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Significance: Both redox and pH are important regulatory processes that underpin cell physiological functions, in addition to influencing cancer cell development and tumor progression. The thioredoxin (Trx) and glutathione redox systems and the carbonic anhydrase (CA) proteins are considered key regulators of cellular redox and pH, respectively, with components of the Trx system and CAs regarded as cancer therapeutic targets. However, the redox and pH axis in cancer cells is an underexplored topic of research. Recent Advances: Structural studies of a CA family member, CA3, localized two of its five cysteine residues to the protein surface. Redox-regulated modifications to CA3 have been identified, including glutathionylation. CA3 has been shown to bind to other proteins, including B cell lymphoma-2-associated athanogene 3, and squalene epoxidase, which can modulate autophagy and proinflammatory signaling, respectively, in cancer cells. Critical Issues: CA3 has also been associated with epithelial–mesenchymal transition processes, which promote cancer cell metastasis, whereas CA3 overexpression activates the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, which upregulates cell growth and inhibits autophagy. It is not yet known if CA3 modulates cancer progression through its reported antioxidant functions. Future Directions: CA3 is one of the least studied CA isozymes. Further studies are required to assess the cellular antioxidant role of CA3 and its impact on cancer progression. Identification of other binding partners is also required, including whether CA3 binds to Trx in human cells. The development of specific CA3 inhibitors will facilitate these functional studies and allow CA3 to be investigated as a cancer therapeutic target. Antioxid. Redox Signal. 41, 957–975. [ABSTRACT FROM AUTHOR]

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 COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the 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 because of increased resistance to collagenase-mediated degradation and fibroblast activation. [ABSTRACT FROM AUTHOR]

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway. [ABSTRACT FROM AUTHOR]

Activation of Nrf2—the master regulator of antioxidative response—at different stages of embryonic development has been shown to result in changes in gene expression, but the tissue-specific and downstream effects of Nrf2 activation during development remain unclear. This work seeks to elucidate the tissue-specific Nrf2 cellular localization and the downstream changes in protein S-glutathionylation during critical windows of zebrafish (Danio rerio) development. Wild-type and mutant zebrafish embryos with a loss-of-function mutation in Nrf2a were treated with two canonical activators, sulforaphane (SFN; 40 µM) or tert-butylhydroquinone (tBHQ; 1 µM), for 6 h at either pharyngula, hatching, or the protruding-mouth stage. Nrf2a protein and S-glutathionylation were visualized in situ using immunohistochemistry. At the hatching stage, Nrf2a protein levels were decreased with SFN, but not tBHQ, exposure. Exposure to both activators, however, decreased downstream S-glutathionylation. Stage- and tissue-specific differences in Nrf2a protein and S-glutathionylation were identified in the pancreatic islet and liver. Protein S-glutathionylation in Nrf2a mutant fish was increased in the liver by both activators, but not the islets, indicating a tissue-specific and Nrf2a-dependent dysregulation. This work demonstrates that critical windows of exposure and Nrf2a activity may influence redox homeostasis and highlights the importance of considering tissue-specific outcomes and sensitivity in developmental redox biology. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) is one of the leading causes of mortality in humans, and oxidative stress plays a pivotal role in disease progression. This phenomenon typically arises from weakening of the cellular antioxidant system or excessive accumulation of peroxides. This review focuses on a specialized form of oxidative stress—disulfide stress—which is triggered by an imbalance in the glutaredoxin and thioredoxin antioxidant systems within the cell, leading to the accumulation of disulfide bonds. The genesis of disulfide stress is usually induced by extrinsic pathological factors that disrupt the thiol-dependent antioxidant system, manifesting as sustained glutathionylation of proteins, formation of abnormal intermolecular disulfide bonds between cysteine-rich proteins, or irreversible oxidation of thiol groups to sulfenic and sulfonic acids. Disulfide stress not only precipitates the collapse of the antioxidant system and the accumulation of reactive oxygen species, exacerbating oxidative stress, but may also initiate cellular inflammation, autophagy, and apoptosis through a cascade of signaling pathways. Furthermore, this review explores the detrimental effects of disulfide stress on the progression of various CVDs including atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, diabetic cardiomyopathy, cardiac hypertrophy, and heart failure. This review also proposes several potential therapeutic avenues to improve the future treatment of CVDs.

Activation of Nrf2—the master regulator of antioxidative response—at different stages of embryonic development has been shown to result in changes in gene expression, but the tissue-specific and downstream effects of Nrf2 activation during development remain unclear. This work seeks to elucidate the tissue-specific Nrf2 cellular localization and the downstream changes in protein S-glutathionylation during critical windows of zebrafish (Danio rerio) development. Wild-type and mutant zebrafish embryos with a loss-of-function mutation in Nrf2a were treated with two canonical activators, sulforaphane (SFN; 40 µM) or tert-butylhydroquinone (tBHQ; 1 µM), for 6 h at either pharyngula, hatching, or the protruding-mouth stage. Nrf2a protein and S-glutathionylation were visualized in situ using immunohistochemistry. At the hatching stage, Nrf2a protein levels were decreased with SFN, but not tBHQ, exposure. Exposure to both activators, however, decreased downstream S-glutathionylation. Stage- and tissue-specific differences in Nrf2a protein and S-glutathionylation were identified in the pancreatic islet and liver. Protein S-glutathionylation in Nrf2a mutant fish was increased in the liver by both activators, but not the islets, indicating a tissue-specific and Nrf2a-dependent dysregulation. This work demonstrates that critical windows of exposure and Nrf2a activity may influence redox homeostasis and highlights the importance of considering tissue-specific outcomes and sensitivity in developmental redox biology.

Annually, peripheral arterial disease is estimated to cost over USD 21 billion and diabetic foot disease an estimated at USD 9–13 billion. Mirabegron is a TGA-approved beta-3 adrenoreceptor agonist, shown to be safe and effective in the treatment of overactive bladder syndrome by stimulating bladder smooth muscle relaxation. In this review, we discuss the potential use of beta-3 adrenoreceptor agonists as therapeutic agents repurposed for peripheral arterial disease and diabetic foot ulcers. The development of both conditions is underpinned by the upregulation of oxidative stress pathways and consequential inflammation and hypoxia. In oxidative stress, there is an imbalance of reactive oxygen species and nitric oxide. Endothelial nitric oxide synthase becomes uncoupled in disease states, producing superoxide and worsening oxidative stress. Agonist stimulation of the beta-3 adrenoreceptor recouples and activates endothelial nitric oxide synthase, increasing the production of nitric oxide. This reduces circulating reactive oxygen species, thus decreasing redox modification and dysregulation of cellular proteins, causing downstream smooth muscle relaxation, improved endothelial function and increased angiogenesis. These mechanisms lead to endothelial repair in peripheral arterial disease and an enhanced perfusion in hypoxic tissue, which will likely improve the healing of chronic ulcers. [ABSTRACT FROM AUTHOR]

Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells. [ABSTRACT FROM AUTHOR]

This Special Issue of Antioxidants on Glutathione (GSH) and Glutaredoxin (Grx) was designed to collect review articles and original research studies focused on advancing the current understanding of the roles of the GSH/Grx system in cellular homeostasis and disease processes. The tripeptide glutathione (GSH) is the most abundant non-enzymatic antioxidant/nucleophilic molecule in cells. In addition to various metabolic reactions involving GSH and its oxidized counterpart GSSG, oxidative post-translational modification (PTM) of proteins has been a focal point of keen interest in the redox field over the last few decades. In particular, the S-glutathionylation of proteins (protein-SSG formation), i.e., mixed disulfides between GSH and protein thiols, has been studied extensively. This reversible PTM can act as a regulatory switch to interconvert inactive and active forms of proteins, thereby mediating cell signaling and redox homeostasis. The unique architecture of the GSH molecule enhances its relative abundance in cells and contributes to the glutathionyl specificity of the primary catalytic activity of the glutaredoxin enzymes, which play central roles in redox homeostasis and signaling, and in iron metabolism in eukaryotes and prokaryotes under physiological and pathophysiological conditions. The class-1 glutaredoxins are characterized as cytosolic GSH-dependent oxidoreductases that catalyze reversible protein S-glutathionylation specifically, thereby contributing to the regulation of redox signal transduction and/or the protection of protein thiols from irreversible oxidation. This Special Issue includes nine other articles: three original studies and six review papers. Together, these ten articles support the central theme that GSH/Grx is a unique system for regulating thiol-redox hemostasis and redox-signal transduction, and the dysregulation of the GSH/Grx system is implicated in the onset and progression of various diseases involving oxidative stress. Within this context, it is important to appreciate the complementary functions of the GSH/Grx and thioredoxin systems not only in thiol-disulfide regulation but also in reversible S-nitrosylation. Several potential clinical applications have emerged from a thorough understanding of the GSH/Grx redox regulatory system at the molecular level, and in various cell types in vitro and in vivo, including, among others, the concept that elevating Grx content/activity could serve as an anti-fibrotic intervention; and discovering small molecules that mimic the inhibitory effects of S-glutathionylation on dimer association could identify novel anti-viral agents that impact the key protease activities of the HIV and SARS-CoV-2 viruses. Thus, this Special Issue on Glutathione and Glutaredoxin has focused attention and advanced understanding of an important aspect of redox biology, as well as spawning questions worthy of future study. [ABSTRACT FROM AUTHOR]

In premature infants receiving parenteral nutrition, oxidative stress is a trigger for the development of bronchopulmonary dysplasia, which is an important factor in the development of adult lung diseases. Neonatal vitamin C and glutathione deficiency is suspected to induce permanent modification of redox metabolism favoring the development of neonatal and adult lung diseases. A total of 64 3-day-old guinea pigs were fed an oral diet that was either complete or deficient in vitamin C (VCD), cysteine (CD) (glutathione-limiting substrate) or both (DD) for 4 days. At 1 week of age, half of the animals were sacrificed while the other started a complete diet until 12 weeks of age. At 1 week, the decrease in lung GSH in all deficient groups was partially explained by the oxidation of liver methionine-adenosyltransferase. mRNA levels of kelch-like ECH-associated protein 1 (Keap1), glutathione-reductase (Gsr) and glutaredoxin-1 (Glrx) were significantly lower only in CD but not in DD. At 12 weeks, glutathione levels were increased in VCD and CD. Keap1, Gsr and Glrx mRNA were increased, while glutathione-reductase and glutaredoxin proteins were lower in CD, favoring a higher glutathionylation status. Both neonatal deficiencies result in a long-term change in glutathione metabolism that could contribute to lung diseases' development. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

Invasive and metastatic tumor cells show an increase in migration and invasion, making the processes contributing to these phenotypes potential therapeutic targets. Lipocalin 2 (LCN2; also known as neutrophil gelatinase‐associated lipocalin) is a putative therapeutic target in multiple tumor types and promotes invasion and migration, although the mechanisms underlying these phenotypes are unclear. The data in this report demonstrate that LCN2 promotes actin polymerization, invasion, and migration by inhibiting actin glutathionylation. LCN2 inhibits actin glutathionylation by decreasing the levels of reactive oxygen species (ROS) and by reducing intracellular iron levels. Inhibiting LCN2 function leads to increased actin glutathionylation, decreased migration, and decreased invasion. These results suggest that LCN2 is a potential therapeutic target in invasive tumors. [ABSTRACT FROM AUTHOR]

In humans, the cytosolic glutathione S-transferase (GST) family of proteins is encoded by 16 genes presented in seven different classes. GSTs exhibit remarkable structural similarity with some overlapping functionalities. As a primary function, GSTs play a putative role in Phase II metabolism by protecting living cells against a wide variety of toxic molecules by conjugating them with the tripeptide glutathione. This conjugation reaction is extended to forming redox sensitive post-translational modifications on proteins: S-glutathionylation. Apart from these catalytic functions, specific GSTs are involved in the regulation of stress-induced signaling pathways that govern cell proliferation and apoptosis. Recently, studies on the effects of GST genetic polymorphisms on COVID-19 disease development revealed that the individuals with higher numbers of risk-associated genotypes showed higher risk of COVID-19 prevalence and severity. Furthermore, overexpression of GSTs in many tumors is frequently associated with drug resistance phenotypes. These functional properties make these proteins promising targets for therapeutics, and a number of GST inhibitors have progressed in clinical trials for the treatment of cancer and other diseases. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is a known inducer of inflammatory signaling which triggers generation of reactive oxygen species (ROS) and cell death in responsive cells like THP-1 promonocytes and freshly isolated human monocytes. A key LPS-responsive metabolic pivot point is the 9 MDa mitochondrial pyruvate dehydrogenase complex (PDC), which provides pyruvate dehydrogenase (E1), lipoamide-linked transacetylase (E2) and lipoamide dehydrogenase (E3) activities to produce acetyl-CoA from pyruvate. While phosphorylation-dependent decreases in PDC activity following LPS treatment or sepsis have been deeply investigated, redox-linked processes have received less attention. Data presented here demonstrate that LPS-induced reversible oxidation within PDC occurs in PDCE2 in both THP-1 cells and primary human monocytes. Knockout of PDCE2 by CRISPR and expression of FLAG-tagged PDCE2 in THP-1 cells demonstrated that LPS-induced glutathionylation is associated with wild type PDCE2 but not mutant protein lacking the lipoamide-linking lysine residues. Moreover, the mitochondrially-targeted electrophile MitoCDNB, which impairs both glutathione- and thioredoxin-based reductase systems, elevates ROS similar to LPS but does not cause PDCE2 glutathionylation. However, LPS and MitoCDNB together are highly synergistic for PDCE2 glutathionylation, ROS production, and cell death. Surprisingly, the two treatments together had differential effects on cytokine production; pro-inflammatory IL-1β production was enhanced by the co-treatment, while IL-10, an important anti-inflammatory cytokine, dropped precipitously compared to LPS treatment alone. This new information may expand opportunities to understand and modulate PDC redox status and activity and improve the outcomes of pathological inflammation.

Several redox modifications have been described during viral infection, including influenza virus infection, but little is known about glutathionylation and this respiratory virus. Glutathionylation is a reversible, post-translational modification, in which protein cysteine forms transient disulfides with glutathione (GSH), catalyzed by cellular oxidoreductases and in particular by glutaredoxin (Grx). We show here that (i) influenza virus infection induces protein glutathionylation, including that of viral proteins such as hemagglutinin (HA); (ii) Grx1-mediated deglutathionylation is important for the viral life cycle, as its inhibition, either with an inhibitor of its enzymatic activity or by siRNA, decreases viral replication. Overall these data contribute to the characterization of the complex picture of redox regulation of the influenza virus replication cycle and could help to identify new targets to control respiratory viral infection. [ABSTRACT FROM AUTHOR]

The tripeptide glutathione is found in all eukaryotic cells, and due to the compartmentalization of biochemical processes, its synthesis takes place exclusively in the cytosol. At the same time, its functions depend on its transport to/from organelles and interorgan transport, in which the liver plays a central role. Glutathione is determined as a marker of the redox state in many diseases, aging processes, and cell death resulting from its properties and reactivity. It also uses other enzymes and proteins, which enables it to engage and regulate various cell functions. This paper approximates the role of these systems in redox and detoxification reactions such as conjugation reactions of glutathione-S-transferases, glyoxylases, reduction of peroxides through thiol peroxidases (glutathione peroxidases, peroxiredoxins) and thiol–disulfide exchange reactions catalyzed by glutaredoxins. [ABSTRACT FROM AUTHOR]

Dimethyl fumarate (DMF) is pharmaceutical activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates of many cellular antioxidant response pathways, and has been used to treat inflammatory diseases such as multiple sclerosis. However, DMF has been shown to produce adverse effects on offspring in animal studies and as such is not recommended for use during pregnancy. The goal of this work is to better understand how these adverse effects are initiated and the role of DMF-induced Nrf2 activation during three critical windows of development in embryonic zebrafish (Danio rerio): pharyngula, hatching, and protruding-mouth stages. To evaluate Nrf2 activation, wildtype zebrafish, and mutant zebrafish (nrf2afh318/fh318) embryos with a loss of function mutation in Nrf2a, the co-ortholog to human Nrf2, were treated for 6 h with DMF (0–20 μM) beginning at the pharyngula, hatching, or protruding-mouth stage and assessed for survival and morphology. Nrf2a mutant fish had an increase in survival, however, morphology studies demonstrated Nrf2a mutant fish had more severe deformities occurring with exposures during the hatching stage. To verify Nrf2 cellular localization and downstream impacts on protein- S- glutathionylation in situ , a concentration below the LOAEL was chosen (7 μM) for immunohistochemistry and S -glutathionylation. Embryos were imaged via epifluorescence microscopy studies, the Nrf2a protein in the body tissue was decreased with DMF only when exposed at the hatching stage, while total protein S -glutathionylation was modulated by Nrf2a activity and DMF during the pharyngula and protruding-mouth stage. The pancreatic islet and liver were further analyzed via confocal microscopy. Pancreatic islets and liver also had tissue specific differences with Nrf2a protein expression and protein S -glutathionylation. This work demonstrates how critical windows of exposure and Nrf2a activity may influence toxicity of DMF and highlights tissue-specific changes in Nrf2a protein levels and S-glutathionylation in pancreatic islet and liver during embryonic development. [Display omitted] • DMF adverse effects on zebrafish morphology were modulated with Nrf2a activity. • Changes at developmental windows with DMF correlated with Nrf2 and glutathionylation. • DMF exposure decreased Nrf2 protein and increased glutathionylation in the islet. • Unlike the islet, liver Nrf2 and glutathionylation results suggests Nrf2 is protective. [ABSTRACT FROM AUTHOR]

Background: It has been four decades since protein S-glutathionylation was proposed to serve as a regulator of cell metabolism. Since then, this redox-sensitive covalent modification has been identified as a cell-wide signaling platform required for embryonic development and regulation of many physiological functions. Scope of the Review: Mitochondria use hydrogen peroxide (H2O2) as a second messenger, but its availability must be controlled to prevent oxidative distress and promote changes in cell behavior in response to stimuli. Experimental data favor the function of protein S-glutathionylation as a feedback loop for the inhibition of mitochondrial H2O2 production. Major conclusions: The glutathione pool redox state is linked to the availability of H2O2, making glutathionylation an ideal mechanism for preventing oxidative distress whilst playing a part in desensitizing mitochondrial redox signals. General Significance: The biological significance of glutathionylation is rooted in redox status communication. The present review critically evaluates the experimental evidence supporting its role in negating mitochondrial H2O2 production for cell signaling and prevention of electrophilic stress. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1 α -dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2 , rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia. Dihydrotanshinone I induces mild ROS generation and facilitates the nuclear translocation of PKM2 via glutathionylation. Glutathionylated PKM2 stabilizes HIF-1 α and potentiates the transcriptional activity of HIF-1 α , contributing to preconditioning protection. [Display omitted] [ABSTRACT FROM AUTHOR]

Redox post-translational modifications are derived from fluctuations in the redox potential and modulate protein function, localization, activity and structure. Amongst the oxidative reversible modifications, the S-glutathionylation of proteins was the first to be characterized as a post-translational modification, which primarily protects proteins from irreversible oxidation. However, a growing body of evidence suggests that S-glutathionylation plays a key role in core cell processes, particularly in mitochondria, which are the main source of reactive oxygen species. S-nitrosylation, another post-translational modification, was identified >150 years ago, but it was re-introduced as a prototype cell-signaling mechanism only recently, one that tightly regulates core processes within the cell's sub-compartments, especially in mitochondria. S-glutathionylation and S-nitrosylation are modulated by fluctuations in reactive oxygen and nitrogen species and, in turn, orchestrate mitochondrial bioenergetics machinery, morphology, nutrients metabolism and apoptosis. In many neurodegenerative disorders, mitochondria dysfunction and oxidative/nitrosative stresses trigger or exacerbate their pathologies. Despite the substantial amount of research for most of these disorders, there are no successful treatments, while antioxidant supplementation failed in the majority of clinical trials. Herein, we discuss how S-glutathionylation and S-nitrosylation interfere in mitochondrial homeostasis and how the deregulation of these modifications is associated with Alzheimer's, Parkinson's, amyotrophic lateral sclerosis and Friedreich's ataxia. [ABSTRACT FROM AUTHOR]

We investigated the nephroprotective effect of D-panthenol in rhabdomyolysis-induced acute kidney injury (AKI). Adult male Wistar rats were injected with 50% glycerol solution to induce rhabdomyolysis. Animals with rhabdomyolysis were injected with D-panthenol (200 mg/kg) for 7 days. On day 8, we examined AKI markers, renal histology, antioxidant capacity, and protein glutathionylation in kidneys to uncover mechanisms of D-panthenol effects. Rhabdomyolysis kidneys were shown to have pathomorphological alterations (mononuclear infiltration, dilatation of tubules, and hyaline casts in Henle's loops and collecting ducts). Activities of skeletal muscle damage markers (creatine kinase and lactate dehydrogenase) increased, myoglobinuria was observed, and creatinine, BUN, and pantetheinase activity in serum and urine rose. Signs of oxidative stress in the kidney tissue of rhabdomyolysis rats, increased levels of lipid peroxidation products, and activities of antioxidant enzymes (SOD, catalase, and glutathione peroxidase) were all alleviated by administration of D-panthenol. Its application improved kidney morphology and decreased AKI markers. Mechanisms of D-panthenol's beneficial effects were associated with an increase in total coenzyme A levels, activity of Krebs cycle enzymes, and attenuation of protein glutathionylation. D-Panthenol protects kidneys from rhabdomyolysis-induced AKI through antioxidant effects, normalization of mitochondrial metabolism, and modulation of glutathione-dependent signaling. [ABSTRACT FROM AUTHOR]

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O2●−/H2O2) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O2●−/H2O2 production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50–1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies [ABSTRACT FROM AUTHOR]

S-nitrosylation of proteins occurs as a consequence of the derivatization of cysteine thiols with nitric oxide (NO) and is often associated with diseases and protein malfunction. Aberrant S-nitrosylation, in addition to other genetic and epigenetic factors, has gained rapid importance as a prime cause of various metabolic, respiratory, and cardiac disorders, with a major emphasis on cancer and neurodegeneration. The S-nitrosoproteome, a term used to collectively refer to the diverse and dynamic repertoire of S-nitrosylated proteins, is relatively less explored in the field of redox biochemistry, in contrast to other covalently modified versions of the same set of proteins. Advancing research is gradually unveiling the enormous clinical importance of S-nitrosylation in the etiology of diseases and is opening up new avenues of prompt diagnosis that harness this phenomenon. Ever since the discovery of the two robust and highly conserved S-nitrosoglutathione reductase and thioredoxin systems as candidate denitrosylases, years of rampant speculation centered around the identification of specific substrates and other candidate denitrosylases, subcellular localization of both substrates and denitrosylases, the position of susceptible thiols, mechanisms of S-denitrosylation under basal and stimulus-dependent conditions, impact on protein conformation and function, and extrapolating these findings towards the understanding of diseases, aging and the development of novel therapeutic strategies. However, newer insights in the ever-expanding field of redox biology reveal distinct gaps in exploring the crucial crosstalk between the redoxins/major denitrosylase systems. Clarifying the importance of the functional overlap of the glutaredoxin, glutathione, and thioredoxin systems and examining their complementary functions as denitrosylases and antioxidant enzymatic defense systems are essential prerequisites for devising a rationale that could aid in predicting the extent of cell survival under high oxidative/nitrosative stress while taking into account the existence of the alternative and compensatory regulatory mechanisms. This review thus attempts to highlight major gaps in our understanding of the robust cellular redox regulation system, which is upheld by the concerted efforts of various denitrosylases and antioxidants. [ABSTRACT FROM AUTHOR]

Seneca virus A (SVA) is a newly discovered small nucleic acid virus, which can cause swine blister disease (PVD). Currently, there is no drug or vaccine. Studies have shown that SVA relies on the endolysosomal pathway to accomplish intracellular transport and release, and can disrupt lysosomal homeostasis, but its specific mechanism has not been revealed. At present, there is limited research on vaccines and antiviral drugs for it. Esterase D (ESD), a serine hydrolase, is active against many substrates and plays a role in inhibiting viral replication. However, the specific mechanism of the thioesterase activity of ESD in the signaling pathway is unknown. In this study, we synthesized and screened a novel small-molecule ESD activator, FPD5 (4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro- 1h -pyrazol-3-yl)phenyl), capable of inhibiting the replication of SVA. To explore the mechanism of action of this process, we demonstrated the direct interaction between ESD and lysosomal membrane protein-1 (LAMP1) by CO-IP; Western blot revealed that FPD5 could activate ESD and exert a protective effect on LAMP1 and lysosomes. The deglutathionization function of ESD was verified by protein glutathione immunoblotting and detection reagents, and the affected cysteine residues were found by point mutation technique. The results showed that FPD5 activated ESD to exert thioesterase activity, reduced glutathionylation cysteine 375 in the LAMP1 and decreased SVA production. This study provides a theoretical basis for the development of small molecule drugs against SVA, and also suggests that the activation of the thioesterase activity of ESD is a new direction for future antiviral drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Redox signaling is a fundamental mechanism that controls all major biological processes partly via protein cysteine oxidations, including S-glutathionylation. Despite over 2000 cysteines identified to form S-glutathionylation in databases, the identification of redox cysteines functionally linked to a biological process of interest remains challenging. Here, we demonstrate a strategy combining glutathionylation proteomic database, bioinformatics, and biological screening, which resulted in the identification of S-glutathionylated proteins, including PP2Cα, as redox players of cell migration. We showed that PP2Cα, a prototypical magnesium-dependent serine/threonine phosphatase, is susceptible to S-glutathionylation selectively at nonconserved C314. PP2Cα glutathionylation causes increased migration and invasion of breast cancer cell lines in oxidative stress or upon hydrogen peroxide production. Mechanistically, PP2Cα glutathionylation modulates its protein-protein interactions, activating c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways to elevate migration and invasion. In addition, PP2Cα glutathionylation occurs in response to epidermal growth factor, supporting a serine/threonine phosphatase PP2Cα as a new redox player in growth factor signal transduction., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This Special Issue of Antioxidants on Glutathione (GSH) and Glutaredoxin (Grx) was designed to collect review articles and original research studies focused on advancing the current understanding of the roles of the GSH/Grx system in cellular homeostasis and disease processes. The tripeptide glutathione (GSH) is the most abundant non-enzymatic antioxidant/nucleophilic molecule in cells. In addition to various metabolic reactions involving GSH and its oxidized counterpart GSSG, oxidative post-translational modification (PTM) of proteins has been a focal point of keen interest in the redox field over the last few decades. In particular, the S-glutathionylation of proteins (protein-SSG formation), i.e., mixed disulfides between GSH and protein thiols, has been studied extensively. This reversible PTM can act as a regulatory switch to interconvert inactive and active forms of proteins, thereby mediating cell signaling and redox homeostasis. The unique architecture of the GSH molecule enhances its relative abundance in cells and contributes to the glutathionyl specificity of the primary catalytic activity of the glutaredoxin enzymes, which play central roles in redox homeostasis and signaling, and in iron metabolism in eukaryotes and prokaryotes under physiological and pathophysiological conditions. The class-1 glutaredoxins are characterized as cytosolic GSH-dependent oxidoreductases that catalyze reversible protein S-glutathionylation specifically, thereby contributing to the regulation of redox signal transduction and/or the protection of protein thiols from irreversible oxidation. This Special Issue includes nine other articles: three original studies and six review papers. Together, these ten articles support the central theme that GSH/Grx is a unique system for regulating thiol-redox hemostasis and redox-signal transduction, and the dysregulation of the GSH/Grx system is implicated in the onset and progression of various diseases involving oxidative stress. Within this context, it is important to appreciate the complementary functions of the GSH/Grx and thioredoxin systems not only in thiol-disulfide regulation but also in reversible S-nitrosylation. Several potential clinical applications have emerged from a thorough understanding of the GSH/Grx redox regulatory system at the molecular level, and in various cell types in vitro and in vivo, including, among others, the concept that elevating Grx content/activity could serve as an anti-fibrotic intervention; and discovering small molecules that mimic the inhibitory effects of S-glutathionylation on dimer association could identify novel anti-viral agents that impact the key protease activities of the HIV and SARS-CoV-2 viruses. Thus, this Special Issue on Glutathione and Glutaredoxin has focused attention and advanced understanding of an important aspect of redox biology, as well as spawning questions worthy of future study.

Acetaminophen (APAP) is one of the most widely used drugs with antipyretic and analgesic effects, and thus hepatotoxicity from the overdose of APAP becomes one of the most common forms of drug-induced liver injury. The reaction towards thiol molecules, such as GSH by APAP metabolite, N-acetyl-p-benzo-quinonimine (NAPQI), is the main cause of APAP-induced hepatotoxicity. However, the role of many other thiol-related regulators in toxicity caused by APAP is still unclear. Here we have found that knockout of the Glrx2 gene, which encodes mitochondrial glutaredoxin2 (Grx2), sensitized mice to APAP-caused hepatotoxicity. Glrx2 deletion hindered Nrf2-mediated compensatory recovery of thiol-dependent redox systems after acetaminophen challenge, resulting in a more oxidized cellular state with a further decrease in GSH level, thioredoxin reductase activity, and GSH/GSSG ratio. The weakened feedback regulation capacity of the liver led to higher levels of protein glutathionylation and thioredoxin (both Trx1 and Trx2) oxidation in Glrx2−/− mice. Following the cellular environment oxidation, nuclear translocation of apoptosis-inducing factor (AIF) was elevated in the liver of Glrx2−/− mice. Taken together, these results demonstrated that mitochondrial Grx2 deficiency deteriorated APAP-induced hepatotoxicity by interrupting thiol-redox compensatory response, enhancing the AIF pathway-mediated oxidative damage. [ABSTRACT FROM AUTHOR]

• AGE-RAGE axis mediated oxidative and inflammatory stress. • Cysteine amino acid of unfolded protein susceptibility to glutathionylation and various amino acids of folded/ nonfolded proteins susceptibility to carbonylation. • Carbonylation and glutathionylation predicted stressed protein activities. • Immunoprecipitation followed by Western blotting inferred glutathionylation measurement of a specific protein. • DNPH derivatization and carbonylated proteins quantification by Western blotting using anti-DNPH antibody. Interaction of advanced glycation end products (AGE) with their receptor i.e. receptor for advanced glycation end products (RAGE), better understood as AGE-RAGE axis, generates oxidative and inflammatory stress. The generated stress extent, in turn aggravates the AGE and RAGE levels through a vicious self propagation cycle. The associated oxidation and inflammation culminates in modifications and subsequent detrimental state of cellular macromolecules, including nucleic acids and proteins, manifesting multiple diseased conditions. Under normal physiological state, fewer carbonyl group(s) and glutathione, a tripeptide antioxidant may be added to proteins during post-translational modifications, recognized as carbonylation and glutathionylation, respectively. However, under oxidative and inflammatory stress conditions, protein carbonylation and glutathionylation are caused to considerably greater extents, leading to numerous diseased complications. Thereby, increased protein carbonylation and glutathionylation could be used as predictive markers of oxidative and inflammatory stress. The AGE-RAGE axis generated oxidatively modified proteins can be screened via assessing the protein carbonylation and glutathionylation. The present article focuses on most widely used protein carbonylation and glutathionylation based assays for quantifying the AGE-RAGE axis mediated oxidative and inflammatory stress. [ABSTRACT FROM AUTHOR]

In premature infants receiving parenteral nutrition, oxidative stress is a trigger for the development of bronchopulmonary dysplasia, which is an important factor in the development of adult lung diseases. Neonatal vitamin C and glutathione deficiency is suspected to induce permanent modification of redox metabolism favoring the development of neonatal and adult lung diseases. A total of 64 3-day-old guinea pigs were fed an oral diet that was either complete or deficient in vitamin C (VCD), cysteine (CD) (glutathione-limiting substrate) or both (DD) for 4 days. At 1 week of age, half of the animals were sacrificed while the other started a complete diet until 12 weeks of age. At 1 week, the decrease in lung GSH in all deficient groups was partially explained by the oxidation of liver methionine-adenosyltransferase. mRNA levels of kelch-like ECH-associated protein 1 (Keap1), glutathione-reductase (Gsr) and glutaredoxin-1 (Glrx) were significantly lower only in CD but not in DD. At 12 weeks, glutathione levels were increased in VCD and CD. Keap1, Gsr and Glrx mRNA were increased, while glutathione-reductase and glutaredoxin proteins were lower in CD, favoring a higher glutathionylation status. Both neonatal deficiencies result in a long-term change in glutathione metabolism that could contribute to lung diseases’ development.

Dysregulated redox balance is involved in the pathogenesis of type 2 diabetes. While the benefit of antioxidants in neutralizing oxidative stress is well characterized, the potential harm of antioxidant-induced reductive stress is unclear. The aim of this study was to investigate the dose-dependent effects of the antioxidant N-acetylcysteine (NAC) on various tissues involved in the regulation of blood glucose and the mechanisms underlying its functions. H2O2 was used as an oxidizing agent in order to compare the outcomes of oxidative and reductive stress on cellular function. Cellular death in pancreatic islets and diminished insulin secretion were facilitated by H2O2-induced oxidative stress but not by NAC. On the other hand, myotubes and adipocytes were negatively affected by NAC-induced reductive stress, as demonstrated by the impaired transmission of insulin signaling and glucose transport, as opposed to H2O2-stimulatory action. This was accompanied by redox balance alteration and thiol modifications of proteins. The NAC-induced deterioration of insulin signaling was also observed in healthy mice, while both insulin secretion and insulin signaling were improved in diabetic mice. This study establishes the tissue-specific effects of NAC and the importance of the delicate maintenance of redox balance, emphasizing the challenge of implementing antioxidant therapy in the clinic. [ABSTRACT FROM AUTHOR]

Glutathionylation and cysteinylation can be involved in the protection of critical cysteines from irreversible oxidative damages. S100A9 long and cystatin B, proteins highly represented in the saliva of preterm and at‐term newborns, can undergo these modifications. Levels of S100A9 long and cystatin B and their glutathionylated and cysteinylated derivatives have been determined by a top‐down platform based on high‐performance liquid chromatography–electrospray ionization–mass spectrometry in 100 salivary samples serially collected from 17 preterm newborns with different postconceptional age at birth (178‐226 days) and in 90 salivary samples collected from at‐term newborns and babies. Results showed that: (1) S100A9 long and cystatin B were mainly present as unmodified forms in extremely preterm newborn; (2) the percentage of the S‐thiolated derivatives of both proteins increased with increasing the postconceptional age; (3) the greatest variation occurred up to about 280 days of postconceptional age. Interestingly, differences in the levels of the S‐thiolated derivatives only depended on the postconceptional age and not on whether the infant was born preterm or at‐term. Inadequate levels of cysteine and glutathione might be responsible for the low level of S‐thiolated derivatives measured in preterm newborns. Data are available via ProteomeXchange with identifier PXD025517. [ABSTRACT FROM AUTHOR]

Background Macrophage polarization affects tumor growth, metabolism, and many other tumor processes. M1 macrophages can promote antitumor immunity response. Signal transducer and activator of transcription 1 (STAT1) is one of the critical transcription factors in this process, which promotes the expression of a series of inflammatory molecules. STAT1 has been reported as a potential target of reactive oxygen species (ROS)‐induced glutathionylation, while the glutathionylation of STAT1 in macrophages and its underlying regulatory mechanism remains unclear. Glutaredoxin 1 (Grx1) functions as a deglutathionylation enzyme, which regulates the activities of many proteins through reversing glutathionylation. Methods GeneChip and RT‐qPCR was first applied to test the mRNA level of Grx1 in M1 macrophages. Western blot was then used to evaluate the variations of the Grx1 protein expression in M1 macrophages. Next, immunoprecipitation was used to investigate the glutathionylated STAT1 in both wild‐type and Grx1−/− mouse macrophages. Finally, the induced alterations of STAT1 activity and function by Grx1 in M1 macrophage were examined by western blot and RT‐qPCR. Results In M1‐type macrophages, the levels of Grx1 were elevated. Glutathionylation of STAT1 was negatively regulated by Grx1. Furthermore, depletion of Grx1 increased the activity of STAT1, and thereby promoted the mRNA level of C‐X‐C motif chemokine ligand 9 (CXCL9) during M1‐type polarization of macrophages. Conclusions Grx1 controlled deglutathionylation of STAT1, which in turn might regulate M1‐type polarization of macrophages.

Our group recently documented that male mice containing a deletion for one copy of the glutaredoxin-2 (Grx2) gene were completely protected from developing diet-induced obesity (DIO). Objectives: Here, we conducted a similar investigation but with female littermates. Results: In comparison to our recent publication using male mice, exposure of WT and GRX2+/- female mice to a HFD from 3-to-10 weeks of age did not induce any changes in body mass, circulating blood glucose, food intake, hepatic glycogen levels, or abdominal fat pad mass. Examination of the bioenergetics of muscle mitochondria revealed no changes in the rate of superoxide (O2●−)/hydrogen peroxide (H2O2) or O2 consumption under different states of respiration or alterations in lipid peroxidation adduct levels regardless of mouse strain or diet. Additionally, we measured the bioenergetics of mitochondria isolated from liver tissue and found that partial loss of GRX2 augmented respiration but did not alter ROS production. Discussion: Overall, our findings demonstrate there are sex differences in the protection of female GRX2+/- mice from DIO, fat accretion, intrahepatic lipid accumulation, and the bioenergetics of mitochondria from muscle and liver tissue.

The tripeptide glutathione is found in all eukaryotic cells, and due to the compartmentalization of biochemical processes, its synthesis takes place exclusively in the cytosol. At the same time, its functions depend on its transport to/from organelles and interorgan transport, in which the liver plays a central role. Glutathione is determined as a marker of the redox state in many diseases, aging processes, and cell death resulting from its properties and reactivity. It also uses other enzymes and proteins, which enables it to engage and regulate various cell functions. This paper approximates the role of these systems in redox and detoxification reactions such as conjugation reactions of glutathione-S-transferases, glyoxylases, reduction of peroxides through thiol peroxidases (glutathione peroxidases, peroxiredoxins) and thiol–disulfide exchange reactions catalyzed by glutaredoxins.

Cardiovascular disease (CVD) is one of the leading causes of mortality in humans, and oxidative stress plays a pivotal role in disease progression. This phenomenon typically arises from weakening of the cellular antioxidant system or excessive accumulation of peroxides. This review focuses on a specialized form of oxidative stress-disulfide stress-which is triggered by an imbalance in the glutaredoxin and thioredoxin antioxidant systems within the cell, leading to the accumulation of disulfide bonds. The genesis of disulfide stress is usually induced by extrinsic pathological factors that disrupt the thiol-dependent antioxidant system, manifesting as sustained glutathionylation of proteins, formation of abnormal intermolecular disulfide bonds between cysteine-rich proteins, or irreversible oxidation of thiol groups to sulfenic and sulfonic acids. Disulfide stress not only precipitates the collapse of the antioxidant system and the accumulation of reactive oxygen species, exacerbating oxidative stress, but may also initiate cellular inflammation, autophagy, and apoptosis through a cascade of signaling pathways. Furthermore, this review explores the detrimental effects of disulfide stress on the progression of various CVDs including atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, diabetic cardiomyopathy, cardiac hypertrophy, and heart failure. This review also proposes several potential therapeutic avenues to improve the future treatment of CVDs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Increased sirtuin deacylase activity is correlated with increased lifespan and healthspan in eukaryotes. Conversely, decreased sirtuin deacylase activity is correlated with increased susceptibility to aging-related diseases. However, the mechanisms leading to decreased sirtuin activity during aging are poorly understood. Recent work has shown that oxidative post-translational modification by reactive oxygen (ROS) or nitrogen (RNS) species results in inhibition of sirtuin deacylase activity through cysteine nitrosation, glutathionylation, sulfenylation, and sulfhydration as well as tyrosine nitration. The prevalence of ROS/RNS (e.g., nitric oxide, S -nitrosoglutathione, hydrogen peroxide, oxidized glutathione, and peroxynitrite) is increased during inflammation and as a result of electron transport chain dysfunction. With age, cellular production of ROS/RNS increases; thus, cellular oxidants may serve as a causal link between loss of sirtuin activity and aging-related disease development. Therefore, the prevention of inhibitory oxidative modification may represent a novel means to increase sirtuin activity during aging. In this review, we explore the role of cellular oxidants in inhibiting individual sirtuin human isoform deacylase activity and clarify the relevance of ROS/RNS as regulatory molecules of sirtuin deacylase activity in the context of health and disease. [ABSTRACT FROM AUTHOR]

Our group has found that protein S-glutathionylation serves as an important feedback inhibitor for superoxide (O 2 ●-)/hydrogen peroxide (H 2 O 2) production by several mitochondrial dehydrogenases. Since cytoplasmic oxidases can also serve as important reactive oxygen species (ROS) sources, we hypothesized that glutathionylation can also inhibit O 2 ●-/H 2 O 2 by these enzymes. We first focused our attention on using a purified xanthine oxidase (XO) of bacterial origin to discern if glutathionylation can shut down ROS production by this enzyme. Incubating XO in glutathione disulfide (GSSG) at a final concentration of 1 mM did not significantly alter ROS production. Additionally, incubating samples in up to 10 mM GSSG increased ROS production. However, diamide and disulfiram titrations in the presence of 1 mM GSH revealed that both glutathionylation catalysts were able to abolish O 2 ●-/H 2 O 2 by XO. Exposure of XO to glutaredoxin-1 (GRX1) and GSSG did not alter the rate of O 2 ●-/H 2 O 2 production. However, incubation with GSH and purified glutathione S-transferase (GST) almost abolished ROS production by XO. Similar results were collected with rat liver cytoplasm. Indeed, diamide and disulfiram significantly decreased ROS production by xanthine oxidoreductase (XOR). Additionally, incubating the cytoplasm in GSH and GST led to a significant decrease in XOR activity. Immunoblot analyses revealed that immunoreactive bands corresponding to XOR were glutathionylated by diamide. Collectively, our findings demonstrate for the first time that cytoplasmic ROS sources, such as XOR, can also be inhibited by glutathionylation and these reactions are enzymatically mediated by GST. Additionally, we found that bacterial XO is also a target for glutathionylation. [Display omitted] • ROS production by bacterial XO is inhibited by glutathionylation. • ROS production by rat liver XOR is inhibited by glutathionylation. • GST glutathionylates XO and XOR. [ABSTRACT FROM AUTHOR]

• Cd2+ inhibited the most of basic metabolism pathways except lipid metabolism. • Seven transcription factors with glutathionylated sites linked to TAG metabolism. • Transcription factors MYB, NF-YB and ABI5 regulated DGAT expression. • Triacylglycerol maintained lipid homeostasis by balancing fatty acids saturation. • Microalgae activated TAG synthesis by GSH then resisted the Cd stress together. Under ultra-high cadmium (Cd) stress, large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341, and the lipid content increases significantly. Glutathione is the best reductant that can effectively remove Cd, but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained. Integrating analyses of the transcriptomes and lipidomes, the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism, transcriptional regulation and protein glutathionylation. Under Cd stress, basic metabolic pathways, such as purine metabolism, translation and pre-mRNA splicing process, were inhibited, while the lipid accumulation pathway was significantly activated. Further analysis revealed that the transcription factors (TFs) and genes related to lipid accumulation were also activated. Analysis of the TF interaction sites showed that ABI5, MYB_rel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation, which led to increase of the triacylglycerol (TAG) content. Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels. This study for the first time indicated that glutathione could activate TAG synthesis in microalga A. protothecoides , leading to TAG accumulation and glutathione accumulation under Cd stress. Therefore, the accumulation of TAG and glutathione can confer resistance to high Cd stress. This study provided insights into a new operation mode of TAG accumulation under heavy metal stress. [Display omitted] [ABSTRACT FROM AUTHOR]

Increased sirtuin deacylase activity is correlated with increased lifespan and healthspan in eukaryotes. Conversely, decreased sirtuin deacylase activity is correlated with increased susceptibility to aging-related diseases. However, the mechanisms leading to decreased sirtuin activity during aging are poorly understood. Recent work has shown that oxidative post-translational modification by reactive oxygen (ROS) or nitrogen (RNS) species results in inhibition of sirtuin deacylase activity through cysteine nitrosation, glutathionylation, sulfenylation, and sulfhydration as well as tyrosine nitration. The prevalence of ROS/RNS (e.g., nitric oxide, S-nitrosoglutathione, hydrogen peroxide, oxidized glutathione, and peroxynitrite) is increased during inflammation and as a result of electron transport chain dysfunction. With age, cellular production of ROS/RNS increases; thus, cellular oxidants may serve as a causal link between loss of sirtuin activity and aging-related disease development. Therefore, the prevention of inhibitory oxidative modification may represent a novel means to increase sirtuin activity during aging. In this review, we explore the role of cellular oxidants in inhibiting individual sirtuin human isoform deacylase activity and clarify the relevance of ROS/RNS as regulatory molecules of sirtuin deacylase activity in the context of health and disease.

Background: It has been four decades since protein S-glutathionylation was proposed to serve as a regulator of cell metabolism. Since then, this redox-sensitive covalent modification has been identified as a cell-wide signaling platform required for embryonic development and regulation of many physiological functions. Scope of the Review: Mitochondria use hydrogen peroxide (H2O2) as a second messenger, but its availability must be controlled to prevent oxidative distress and promote changes in cell behavior in response to stimuli. Experimental data favor the function of protein S-glutathionylation as a feedback loop for the inhibition of mitochondrial H2O2 production. Major conclusions: The glutathione pool redox state is linked to the availability of H2O2, making glutathionylation an ideal mechanism for preventing oxidative distress whilst playing a part in desensitizing mitochondrial redox signals. General Significance: The biological significance of glutathionylation is rooted in redox status communication. The present review critically evaluates the experimental evidence supporting its role in negating mitochondrial H2O2 production for cell signaling and prevention of electrophilic stress.

Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O2●−/H2O2) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O2●−/H2O2 production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50–1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.