such as Glutathione S-Transferase 6 (GST6) and GST8, and actuating defense responses against various ecological constraints such as salinity, excess oxidants and light, as well as mechanical wounding. We thus propose that OPDA regulates a unique metabolic switch that interfaces light and defense signaling, where it links cellular and environmental cues to a multitude of plant physiological, e.g., growth, development, recovery, and acclimation, processes.

14.
Antioxidants (Basel) ; 12(11)2023 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-38001829

RESUMO

Hemoglobin is one of the proteins that are more susceptible to S-glutathionylation and the levels of its modified form, glutathionyl hemoglobin (HbSSG), increase in several human pathological conditions. The scope of the present review is to provide knowledge about how hemoglobin is subjected to S-glutathionylation and how this modification affects its functionality. The different diseases that showed increased levels of HbSSG and the methods used for its quantification in clinical investigations will be also outlined. Since there is a growing need for precise and reliable methods for markers of oxidative stress in human blood, this review highlights how HbSSG is emerging more and more as a good indicator of severe oxidative stress but also as a key pathogenic factor in several diseases.

15.
Curr Protoc ; 3(10): e907, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37818879

RESUMO

Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells.


Assuntos
Cisteína , Proteômica , Cisteína/metabolismo , Proteômica/métodos , Processamento de Proteína Pós-Traducional , Glutationa/química , Glutationa/metabolismo , Proteínas/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/química , Glutationa Sintase/metabolismo
16.
Int J Mol Sci ; 24(19)2023 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-37834470

RESUMO

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.


Assuntos
Cisteína , Glutationa , Cisteína/metabolismo , Glutationa/metabolismo , Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Oxirredução
17.
Biochemistry (Mosc) ; 88(7): 924-943, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37751864

RESUMO

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.


Assuntos
Apoptose , Neoplasias , Espécies Reativas de Oxigênio , Oxirredução , Morte Celular , Proliferação de Células , Oxigênio , Espécies Reativas de Nitrogênio
18.
Cell Chem Biol ; 30(12): 1542-1556.e9, 2023 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-37714153

RESUMO

Identification of cysteines with high oxidation susceptibility is important for understanding redox-mediated biological processes. In this report, we report a chemical proteomic strategy that finds cysteines with high susceptibility to S-glutathionylation. Our proteomic strategy, named clickable glutathione-based isotope-coded affinity tag (G-ICAT), identified 1,518 glutathionylated cysteines while determining their relative levels of glutathionylated and reduced forms upon adding hydrogen peroxide. Among identified cysteines, we demonstrated that CTNND1 (p120) C692 has high susceptibility to glutathionylation. Also, p120 wild type (WT), compared to C692S, induces its dissociation from E-cadherin under oxidative stress, such as glucose depletion. p120 and E-cadherin dissociation correlated with E-cadherin destabilization via its proteasomal degradation. Lastly, we showed that p120 WT, compared to C692S, increases migration and invasion of MCF7 cells under glucose depletion, supporting a model that p120 C692 glutathionylation increases cell migration and invasion by destabilization of E-cadherin, a core player in cell-cell adhesion.


Assuntos
Cateninas , delta Catenina , Humanos , Cateninas/metabolismo , Proteômica , Caderinas/metabolismo , Movimento Celular , Glucose
19.
Biochim Biophys Acta Gen Subj ; 1867(9): 130418, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37355052

RESUMO

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation. Incubation of HEK 293 T cells in the presence of H2O2 or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H2O2 or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.


Assuntos
Cisteína , Peróxido de Hidrogênio , Humanos , Cisteína/metabolismo , Actinas/metabolismo , Células HEK293 , Oxirredução , Gliceraldeído-3-Fosfato Desidrogenases/química
20.
Toxicol In Vitro ; 91: 105616, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37279824

RESUMO

Cellulose nanocrystals (CNCs) display remarkable strength and physicochemical properties with significant potential applications. To better understand the potential adjuvanticity of a nanomaterial, it is important to investigate the extent of the immunological response, the mechanisms by which they elicit this response, and how this response is associated with their physicochemical characteristics. In this study, we investigated the potential mechanisms of immunomodulation and redox activity of two chemically related cationic CNC derivatives (CNC-METAC-1B and CNC-METAC-2B), using human peripheral blood mononuclear cells and mouse macrophage cells (J774A.1). Our data demonstrated that the biological effects caused by these nanomaterials occurred mainly with short term exposure. We observed opposite immunomodulatory activity between the tested nanomaterials. CNC-METAC-2B, induced IL-1ß secretion at 2 h while CNC-METAC-1B decreased it at 24 h of treatment. In addition, both nanomaterials caused more noticeable increases in mitochondrial reactive oxygen species (ROS) at early time. The differences in apparent sizes of the two cationic nanomaterials could explain, at least in part, the discrepancies in biological effects, despite their closely related surface charges. This work provides initial insights about the complexity of the in vitro mechanism of action of these nanomaterials as well as foundation knowledge for the development of cationic CNCs as potential immunomodulators.


Assuntos
Celulose , Nanoestruturas , Animais , Humanos , Camundongos , Celulose/toxicidade , Leucócitos Mononucleares , Nanopartículas/toxicidade , Nanopartículas/química , Nanoestruturas/toxicidade , Espécies Reativas de Oxigênio
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