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1.  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
2.  Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space 
Antioxidants & Redox Signaling  2011;15(10):2645-2661.
Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear.
The goal of this work focused on the identification of mechanisms underlying UCP3 functions.
Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3–Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3–Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion.
This is the first report of a mitochondrial protein–protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2.
These studies identify a novel UCP3–Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production. Antioxid. Redox Signal. 15, 2645–2661.
PMCID: PMC3183655  PMID: 21619484
3.  Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid–Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin 
Diabetes  2009;58(10):2246-2257.
Oxidative stress induced by free fatty acids contributes to the development of cardiovascular diseases in patients with metabolic syndrome. Reducing oxidative stress may attenuate these pathogenic processes. Activation of AMP-activated protein kinase (AMPK) has been reported to reduce intracellular reactive oxygen species (ROS) levels. The thioredoxin (Trx) system is a major antioxidant system. In this study, we investigated the mechanisms involved in the AMPK-mediated regulation of Trx expression and the reduction of intracellular ROS levels.
We observed that activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) significantly reduced ROS levels induced by palmitic acid in human aortic endothelial cells. Activation of AMPK increased expression of the antioxidant Trx, which mediated the ROS reduction. RT-PCR showed that AMPK regulated Trx at the transcriptional level.
Forkhead transcription factor 3 (FOXO3) was identified as the target transcription factor involved in the upregulation of Trx expression. FOXO3 bound to the Trx promoter, recruited the histone acetylase p300 to the Trx promoter, and formed a transcription activator complex, which was enhanced by AICAR treatment. AMPK activated FOXO3 by promoting its nuclear translocation. We further showed that AICAR injection increased the expression of Trx and decreased ROS production in the aortic wall of ApoE−/− mice fed a high-fat diet.
These results suggest that activation of the AMPK-FOXO3 pathway reduces ROS levels by inducing Trx expression. Thus, the AMPK-FOXO3-Trx axis may be an important defense mechanism against excessive ROS production induced by metabolic stress and could be a therapeutic target in treating cardiovascular diseases in metabolic syndrome.
PMCID: PMC2750236  PMID: 19592618
4.  Acetaminophen Reactive Intermediates Target Hepatic Thioredoxin Reductase 
Chemical Research in Toxicology  2014;27(5):882-894.
Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 and 6 h post-APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP-reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 μM) than the oxidized enzyme (IC50 = 1.0 μM) or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 μM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497, and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP-reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.
PMCID: PMC4033643  PMID: 24661219
5.  Acetaminophen Reactive Intermediates Target Hepatic Thioredoxin Reductase 
Chemical research in toxicology  2014;27(5):882-894.
Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 h and 6 h post APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 µM) than the oxidized enzyme (IC50 = 1.0 µM), or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 µM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497 and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.
PMCID: PMC4033643  PMID: 24661219
6.  In vitro susceptibility of thioredoxins and glutathione to redox modification and aging-related changes in skeletal muscle 
Free Radical Biology & Medicine  2012;53(11):2017-2027.
Thioredoxins (Trx's) regulate redox signaling and are localized to various cellular compartments. Specific redox-regulated pathways for adaptation of skeletal muscle to contractions are attenuated during aging, but little is known about the roles of Trx's in regulating these pathways. This study investigated the susceptibility of Trx1 and Trx2 in skeletal muscle to oxidation and reduction in vitro and the effects of aging and contractions on Trx1, Trx2, and thioredoxin reductase (TrxR) 1 and 2 contents and nuclear and cytosolic Trx1 and mitochondrial Trx2 redox potentials in vivo. The proportions of cytosolic and nuclear Trx1 and mitochondrial Trx2 in the oxidized or reduced forms were analyzed using redox Western blotting. In myotubes, the mean redox potentials were nuclear Trx1, −251 mV; cytosolic Trx1, −242 mV; mitochondrial Trx2, −346 mV, data supporting the occurrence of differing redox potentials between cell compartments. Exogenous treatment of myoblasts and myotubes with hydrogen peroxide or dithiothreitol modified glutathione redox status and nuclear and cytosolic Trx1, but mitochondrial Trx2 was unchanged. Tibialis anterior muscles from young and old mice were exposed to isometric muscle contractions in vivo. Aging increased muscle contents of Trx1, Trx2, and TrxR2, but neither aging nor endogenous ROS generated during contractions modified Trx redox potentials, although oxidation of glutathione and other thiols occurred. We conclude that glutathione redox couples in skeletal muscle are more susceptible to oxidation than Trx and that Trx proteins are upregulated during aging, but do not appear to modulate redox-regulated adaptations to contractions that fail during aging.
Graphical Abstract
► Muscle Trx1 and Trx2 redox potentials vary between nucleus, cytosol, and mitochondria. ► Oxidants modified nuclear and cytosolic Trx1 and GSH but not mitochondrial Trx2. ► Aging increased muscle Trx contents, but did not affect Trx redox potentials. ► Contractile activity affected muscle GSH but not Trx content or redox potentials. ► Glutathione in muscle is more susceptible to physiological oxidation than Trx's.
PMCID: PMC3657158  PMID: 23022873
Thioredoxin; Redox Western blotting; Aging; Skeletal muscle; Free radicals
7.  Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants 
Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.
PMCID: PMC3797462  PMID: 24137166
antioxidant mechanisms; oxidative stress; photosynthesis; redox homeostasis; thioredoxin; tobacco
8.  High-fat diet-induced changes in liver thioredoxin and thioredoxin reductase as a novel feature of insulin resistance 
FEBS Open Bio  2014;4:928-935.
•Liver thioredoxin and its reductase are decreased after consuming a high-fat diet.•S-acylation may mediate the negative effect of a high-fat diet on these proteins.•High-fat diet also elevates 4-hydroxynonenal, another inhibitor of these proteins.•Dysfunction of these proteins is associated with insulin resistance.
High-fat diet (HFD) can induce oxidative stress. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are critical antioxidant proteins but how they are affected by HFD remains unclear. Using HFD-induced insulin-resistant mouse model, we show here that liver Trx and TrxR are significantly decreased, but, remarkably, the degree of their S-acylation is increased after consuming HFD. These HFD-induced changes in Trx/TrxR may reflect abnormalities of lipid metabolism and insulin signaling transduction. HFD-driven accumulation of 4-hydroxynonenal is another potential mechanism behind inactivation and decreased expression of Trx/TrxR. Thus, we propose HFD-induced impairment of liver Trx/TrxR as major contributor to oxidative stress and as a novel feature of insulin resistance.
PMCID: PMC4239481  PMID: 25426412
ASK-1, apoptosis signal-regulating kinase-1; HFD, high-fat diet; 4-HNE, 4-hydroxynonenal; IRS-1, insulin receptor substrate-1; ITT, insulin tolerance test; Gpx, glutathione peroxidase; OGTT, oral glucose tolerance test; PTP-1B, protein-tyrosine phophatase-1B; Trx, thioredoxin; TrxR, thioredoxin reductase; High-fat diet; Insulin resistance; S-acylation; Thioredoxin; Thioredoxin reductase
9.  The Effects of Acrolein on Peroxiredoxins, Thioredoxins, and Thioredoxin Reductase in Human Bronchial Epithelial Cells 
Toxicology  2008;257(1-2):95-104.
Inhalation is a common form of exposure to acrolein, a toxic reactive volatile aldehyde that is a ubiquitous environmental pollutant. Bronchial epithelial cells would be directly exposed to inhaled acrolein. The thioredoxin (Trx) system is essential for the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, thioredoxin reductase (TrxR) maintains the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins in the reduced state, and the thioredoxins keep the peroxiredoxins (Prx) reduced, thereby supporting their peroxidase function. The effects of acrolein on TrxR, Trx and Prx in human bronchial epithelial (BEAS-2B) cells were determined. A 30-min exposure to 5 μM acrolein oxidized both Trx1 and Trx2, although significant effects were noted for Trx1 at even lower acrolein concentrations. The effects on Trx1 and Trx2 could not be reversed by treatment with disulfide reductants. TrxR activity was inhibited 60% and >85% by 2.5 and 5 μM acrolein, respectively. The endogenous electron donor for TrxR, NADPH, could not restore its activity, and activity did not recover in cells during a 4-hr acrolein-free period in complete medium. The effects of acrolein on TrxR and Trx therefore extend beyond the duration of exposure. While there was a strong correlation between TrxR inhibition and Trx1 oxidation, the irreversible effects on Trx1 suggest direct effects of acrolein rather than loss of reducing equivalents from TrxR. Trx2 did not become oxidized until ≥90% of TrxR was inhibited, but irreversible effects on Trx2 also suggest direct effects of acrolein. Prx1 (cytosolic) and Prx3 (mitochondrial) shifted to a largely oxidized state only when >90 and 100% of their respective Trxs were oxidized. Prx oxidation was readily reversed with a disulfide reductant, suggesting that Prx oxidation resulted from lack of reducing equivalents from Trx and not direct reaction with acrolein. The effects of acrolein on the thioredoxin system and peroxiredoxins could have important implications for cell survival, redox-sensitive cell signaling, and tolerance to other oxidant insults.
PMCID: PMC2668956  PMID: 19135121
Acrolein; Peroxiredoxin-1; Peroxiredoxin-3; Thioredoxin; Thioredoxin Reductase; BEAS-2B cells
10.  Overlapping Roles of the Cytoplasmic and Mitochondrial Redox Regulatory Systems in the Yeast Saccharomyces cerevisiae 
Eukaryotic Cell  2005;4(2):392-400.
Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.
PMCID: PMC549330  PMID: 15701801
11.  Suppressive effect of administration of recombinant human thioredoxin on cutaneous  inflammation caused by UV 
Bioengineered  2013;4(4):254-257.
Thioredoxin (TRX) is small ubiquitous protein, which regulates cellular redox status and scavenges reactive oxygen species (ROS). TRX has been shown to exert suppressive effect on skin inflammation where oxidative stress is involved in its pathogenesis. We investigated the effect of TRX on UVB response. Ear swelling after UVB irradiation was significantly reduced in TRX-transgenic mouse compared with wild-type mouse. Furthermore, we have demonstrated that intraperitoneal administration of recombinant human thioredoxin (rhTRX) also reduced acute skin inflammatory reaction, such as skin erythema and edema. Histologically, inflammatory cells including neutrophils and lymphocytes were significantly reduced and average size of the caliber of blood vessels were also reduced in rhTRX-injected mice. The number of apoptotic keratinocytes, were significantly reduced in rhTRX-injected mice. Immunohistochemical intensity of 8-hydroxy-2'-deoxyguanosine was strikingly reduced in rhTRX-injected mouse. Western blotting showed that administration of rhTRX inhibited phosphorylation of p38 mitogen-activated protein kinases and c-Jun NH2-terminal kinase, which play important roles in inflammatory and apoptotic signaling. These findings indicated that rhTRX attenuated inflammatory and apoptotic responses by UVB. Possible mechanisms for this might be via redox regulation of stress signaling and reduction of reactive oxygen species. We discussed the future use of TRX for sedative use of skin inflammation.
PMCID: PMC3728197  PMID: 23328539
UVB; thioredoxin; oxidative stress; cytokine; nerutrophil; JNK; 8-OHdG; p38 MAPK; redox
12.  Thioredoxin 1 Enhances Neovascularization and Reduces Ventricular Remodeling During Chronic Myocardial Infarction: A Study Using Thioredoxin 1 Transgenic Mice 
Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin-1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild type (W) and Trx1 transgenic (Trx1Tg/+) mice were randomized into W Sham (WS), Trx1Tg/+ Sham (TS), WMI and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress, and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS and p-GSK-3β, HIF-1α, β-catenin, VEGF, Bcl-2 and survivin expression in TMI compared to WMI. Echocardiography performed 30 days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12 and subsequent activation of Akt/GSK-3β/β-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.
PMCID: PMC3397477  PMID: 21074540
Apoptosis; Myocardial infarction; Neovascularization; Oxidative stress; Thioredoxin1 and Ventricular remodeling
13.  Essential Role for Mitochondrial Thioredoxin Reductase in Hematopoiesis, Heart Development, and Heart Function 
Molecular and Cellular Biology  2004;24(21):9414-9423.
Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.
PMCID: PMC522221  PMID: 15485910
14.  Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance 
Antioxidants & Redox Signaling  2013;18(10):1165-1207.
The thioredoxin (Trx) system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through Trx reductase to Trx, which reduces its target proteins using highly conserved thiol groups. While the importance of protecting cells from the detrimental effects of reactive oxygen species is clear, decades of research in this field revealed that there is a network of redox-sensitive proteins forming redox-dependent signaling pathways that are crucial for fundamental cellular processes, including metabolism, proliferation, differentiation, migration, and apoptosis. Trx participates in signaling pathways interacting with different proteins to control their dynamic regulation of structure and function. In this review, we focus on Trx target proteins that are involved in redox-dependent signaling pathways. Specifically, Trx-dependent reductive enzymes that participate in classical redox reactions and redox-sensitive signaling molecules are discussed in greater detail. The latter are extensively discussed, as ongoing research unveils more and more details about the complex signaling networks of Trx-sensitive signaling molecules such as apoptosis signal-regulating kinase 1, Trx interacting protein, and phosphatase and tensin homolog, thus highlighting the potential direct and indirect impact of their redox-dependent interaction with Trx. Overall, the findings that are described here illustrate the importance and complexity of Trx-dependent, redox-sensitive signaling in the cell. Our increasing understanding of the components and mechanisms of these signaling pathways could lead to the identification of new potential targets for the treatment of diseases, including cancer and diabetes. Antioxid. Redox Signal. 18, 1165–1207.
I. Introduction
A. Redox control and signaling in the cell
B. Thioredoxin
C. Trx reductase
1. Background
2. Regulation
3. Clinical significance
D. Trx target proteins
II. Reductive Enzymes
A. Peroxiredoxins
1. Background
2. Regulation
3. Clinical significance
B. Ribonucleotide reductase
1. Background
2. Regulation
3. Clinical significance
C. Methionine sulfoxide reductase
1. Background
2. Regulation
3. Clinical significance
III. Trx-Sensitive Signaling Molecules
A. Apoptosis signal-regulating kinase-1
1. Background
a. Mitogen-activated protein kinase signaling cascades
(1) ERK signaling pathway
(2) JNK and p38 signaling pathways
b. Structure and function
c. ASK1 signalosome
2. Regulation
a. Post-translational regulation
(1) Phosphorylation
(2) Ubiquitination
(3) S-nitrosylation
b. Protein-protein interactions
(1) Thioredoxin
(2) Trx interacting protein
(3) Glutaredoxin
(4) Ser/Thr protein phosphatase 5
3. ASK1 in health and diseases
a. Innate immune response signaling
b. Cardiac hypertrophy and remodeling
c. Neurodegenerative diseases and ER stress
d. Cancer
e. Diabetes
4. Conclusion
B. Trx interacting protein
1. Background
2. Regulation
a. Transcriptional regulation
b. Post-transcriptional regulation
c. Post-translational regulation
3. Txnip in health and disease
a. Development, differentiation, and proliferation
b. Metabolism
c. Cardiovascular system
d. Other organ systems
(1) Kidney
(2) Eye
(3) Peripheral nervous system
(4) Liver
4. Conclusion
C. Phosphatase and tensin homolog
1. Background
a. Phosphatidylinositol 3-kinase signaling pathway
b. Structure and function
2. Regulation
a. Transcriptional regulation
b. Post-transcriptional regulation
c. Post-translational regulation
(1) Phosphorylation and protein oxidation
(2) Ubiquitination
(3) Acetylation
d. Localization
e. Protein-protein interactions
3. PTEN in health and disease
a. PTEN hamartoma tumor syndrome
b. Embryonic development
c. Cancer
(1) Lung cancer
(2) Hepatocellular carcinoma
(3) Prostate cancer
(4) Breast cancer
d. Diabetes
4. Conclusion
PMCID: PMC3579385  PMID: 22607099
15.  Chaperone-like properties of tobacco plastid thioredoxins f and m 
Journal of Experimental Botany  2011;63(1):365-379.
Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein.
PMCID: PMC3245471  PMID: 21948853
Chaperone; folding; oligomerization; plastid; thioredoxin; tobacco
16.  DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway 
Human Molecular Genetics  2012;21(13):3013-3024.
DJ-1, which is linked to recessively inherited Parkinson's disease when mutated, is a multi-functional protein with anti-oxidant and transcription regulatory activities. However, the mechanism(s) through which DJ-1 and the genes it regulates provide neuroprotection is not fully understood. Here, we show that wild-type DJ-1 induces the expression of thioredoxin 1 (Trx1), a protein disulfide oxidoreductase, whereas pathogenic mutant isoforms L166P and M26I cannot. Conversely, DJ-1 knockdown in SH-SY5Y cells and DJ-1 knockout in mice result in significant decrease in Trx1 protein and mRNA expression levels. The importance of Trx1 in the cytoprotective function of DJ-1 is confirmed using a pharmacological inhibitor of Trx reductase, 1-chloro-2,4-dinitrobenzene, and Trx1 siRNA. Both approaches result in partial loss of DJ-1-mediated protection. Additionally, knockdown of Trx1 significantly abrogates DJ-1-dependent, hydrogen peroxide-induced activation of the pro-survival factor AKT. Promoter analysis of the human Trx1 gene identified an antioxidant response element (ARE) that is required for DJ-1-dependent induction of Trx1 expression. The transcription factor Nuclear factor erythroid-2 related factor 2 (Nrf2), which is a critical inducer of ARE-mediated expression, is regulated by DJ-1. Overexpression of DJ-1 results in increased Nrf2 protein levels, promotes its translocation into the nucleus and enhances its recruitment onto the ARE site in the Trx1 promoter. Further, Nrf2 knockdown abolishes DJ-1-mediated Trx1 induction and cytoprotection against hydrogen peroxide, indicating the critical role of Nrf2 in carrying out the protective functions of DJ-1 against oxidative stress. These findings provide a new mechanism to support the antioxidant function of DJ-1 by increasing Trx1 expression via Nrf2-mediated transcriptional induction.
PMCID: PMC3373246  PMID: 22492997
17.  Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice 
Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX) using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD) mice.
Islets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX) or enhanced green fluorescence protein (Lt-eGFP), respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated.
The lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p < 0.05).
We successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS) scavenger and antiapoptotic functions of TRX are critical to islet survival after transplantation.
PMCID: PMC2736160  PMID: 19671194
18.  Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1 
Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1.
We have identified the trxB1-encoded thioredoxin reductase (TR) as a key enzyme in the oxidative stress response of Lactobacillus plantarum WCFS1.
Overexpression of the trxB1 gene resulted in a 3-fold higher TR activity in comparison to the wild-type strain. Subsequently, higher TR activity was associated with an increased resistance towards oxidative stress. We further determined the global transcriptional response to hydrogen peroxide stress in the trxB1-overexpression and wild-type strains grown in continuous cultures. Hydrogen peroxide stress and overproduction of TR collectively resulted in the up-regulation of 267 genes. Additionally, gene expression profiling showed significant differential expression of 27 genes in the trxB1-overexpression strain. Over expression of trxB1 was found to activate genes associated with DNA repair and stress mechanisms as well as genes associated with the activity of biosynthetic pathways for purine and sulfur-containing amino acids. A total of 16 genes showed a response to both TR overproduction and hydrogen peroxide stress. These genes are involved in the purine metabolism, energy metabolism (gapB) as well as in stress-response (groEL, npr2), and manganese transport (mntH2).
Based on our findings we propose that overproduction of the trxB1-encoded TR in L. plantarum improves tolerance towards oxidative stress. This response coincides with simultaneous induction of a group of 16 transcripts of genes. Within this group of genes, most are associated with oxidative stress response. The obtained crossover between datasets may explain the phenotype of the trxB1-overexpression strain, which appears to be prepared for encountering oxidative stress. This latter property can be used for engineering robustness towards oxidative stress in industrial strains of L. plantarum.
PMCID: PMC2174512  PMID: 17725816
19.  The Effects of Hexavalent Chromium on Thioredoxin Reductase and Peroxiredoxins in Human Bronchial Epithelial Cells 
Free radical biology & medicine  2009;47(10):1477-1485.
Inhalational exposure to hexavalent chromium [Cr(VI)] compounds (e.g. chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells where they are reduced to reactive Cr species which may also contribute to the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trxs keep many intracellular proteins reduced including the peroxiredoxins (Prx). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trxs. Prx oxidation is therefore likely the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR) maintain the Trxs largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 were reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trxs and Prxs. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trxs, Prxs, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance to oxidant insults.
PMCID: PMC2767428  PMID: 19703554
Chromate; Chromium; Thioredoxin Reductase; Peroxiredoxin; BEAS-2B cells
20.  Thioredoxin from Brugia malayi: Defining a 16-Kilodalton Class of Thioredoxins from Nematodes 
Infection and Immunity  2003;71(7):4119-4126.
Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and ≈20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.
PMCID: PMC162031  PMID: 12819103
21.  Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy 
Journal of Clinical Investigation  2003;112(9):1395-1406.
Thioredoxin 1 (Trx1) has redox-sensitive cysteine residues and acts as an antioxidant in cells. However, the extent of Trx1 contribution to overall antioxidant mechanisms is unknown in any organs. We generated transgenic mice with cardiac-specific overexpression of a dominant negative (DN) mutant (C32S/C35S) of Trx1 (Tg-DN-Trx1 mice), in which the activity of endogenous Trx was diminished. Markers of oxidative stress were significantly increased in hearts from Tg-DN-Trx1 mice compared with those from nontransgenic (NTg) mice. Tg-DN-Trx1 mice exhibited cardiac hypertrophy with maintained cardiac function at baseline. Intraperitoneal injection of N-2-mercaptopropionyl glycine, an antioxidant, normalized cardiac hypertrophy in Tg-DN-Trx1 mice. Thoracic aortic banding caused greater increases in myocardial oxidative stress and enhanced hypertrophy in Tg-DN-Trx1 compared with NTg mice. In contrast, transgenic mice with cardiac-specific overexpression of wild-type Trx1 did not show cardiac hypertrophy at baseline but exhibited reduced levels of hypertrophy and oxidative stress in response to pressure overload. These results demonstrate that endogenous Trx1 is an essential component of the cellular antioxidant mechanisms and plays a critical role in regulating oxidative stress in the heart in vivo. Furthermore, inhibition of endogenous Trx1 in the heart primarily stimulates hypertrophy, both under basal conditions and in response to pressure overload through redox-sensitive mechanisms.
PMCID: PMC228400  PMID: 14597765
22.  Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver 
Hepatology (Baltimore, Md.)  2009;49(5):1709-1717.
Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and anti-inflammatory molecule with anti-apoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate buffered saline (PBS).
Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal (4-HNE) protein adducts, expression of hepatic tumor necrosis factor α (TNFα) and resulted in hepatic steatosis and increased plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In ethanol-fed mice, treatment with rhTrx reduced 4-HNE adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride and improved liver enzyme profiles. Ethanol feeding also increased TUNEL positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.
PMCID: PMC2895317  PMID: 19205032
alcohol; antioxidant; cytokines; TUNEL; inflammation
23.  Salmonella enterica Serovar Typhimurium trxA Mutants Are Protective against Virulent Challenge and Induce Less Inflammation than the Live-Attenuated Vaccine Strain SL3261 ▿  
Infection and Immunity  2009;78(1):326-336.
In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4+ and CD8+ T cells and B lymphocytes in the spleen and reduced infiltration by CD11b+ cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.
PMCID: PMC2798184  PMID: 19884329
24.  Thioredoxin 1 Overexpression Extends Mainly the Earlier Part of Life Span in Mice 
We examined the effects of increased levels of thioredoxin 1 (Trx1) on resistance to oxidative stress and aging in transgenic mice overexpressing Trx1 [Tg(TRX1)+/0]. The Tg(TRX1)+/0 mice showed significantly higher Trx1 protein levels in all the tissues examined compared with the wild-type littermates. Oxidative damage to proteins and levels of lipid peroxidation were significantly lower in the livers of Tg(TRX1)+/0 mice compared with wild-type littermates. The survival study demonstrated that male Tg(TRX1)+/0 mice significantly extended the earlier part of life span compared with wild-type littermates, but no significant life extension was observed in females. Neither male nor female Tg(TRX1)+/0 mice showed changes in maximum life span. Our findings suggested that the increased levels of Trx1 in the Tg(TRX1)+/0 mice were correlated to increased resistance to oxidative stress, which could be beneficial in the earlier part of life span but not the maximum life span in the C57BL/6 mice.
PMCID: PMC3210956  PMID: 21873593
Thioredoxin; Transgenic mouse; Oxidative stress; Protein carbonylation; Aging
25.  Attenuation of Angiotensin II-induced Vascular Dysfunction and Hypertension by Overexpression of Thioredoxin-2 
Hypertension  2009;54(2):338-344.
Reactive oxygen species (ROS) increase in the cardiovascular system during hypertension and in response to angiotensin II. As mitochondria contribute to ROS-generation we sought to investigate the role of thioredoxin-2, a mitochondria specific antioxidant enzyme.
Mice were created with overexpression of human thioredoxin-2 (TghTrx2 mice) and backcrossed to C57BL/6J mice for at least 6 generations. 12 week old male TghTrx2 or littermate wild-type mice were made hypertensive by infusion of angiotensin II (400ng/kg per min) for 14 days using osmotic minipumps. Systolic arterial blood pressure was not different between TghTrx2 and wild-type animals under baseline conditions (101±1 resp. 102±1 mmHg). The angiotensin II-induced hypertension in wild-type (145±2 mmHg) was significantly attenuated in TghTrx2 mice (124±1 mmHg, p<0.001). Aortic endothelium-dependent relaxation was significantly reduced in wild-type following angiotensin II infusion, but nearly unchanged in transgenic mice. Elevated vascular superoxide and hydrogen peroxide levels as well as expression of NADPH oxidase subunits in response to angiotensin II infusion were significantly attenuated in TghTrx2 mice. Mitochondrial superoxide anion levels were augmented after angiotensin II infusion in wild-type mice, this was blunted in TghTrx2 mice. Angiotensin II infusion significantly increased myocardial superoxide formation, heart weight and cardiomyocyte size in wild-type, but not in TghTrx2 mice.
These data indicate a major role for mitochondrial thioredoxin-2 in the development of cardiovascular alterations and hypertension during chronic angiotensin II infusion. Thioredoxin-2 may represent an important therapeutic target for the prevention and treatment of hypertension and oxidative stress.
PMCID: PMC2752391  PMID: 19506101
angiotensin II; endothelial function; hypertension; mitochondria; thioredoxin-2; reactive oxygen species

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