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1.  Modulation of Glutaredoxin-1 Expression in a Mouse Model of Allergic Airway Disease 
Glutaredoxins (GRX) are antioxidant enzymes that preferentially catalyze the reduction of protein-glutathione mixed disulfides. The formation of mixed disulfides with GSH is known as S-glutathionylation, a post-translational modification that is emerging as an important mode of redox signaling. Since asthma is a disease that is associated with increased oxidative stress and altered antioxidant defenses, we investigated the expression of GRX in a murine model of allergic airway disease. Sensitization and challenge of C57BL/6 mice with ovalbumin resulted in increased expression of GRX1 mRNA, as well as increased amounts of GRX1 protein and total GRX activity in the lung. Because GRX1 expression is prominent in bronchial epithelium, we isolated primary epithelial cells from mouse trachea to investigate the presence of GRX. Primary tracheal epithelial cells were found to express both GRX1 and 2 mRNA and detectable GRX activity. Treatment with IFN-γ increased the expression of GRX1 and overall GRX activity, resulting in attenuation of protein S-glutathionylation. In contrast, TGF-β1 caused decreased GRX1 expression and overall GRX activity, leading to markedly enhanced protein S-glutathionylation. GRX1 joins the cadre of antioxidant defenses known to be modulated during allergic airway inflammation.
PMCID: PMC1899315  PMID: 16980552
glutaredoxin; asthma; epithelium; IFN-γ; TGF-β
2.  Molecular Mechanisms and Clinical Implications of Reversible Protein S-Glutathionylation 
Antioxidants & Redox Signaling  2008;10(11):1941-1988.
Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol–disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein–SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein–SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein–SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein–thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases. Antioxid. Redox Signal, 10, 1941–1988.
Potential Mechanisms of Protein–SSG Formation
Thiol-disulfide exchange
Sulfenic acid intermediates
Sulfenylamide intermediates
Thiyl radical intermediates
Thiosulfinate intermediates
S-Nitrosylated intermediates
Potential Catalysis of Protein Glutathionylation
Flavoprotein sulfhydryl oxidease (QSOX)
Other potential mechanisms of catalysis/control of protein S-glutathionylation
Proteomics of Discovery of Potential Protein–SSG Intermediates
Deglutathionylation (Reversal) of Protein–SSG: Properties of the Glutaredoxin Enzymes
Glutaredoxin Mechanism of Action
Modualtion of Grx Expression
Diabetes and Implications of Changes in S-Glutathionylation Status
Mechanism of hyperglycemic damage and ROS
Insulin-glucose dynamics and diabetes complications
Glucose metabolism: aldose reductase–SSG (Fig. 3, step 1a)
K+ channels: Grx regulated (Fig. 3, step 2a)
ATP-sensitive potassium channels
Voltage-gated potassium channels
Ca2+ channels: SERCA-SSG and Grx-reversible RyR-SSG (Fig. 3, step 3a)
Insulin exocytosis: Grx regulated (Fig. 3 step 6a)
Insulin receptor: Grx-reversible PTP1B-SSG (Fig. 3, step 6b)
Signal transduction [Fig. 3, Ras-SSG (step 7b), MEKK-SSG (step 8b), c-Jun-SSG (step 9b), Akt-SSG (step 10b), IKK-SSG (step 11b), NF-κB(p50)-SSG (steps 5a and 12b), and PKC-SSG (step 4a)]
Summary and discussion: Grx as a therapeutic target in diabetic complications
Cardiovascular Diseases and Alterations in Protein-S-Glutathionylation Status
Myocardial infarction
Protein kinase C (PKC)
Protein kinase A (PKA)
Nuclear factor κB (NF-κB)
Nonspecific oxidative injury
Cardiac hypertrophy
Implications of Protein S-Glutathionylation in Lung Disease
Tobacco exposure
Hyperoxic injury
Fibrotic and granulomatous diseases
Chronic obstructuve pulmonary disease (COPD)
Implications of Reversible Protein S-Glutathionylation in Cancer
Thiol oxidation and cancer
S-Glutathionylation and signal transduction in cancer
S-Glutathionylation and modulation of kinase/phosphatase signaling pathways
Protein kinase C (PKC)
I3 kinase and Akt
Protein tryosine phosphatase
c-Jun N-terminal kinase (JKN)
S-Glutathionylation and modulation of the proteasome pathway
S-Glutathionylation and modulation of transcription factors (c-Jun, NF-κB, p53, AP-1)
AP-1, c-Jun
Modulation of S-glutathionylation as a chemotherapeutic strategy for cancer
Implications of Protein S-Glutathionylation in Neurodegenerative Diseases
Oxidative stress and neurodegeneration
Sources of reactive oxygen and nitrogen species in brain
Alzheimer's disease
Parkinson's disease
Huntington's disease
Amyotrophic lateral sclerosis
Freidreich's ataxia
Glutaredoxin and neurodegeneration
Proteins associated with neurodegeneration that are redox regulated through S-glutathionylation
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm)
Tyrosine hydroxylase
Cytosolic calcium regulators
Proteasome degradation pathway
α-Ketoglutarate dehydrogenase
Summary and Conclusions
Frontier Areas of Investigation
PMCID: PMC2774718  PMID: 18774901
3.  Redox Modulation of eNOS by Glutaredoxin-1 through Reversible Oxidative Post-translational Modification† 
Biochemistry  2013;52(38):10.1021/bi400404s.
S-glutathionylation is a redox-regulated modification that uncouples eNOS, switching its function from NO synthesis to •O2− generation, and serves to regulate vascular function. While in vitro or in vivo eNOS S-glutathionylation with modification of Cys689 and Cys908 of its reductase domain is triggered by high levels of GSSG or oxidative thiyl radical formation, it remains unclear how this process may be reversed. Glutaredoxin-1 (Grx1), a cytosolic/glutathione-dependent enzyme, can reverse protein S-glutathionylation; however, its role in regulating eNOS S-glutathionylation remains unknown. We demonstrate that Grx1 in the presence of GSH (1 mM) reverses GSSG-mediated eNOS S-glutathionylation with restoration of NO synthase activity. Since Grx1 also catalyzes protein S-glutathionylation with increased [GSSG]/[GSH], we measured its effect on eNOS-S-glutathionylation when [GSSG]/[GSH] was > 0.2, as can occur in cells and tissues under oxidative stress, and observed increased eNOS S-glutathionylation with a marked decrease in eNOS activity without uncoupling. This eNOS S-glutathionylation was reversed with decrease in [GSSG]/[GSH] to < 0.1. LC/MS/MS identified a new site of eNOS S-glutathionylation by Grx1 at Cys382, on the surface of the oxygenase domain, without modification of Cys689 or Cys908 that are buried within the reductase. Furthermore, Grx1 was demonstrated to be a protein partner of eNOS in vitro and in normal endothelial cells, supporting its role in eNOS redox-regulation. In endothelial cells, Grx1 inhibition or gene silencing increased eNOS S-glutathionylation and decreased cellular NO generation. Thus, Grx1 can exert an important role in the redox-regulation of eNOS in cells.
PMCID: PMC3813969  PMID: 23977830
4.  Glutaredoxin 1 Protects Dopaminergic Cells by Increased Protein Glutathionylation in Experimental Parkinson's Disease 
Antioxidants & Redox Signaling  2012;17(12):1676-1693.
Aims: Chronic exposure to environmental toxicants, such as paraquat, has been suggested as a risk factor for Parkinson's disease (PD). Although dopaminergic cell death in PD is associated with oxidative damage, the molecular mechanisms involved remain elusive. Glutaredoxins (GRXs) utilize the reducing power of glutathione to modulate redox-dependent signaling pathways by protein glutathionylation. We aimed to determine the role of GRX1 and protein glutathionylation in dopaminergic cell death. Results: In dopaminergic cells, toxicity induced by paraquat or 6-hydroxydopamine (6-OHDA) was inhibited by GRX1 overexpression, while its knock-down sensitized cells to paraquat-induced cell death. Dopaminergic cell death was paralleled by protein deglutathionylation, and this was reversed by GRX1. Mass spectrometry analysis of immunoprecipitated glutathionylated proteins identified the actin binding flightless-1 homolog protein (FLI-I) and the RalBP1-associated Eps domain-containing protein 2 (REPS2/POB1) as targets of glutathionylation in dopaminergic cells. Paraquat induced the degradation of FLI-I and REPS2 proteins, which corresponded with the activation of caspase 3 and cell death progression. GRX1 overexpression reduced both the degradation and deglutathionylation of FLI-I and REPS2, while stable overexpression of REPS2 reduced paraquat toxicity. A decrease in glutathionylated proteins and REPS2 levels was also observed in the substantia nigra of mice treated with paraquat. Innovation: We have identified novel protein targets of glutathionylation in dopaminergic cells and demonstrated the protective role of GRX1-mediated protein glutathionylation against paraquat-induced toxicity. Conclusions: These results demonstrate a protective role for GRX1 and increased protein glutathionylation in dopaminergic cell death induced by paraquat, and identify a novel protective role for REPS2. Antioxid. Redox Signal. 17, 1676–1693.
PMCID: PMC3474191  PMID: 22816731
5.  Altered Cigarette Smoke-Induced Lung Inflammation Due to Ablation of Grx1 
PLoS ONE  2012;7(6):e38984.
Glutaredoxins (Grx) are redox enzymes that remove glutathione bound to protein thiols, know as S-glutathionylation (PSSG). PSSG is a reservoir of GSH and can affect the function of proteins. It inhibits the NF-κB pathway and LPS aspiration in Grx1 KO mice with decreased inflammatory cytokine levels. In this study we investigated whether absence of Grx1 similarly repressed cigarette smoke-induced inflammation in an exposure model in mice. Cigarette smoke exposure for four weeks decreased lung PSSG levels, but increased PSSG in lavaged cells and lavage fluid (BALF). Grx1 KO mice had increased levels of PSSG in lung tissue, BALF and BAL cells in response to smoke compared to wt mice. Importantly, levels of multiple inflammatory mediators in the BALF were decreased in Grx1 KO animals following cigarette smoke exposure compared to wt mice, as were levels of neutrophils, dendritic cells and lymphocytes. On the other hand, macrophage numbers were higher in Grx1 KO mice in response to smoke. Although cigarette smoke in vivo caused inverse effects in inflammatory and resident cells with respect to PSSG, primary macrophages and epithelial cells cultured from Grx1 KO mice both produced less KC compared to cells isolated from WT mice after smoke extract exposure. In this manuscript, we provide evidence that Grx1 has an important role in regulating cigarette smoke-induced lung inflammation which seems to diverge from its effects on total PSSG. Secondly, these data expose the differential effect of cigarette smoke on PSSG in inflammatory versus resident lung cells.
PMCID: PMC3377591  PMID: 22723915
6.  Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas 
The Journal of Cell Biology  2009;184(2):241-252.
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase–induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.
PMCID: PMC2654302  PMID: 19171757
7.  Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: from Cofactors to Antioxidants to Redox Signaling 
Antioxidants & Redox Signaling  2013;19(13):1539-1605.
Thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs) have been characterized as electron donors, guards of the intracellular redox state, and “antioxidants”. Today, these redox catalysts are increasingly recognized for their specific role in redox signaling. The number of publications published on the functions of these proteins continues to increase exponentially. The field is experiencing an exciting transformation, from looking at a general redox homeostasis and the pathological oxidative stress model to realizing redox changes as a part of localized, rapid, specific, and reversible redox-regulated signaling events. This review summarizes the almost 50 years of research on these proteins, focusing primarily on data from vertebrates and mammals. The role of Trx fold proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners. On the basis of this analysis, the specific regulatory functions are exemplified for the cellular processes of apoptosis, proliferation, and iron metabolism. The importance of Trxs, Grxs, and Prxs for human health is addressed in the second part of this review, that is, their potential impact and functions in different cell types, tissues, and various pathological conditions. Antioxid. Redox Signal. 19, 1539–1605.
I. Introduction
A. Trx family of proteins
1. Structure and reaction mechanisms
2. Trx, Grx, and Prx family proteins in mammals
a. Trx systems
b. Grx systems
c. Peroxiredoxins
d. Trx-like proteins
B. The concept of redox signaling
C. Reversible post-translational redox modifications of protein thiols
1. Sulfenylation
2. Protein disulfides
3. Glutathionylation and cysteinylation
4. S-nitrosylation
5. Other reversible redox modifications
a. Persulfide formation
b. Methionine sulfoxidation
D. Oxidative stress in the concept of redox signaling
II. Mammalian Trx Family Proteins in Health and Disease
A. Specific pathways
1. Apoptosis
a. Cytosolic pathways
b. Mitochondrial pathways
2. Proliferation
3. Iron metabolism
a. Iron sulfur Grxs
b. Biogenesis of iron-sulfur centers
c. Regulation of iron metabolism
d. Intracellular iron distribution
B. Tissues, organ systems, and diseases
1. Development
2. Central nervous system
a. Expression profile of Trxs, Grxs, Prxs, and related proteins in the CNS
b. Trxs, Grxs, Prxs, and pathologies of the CNS
3. Sensory organs
a. Expression profile of Trx-related proteins in sensory organs
b. Pathologies of the eye
c. Pathologies related to tongue, olfactory system, and ear
4. Cardiovascular system
a. Expression pattern of Trxs, Grxs, and Prxs in cardiovascular tissue
b. Trxs, Grxs, and Prxs in pathologies of the cardiovascular system
5. Skin
6. Skeletal muscle
7. Respiratory system
a. Expression of Trx family proteins in the respiratory system
b. Trxs, Grxs, and Prxs in pathologies of the lung—interplay between ROS and inflammation
8. Infection, inflammation, and immune response
a. Expression pattern of Trx-related proteins in lymphoid tissues
b. Immune system
c. Infectious diseases
9. Metabolic and digestive system
a. Diabetes mellitus
10. Urinary tract and reproductive systems
a. Kidney
b. Urinary bladder
c. Male reproductive system
d. Female reproductive system
11. Ischemia and hypoxia
12. Cancer
a. Carcinogenesis
13. Aging
C. Therapeutic approaches
III. Concluding Remarks
PMCID: PMC3797455  PMID: 23397885
8.  Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia† 
Free radical biology & medicine  2007;43(9):1299-1312.
To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione (protein-SSG), we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively; suggesting that changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury. On the other hand, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H2O2) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H2O2 treatment and retarded deglatuthionylation of the S-glutathionylated proteins, especially evident for an unspecified protein of approximately 44 kDa. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor α plus actinomycin D. These findings suggest that different oxidants may damage the cells via distinct mechanisms in which Grx1-dependent de-glutathionylation may or may not be protective, and Grx1 may exert its function on specific target proteins.
PMCID: PMC2196211  PMID: 17893043
Reaction oxygen species; protein glutathionylation; thiol oxidation; cell death; gene targeting
9.  Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease 
Respiratory Research  2006;7(1):133.
One typical feature in chronic obstructive pulmonary disease (COPD) is the disturbance of the oxidant/antioxidant balance. Glutaredoxins (Grx) are thiol disulfide oxido-reductases with antioxidant capacity and catalytic functions closely associated with glutathione, the major small molecular weight antioxidant of human lung. However, the role of Grxs in smoking related diseases is unclear.
Immunohistochemical and Western blot analyses were conducted with lung specimens (n = 45 and n = 32, respectively) and induced sputum (n = 50) of healthy non-smokers and smokers without COPD and at different stages of COPD.
Grx1 was expressed mainly in alveolar macrophages. The percentage of Grx1 positive macrophages was significantly lower in GOLD stage IV COPD than in healthy smokers (p = 0.021) and the level of Grx1 in total lung homogenate decreased both in stage I–II (p = 0.045) and stage IV COPD (p = 0.022). The percentage of Grx1 positive macrophages correlated with the lung function parameters (FEV1, r = 0.45, p = 0.008; FEV1%, r = 0.46, p = 0.007, FEV/FVC%, r = 0.55, p = 0.001). Grx1 could also be detected in sputum supernatants, the levels being increased in the supernatants from acute exacerbations of COPD compared to non-smokers (p = 0.013) and smokers (p = 0.051).
The present cross-sectional study showed that Grx1 was expressed mainly in alveolar macrophages, the levels being decreased in COPD patients. In addition, the results also demonstrated the presence of Grx1 in extracellular fluids including sputum supernatants. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH in human lung.
PMCID: PMC1633737  PMID: 17064412
10.  Glutaredoxin-1 Attenuates S-Glutathionylation of the Death Receptor Fas and Decreases Resolution of Pseudomonas aeruginosa Pneumonia 
Rationale: The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection.
Objectives: Fas ligand (FasL)–induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection.
Methods: Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1−/−), Glrx1−/− mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa.
Measurements and Main Results: Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1−/− cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1−/− mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals.
Conclusions: These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.
PMCID: PMC3977722  PMID: 24325366
Pseudomonas; glutaredoxin-1; protein S-glutathionylation; Fas; apoptosis
11.  Kinetic and Mechanistic Characterization and Versatile Catalytic Properties of Mammalian Glutaredoxin 2: Implications for Intracellular Roles† 
Biochemistry  2008;47(42):11144-11157.
Glutaredoxin (Grx)-catalyzed deglutathionylation of protein–glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix [Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027–2033]. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency (kcat/KM) of Grx2 for protein deglutathionylation (due primarily to a decreased kcat), reflecting a higher pKa of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 [Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607–14613], hGrx2 catalyzes glutathione-thiyl radical (GS•) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS• serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower kcat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.
PMCID: PMC3569056  PMID: 18816065
12.  S-Glutathionylation: From Molecular Mechanisms to Health Outcomes 
Antioxidants & Redox Signaling  2011;15(1):233-270.
Redox homeostasis governs a number of critical cellular processes. In turn, imbalances in pathways that control oxidative and reductive conditions have been linked to a number of human disease pathologies, particularly those associated with aging. Reduced glutathione is the most prevalent biological thiol and plays a crucial role in maintaining a reduced intracellular environment. Exposure to reactive oxygen or nitrogen species is causatively linked to the disease pathologies associated with redox imbalance. In particular, reactive oxygen species can differentially oxidize certain cysteine residues in target proteins and the reversible process of S-glutathionylation may mitigate or mediate the damage. This post-translational modification adds a tripeptide and a net negative charge that can lead to distinct structural and functional changes in the target protein. Because it is reversible, S-glutathionylation has the potential to act as a biological switch and to be integral in a number of critical oxidative signaling events. The present review provides a comprehensive account of how the S-glutathionylation cycle influences protein structure/function and cellular regulatory events, and how these may impact on human diseases. By understanding the components of this cycle, there should be opportunities to intervene in stress- and aging-related pathologies, perhaps through prevention and diagnostic and therapeutic platforms. Antioxid. Redox Signal. 15, 233–270.
I. Introduction
A. Glutathione homeostasis
B. Proximal donors for S-glutathionylation reactions
II. Detection of S-Glutathionylation
A. Antibody detection of S-glutathionylation
B. Analytical detection and quantification of P-SSG
III. Enzymes That Catalyze the S-Glutathionylation Cycle
A. Proteins with S-glutathionylase activity
1. Glutathione S-transferases
2. Gamma-glutamyl transpeptidase
3. Grx1 and Grx2
B. Proteins with deglutathionylase activity
IV. Redox Regulation of Kinase Signaling Pathways
A. S-glutathionylation and modulation of mitogenic signaling
1. Ras-MEK-ERK pathway
2. Protein tyrosine phosphatases
3. Protein kinase A
B. Phosphatidylinositol 3-kinase-Akt-p53 pathway
C. I kappa B kinase-nuclear factor kappa B pathway
D. JNK-c-Jun pathway
V. S-Glutathionylation and Modulation of Survival/Apoptosis
A. S-glutathionylation of death receptors
B. S-glutathionylation of caspases
VI. Redox Regulation of Calcium-Dependent Proteins
A. Protein kinase C
B. Sarco/ER calcium ATPase
C. Nitric oxide synthase
VII. S-Glutathionylation and Ubiquitin-Proteasome Pathway
VIII. S-Glutathionylation and Unfolded Protein Response
A. Signaling pathways in the unfolded protein response
B. Protein disulfide isomerase
IX. Redox Regulation of Cell Migration and Mobilization
A. S-glutathionylation of cytoskeletal proteins
B. Redox regulation of bone marrow mobilization
X. Cancer and Redox Homeostasis
A. Energy metabolism
B. S-glutathionylation and tumor metastasis
C. S100 proteins in cancer and leukocyte migration
1. S100B
2. S100A8 and S100A9
XI. Redox Dysregulation in Pathophysiology
A. Liver injury
B. Diabetes mellitus
C. Cardiovascular disease
D. Traumatic brain injury
XII. Neurological Diseases and Redox Pathways
A. Parkinson's disease
B. Alzheimer's disease
C. Huntington's disease
D. Friedreich's ataxia
E. Amylotrophic lateral sclerosis
XIII. Conclusions
PMCID: PMC3110090  PMID: 21235352
13.  Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases—Focus on S-Glutathionylation 
Antioxidants & Redox Signaling  2012;16(6):543-566.
Significance: Neurodegenerative diseases are characterized by progressive loss of neurons. A common feature is oxidative stress, which arises when reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) exceed amounts required for normal redox signaling. An imbalance in ROS/RNS alters functionality of cysteines and perturbs thiol–disulfide homeostasis. Many cysteine modifications may occur, but reversible protein mixed disulfides with glutathione (GSH) likely represents the common steady-state derivative due to cellular abundance of GSH and ready conversion of cysteine-sulfenic acid and S-nitrosocysteine precursors to S-glutathionylcysteine disulfides. Thus, S-glutathionylation acts in redox signal transduction and serves as a protective mechanism against irreversible cysteine oxidation. Reversal of protein-S-glutathionylation is catalyzed specifically by glutaredoxin which thereby plays a critical role in cellular regulation. This review highlights the role of oxidative modification of proteins, notably S-glutathionylation, and alterations in thiol homeostatic enzyme activities in neurodegenerative diseases, providing insights for therapeutic intervention. Recent Advances: Recent studies show that dysregulation of redox signaling and sulfhydryl homeostasis likely contributes to onset/progression of neurodegeneration. Oxidative stress alters the thiol–disulfide status of key proteins that regulate the balance between cell survival and cell death. Critical Issues: Much of the current information about redox modification of key enzymes and signaling intermediates has been gleaned from studies focused on oxidative stress situations other than the neurodegenerative diseases. Future Directions: The findings in other contexts are expected to apply to understanding neurodegenerative mechanisms. Identification of selectively glutathionylated proteins in a quantitative fashion will provide new insights about neuropathological consequences of this oxidative protein modification. Antioxid. Redox Signal. 16, 543–566.
I. Introduction
II. Neurodegenerative Diseases
A. Alzheimer 's disease
B. Parkinson's disease
C. Huntington's disease
D. Amyotrophic lateral sclerosis
E. Friedreich's ataxia
III. Production of Oxidants Within the Brain
A. Cytoplasmic sources of ROS
B. Mitochondrial sources of ROS
IV. Inflammation, Oxidative Stress, and Neurodegenerative Diseases
A. Inflammation and Parkinson's disease
B. Potential roles of glutaredoxin in inflammatory responses
V. Cellular Oxidant Defense and Sulfhydryl Homeostasis
A. Cellular functions of Grx
B. Glutaredoxin and neurodegeneration
C. Paradoxical pro-oxidant effects of therapy of Parkinson's disease
VI. Oxidative Stress and Apoptosis
A. Apoptosis signaling kinase 1 may be regulated directly or indirectly by Grx1, Trx1, and other effectors
1. Oxidation of negative and positive effectors of ASK1
B. Redox sensitivity of cytosolic proteins implicated in neuronal cell death
1. Glyceraldehyde-3-phosphate dehydrogenase
2. Tyrosine hydroxylase
3. p53
C. Apoptosis and modification of mitochondrial permeability pore proteins
1. Voltage-dependent anion channel
2. Adenosine nucleotide transporter
3. Redox sensitivity of calcium transporters
D. Oxidative modifications affecting the proteasome system, protein aggregation, and mitochondrial dynamics in neurodegeneration
VII. S-Glutathionylation and Plaque Formation
A. Actin
B. Tau
VIII. S-Glutathionylation of Proteins Involved with Mitochondrial Respiration
A. α-Ketoglutarate dehydrogenase
B. Mitochondrial NADP+-dependent isocitrate dehydrogenase
C. Complex 1
D. Complex 2
E. ATP synthase
F. Succinyl CoA transferase
IX. Potential Approaches to Therapy of the Neurodegenerative Diseases
X. Conclusions
PMCID: PMC3270051  PMID: 22066468
14.  Glutaredoxin Regulates Apoptosis in Cardiomyocytes via NFκB Targets Bcl-2 and Bcl-xL: Implications for Cardiac Aging 
Antioxidants & Redox Signaling  2010;12(12):1339-1353.
Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by ∼40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFκB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFκB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent (∼50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFκB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFκB function. Antioxid. Redox Signal. 12, 1339–1353.
PMCID: PMC2864653  PMID: 19938943
15.  Ablation of Glutaredoxin-1 Attenuates Lipopolysaccharide-Induced Lung Inflammation and Alveolar Macrophage Activation 
Protein S-glutathionylation (PSSG), a reversible posttranslational modification of reactive cysteines, recently emerged as a regulatory mechanism that affects diverse cell-signaling cascades. The extent of cellular PSSG is controlled by the oxidoreductase glutaredoxin-1 (Grx1), a cytosolic enzyme that specifically de-glutathionylates proteins. Here, we sought to evaluate the impact of the genetic ablation of Grx1 on PSSG and on LPS-induced lung inflammation. In response to LPS, Grx1 activity increased in lung tissue and bronchoalveolar lavage (BAL) fluid in WT (WT) mice compared with PBS control mice. Glrx1−/− mice consistently showed slight but statistically insignificant decreases in total numbers of inflammatory cells recovered by BAL. However, LPS-induced concentrations of IL-1β, TNF-α, IL-6, and Granulocyte/Monocyte Colony–Stimulating Factor (GM-CSF) in BAL were significantly decreased in Glrx1−/− mice compared with WT mice. An in situ assessment of PSSG reactivity and a biochemical evaluation of PSSG content demonstrated increases in the lung tissue of Glrx1−/− animals in response to LPS, compared with WT mice or PBS control mice. We also demonstrated that PSSG reactivity was prominent in alveolar macrophages (AMs). Comparative BAL analyses from WT and Glrx1−/− mice revealed fewer and smaller AMs in Glrx1−/− mice, which showed a significantly decreased expression of NF-κB family members, impaired nuclear translocation of RelA, and lower levels of NF-κB–dependent cytokines after exposure to LPS, compared with WT cells. Taken together, these results indicate that Grx1 regulates the production of inflammatory mediators through control of S-glutathionylation–sensitive signaling pathways such as NF-κB, and that Grx1 expression is critical to the activation of AMs.
PMCID: PMC3095922  PMID: 20539014
lipopolysaccharide; glutaredoxin-1; protein S-glutathionylation; nuclear factor-κB
16.  Activation of the glutaredoxin-1 gene by Nuclear Factor kappa B enhances signaling 
Free radical biology & medicine  2011;51(6):1249-1257.
The transcription factor, Nuclear Factor kappa B (NF-κB) is a critical regulator of inflammation and immunity, and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyses deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice which express a doxycyclin-inducible constitutively active version of inhibitory kappa B kinase-beta (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2kb region Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression, and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of pro-inflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB, and suggests a feed forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.
PMCID: PMC3181077  PMID: 21762778
S-glutathionylation; Nuclear Factor kappa B; Glutaredoxin; Lung; Inhibitory kappa B kinase
17.  Protein-Thiol Oxidation and Cell Death: Regulatory Role of Glutaredoxins 
Antioxidants & Redox Signaling  2012;17(12):1748-1763.
Significance: Glutaredoxin (Grx) is the primary enzyme responsible for catalysis of deglutathionylation of protein-mixed disulfides with glutathione (GSH) (protein-SSG). This reversible post-translational modification alters the activity and function of many proteins important in regulation of critical cellular processes. Aberrant regulation of protein glutathionylation/deglutathionylation reactions due to changes in Grx activity can disrupt both apoptotic and survival signaling pathways. Recent Advances: Grx is known to regulate the activity of many proteins through reversible glutathionylation, such as Ras, Fas, ASK1, NFκB, and procaspase-3, all of which play important roles in control of apoptosis. Reactive oxygen species and/or reactive nitrogen species mediate oxidative modifications of critical Cys residues on these apoptotic mediators, facilitating protein-SSG formation and thereby altering protein function and apoptotic signaling. Critical Issues: Much of what is known about the regulation of apoptotic mediators by Grx and reversible glutathionylation has been gleaned from in vitro studies of discrete apoptotic pathways. To relate these results to events in vivo it is important to examine changes in protein-SSG status in situ under natural cellular conditions, maintaining relevant GSH:GSSG ratios and using appropriate inducers of apoptosis. Future Directions: Apoptosis is a highly complex, tightly regulated process involving many different checks and balances. The influence of Grx activity on the interconnectivity among these various pathways remains unknown. Knowledge of the effects of Grx is essential for developing novel therapeutic approaches for treating diseases involving dysregulated apoptosis, such as cancer, heart disease, diabetes, and neurodegenerative diseases, where alterations in redox homeostasis are hallmarks for pathogenesis. Antioxid. Redox Signal. 17, 1748–1763.
PMCID: PMC3474186  PMID: 22530666
18.  Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation☆ 
Redox Biology  2013;1(1):586-598.
Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50–60% in the IMS, but Grx2 is increased by 1.4–2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron–sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron–sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.
Graphical abstract
•Glutaredoxins play key roles in cellular redox regulation, which is sensitive to aging-dependent dysregulation.•Grx1 is diminished in the intermembrane space of mitochondria from aged heart; matrix Grx2 is increased but mostly in an inactive form.•The inactive Grx2 is selectively activated by superoxide.•Mitochondrial glutaredoxin changes may contribute to dysregulation of redox homeostasis during aging.•Changes in in situ activities of heart mitochondrial Grx1 and Grx2 with aging provide mechanistic insights for future studies.
PMCID: PMC4127417  PMID: 25126518
GSH, reduced glutathione; GSSG, glutathione disulfide; Cys-SSG, l-cysteine–glutathione mixed disulfide; Mn-TMPyP, Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin; tetratosylate, hydroxide; DT, sodium dithionite; SSM, heart subsarcolemmal mitochondria; IFM, Heart interfibrillar mitochondria; t-Bid, caspase-8-cleaved human BID; Grx, glutaredoxin; Aging; Glutaredoxin; Glutathionylation; Iron–sulfur cluster; Mitochondria; Reactive oxygen species (ROS); Redox regulation
19.  A mammalian monothiol glutaredoxin, Grx3, is critical for cell cycle progression during embryogenesis 
The FEBS journal  2011;278(14):2525-2539.
Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2 / PICOT) with high similarity to yeast ScGrx3 / ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3−/− cells had impaired growth and cell cycle progression at the G2/M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3-knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.
PMCID: PMC4038268  PMID: 21575136
cell cycle; embryogenesis; glutaredoxin; mouse; oxidative stress
20.  Grx5 Glutaredoxin Plays a Central Role in Protection against Protein Oxidative Damage in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(12):8180-8190.
Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.
PMCID: PMC84902  PMID: 10567543
21.  Knockdown of Cytosolic Glutaredoxin 1 Leads to Loss of Mitochondrial Membrane Potential: Implication in Neurodegenerative Diseases 
PLoS ONE  2008;3(6):e2459.
Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, α-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of β-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.
PMCID: PMC2426930  PMID: 18560520
Free radical biology & medicine  2011;51(11):2108-2117.
Glutaredoxin belongs to the oxidoreductase family with cytosolic glutaredoxin 1 (Grx1) and mitochondrial gluraredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper studied the effect of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild type (WT) mice. We found that both cell types showed similar growth patterns and morphology, and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin (Trx) expression but exhibited high sensitivity to oxidative stress. Under treatment of H2O2, the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H2O2 in comparison with that of the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I, and restored most of its activity. We conclude that Grx2 has a function to protect cells against H2O2-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.
PMCID: PMC3235406  PMID: 21983434
glutaredoxin 2; oxidative stress; complex I; mitochondira; glutathionylation
23.  Glutaredoxin 2 Reduces Both Thioredoxin 2 and Thioredoxin 1 and Protects Cells from Apoptosis Induced by Auranofin and 4-Hydroxynonenal 
Antioxidants & Redox Signaling  2014;21(5):669-681.
Aims: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. Results: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. Innovation: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. Conclusion: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling. Antioxid. Redox Signal. 21, 669–681.
PMCID: PMC4098818  PMID: 24295294
24.  Regulation by Reversible S-Glutathionylation: Molecular Targets Implicated in Inflammatory Diseases 
Molecules and Cells  2008;25(3):332-346.
S-glutathionylation is a reversible post-translational modification that continues to gain eminence as a redox regulatory mechanism of protein activity and associated cellular functions. Many diverse cellular proteins such as transcription factors, adhesion molecules, enzymes, and cytokines are reported to undergo glutathionylation, although the functional impact has been less well characterized. De-glutathionylation is catalyzed specifically and efficiently by glutaredoxin (GRx, aka thioltransferase), and facile reversibility is critical in determining the physiological relevance of glutathionylation as a means of protein regulation. Thus, studies with cohesive themes addressing both the glutathionylation of proteins and the corresponding impact of GRx are especially useful in advancing understanding. Reactive oxygen species (ROS) and redox regulation are well accepted as playing a role in inflammatory processes, such as leukostasis and the destruction of foreign particles by macrophages. We discuss in this review the current implications of GRx and/or glutathionylation in the inflammatory response and in diseases associated with chronic inflammation, namely diabetes, atherosclerosis, inflammatory lung disease, cancer, and Alzheimer’s disease, and in viral infections.
PMCID: PMC3367451  PMID: 18483468
25.  Glutaredoxin-1 Overexpression Enhances Neovascularization and Diminishes Ventricular Remodeling in Chronic Myocardial Infarction 
PLoS ONE  2012;7(3):e34790.
Oxidative stress plays a critical role in the pathophysiology of cardiac failure, including the modulation of neovascularization following myocardial infarction (MI). Redox molecules thioredoxin (Trx) and glutaredoxin (Grx) superfamilies actively maintain intracellular thiol-redox homeostasis by scavenging reactive oxygen species. Among these two superfamilies, the pro-angiogenic function of Trx-1 has been reported in chronic MI model whereas similar role of Grx-1 remains uncertain. The present study attempts to establish the role of Grx-1 in neovascularization and ventricular remodeling following MI. Wild-type (WT) and Grx-1 transgenic (Grx-1Tg/+) mice were randomized into wild-type sham (WTS), Grx-1Tg/+ Sham (Grx-1Tg/+S), WTMI, Grx-1Tg/+MI. MI was induced by permanent occlusion of the LAD coronary artery. Sham groups underwent identical time-matched surgical procedures without LAD ligation. Significant increase in arteriolar density was observed 7 days (d) after surgical intervention in the Grx-1Tg/+MI group as compared to the WTMI animals. Further, improvement in myocardial functional parameters 30 d after MI was observed including decreased LVIDs, LVIDd, increased ejection fraction and, fractional shortening was also observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Moreover, attenuation of oxidative stress and apoptotic cardiomyocytes was observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Increased expression of p-Akt, VEGF, Ang-1, Bcl-2, survivin and DNA binding activity of NF-κB were observed in the Grx-1Tg/+MI group when compared to WTMI animals as revealed by Western blot analysis and Gel-shift analysis, respectively. These results are the first to demonstrate that Grx-1 induces angiogenesis and diminishes ventricular remodeling apparently through neovascularization mediated by Akt, VEGF, Ang-1 and NF-κB as well as Bcl-2 and survivin-mediated anti-apoptotic pathway in the infarcted myocardium.
PMCID: PMC3327713  PMID: 22523530

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