The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Cheng, Qing; Antholine, William E.; Myers, Judith M.; Kalyanaraman, Balaraman; Arnér, Elias S.J.; Myers, Charles R.

doi:10.1074/jbc.m110.117259

Cited by 61 publications

(56 citation statements)

References 69 publications

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“…This is in agreement with findings with other inhibitors including auranofin that cause decreases in the formation of superoxide anion and hydroxyl radicals mediated by TrxR. 46 The differential effects of HN2 on disulfide reduction and chemical redox cycling suggest that TrxR mediates these reactions via distinct mechanisms. It is likely that redox cycling occurs on sites on the enzyme in proximity to the flavin that are not modified by HN2.…”

Section: Discussionsupporting

confidence: 90%

Cross-Linking of Thioredoxin Reductase by the Sulfur Mustard Analogue Mechlorethamine (Methylbis(2-chloroethyl)amine) in Human Lung Epithelial Cells and Rat Lung: Selective Inhibition of Disulfide Reduction but Not Redox Cycling

Jan

Heck

Malaviya

et al. 2013

Chem. Res. Toxicol.

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Oxidative stress plays a key role in mechlorethamine (methyl bis(2-chloroethyl) amine, HN2) toxicity. The thioredoxin system, consisting of thioredoxin reductase (TrxR), thioredoxin, and NADPH, is important in redox regulation and protection against oxidative stress. HN2 contains two electrophilic side chains that can react with nucleophilic sites in proteins leading to changes in their structure and function. We report that HN2 inhibits the cytosolic (TrxR1) and mitochondrial (TrxR2) forms of TrxR in A549 lung epithelial cells. TrxR exists as homodimers under native conditions; monomers can be detected by denaturing and reducing SDS-PAGE followed by Western blotting. HN2 treatment caused marked decreases in TrxR1 and TrxR2 monomers along with increases in dimers and oligomers under reducing conditions, indicating that HN2 cross-links TrxR. Cross-links were also observed in rat lung after HN2 treatment. Using purified TrxR1, NADPH reduced, but not oxidized, enzyme was inhibited and cross-linked by HN2. LC-MS/MS analysis of TrxR1 demonstrated that HN2 adducted cysteine- and selenocysteine-containing redox centers forming monoadducts, intramolecule and intermolecule cross-links, resulting in enzyme inhibition. HN2 cross-links two dimeric subunits through intermolecular binding to cysteine 59 in one subunit of the dimer and selenocysteine 498 in the other subunit, confirming the close proximity of the N- and C-terminal redox centers of adjacent subunits. Despite cross-linking and inhibition of TrxR activity by HN2, TrxR continued to mediate menadione redox cycling and generated reactive oxygen species. These data suggest that disruption of the thioredoxin system contributes to oxidative stress and tissue injury induced by HN2.

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Section: Discussionsupporting

confidence: 90%

Cross-Linking of Thioredoxin Reductase by the Sulfur Mustard Analogue Mechlorethamine (Methylbis(2-chloroethyl)amine) in Human Lung Epithelial Cells and Rat Lung: Selective Inhibition of Disulfide Reduction but Not Redox Cycling

Jan

Heck

Malaviya

et al. 2013

Chem. Res. Toxicol.

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“…Furthermore, we determined increased NADPH-oxidation with high concentrations of hTrxR1 W114A,U498C in the presence and absence of hTrx1 C73S in the assay (Table 2). Our results are in accordance with the previously described findings that TrxR1 shows an enhanced NADPH oxidation if there is no electron transport to the substrate 20,21 . Our notion was further supported by the fact that with DTNB, a small artificial disulphide substrate 22 , hTrxR1…”

Section: Htrxr1supporting

confidence: 94%

Crystal structure of the human thioredoxin reductase–thioredoxin complex

et al. 2011

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“…Regarding the detection of intracellular superoxide production, the spin adducts from cyclic nitrones display weaknesses that reside in the hydroperoxide function that is the target of peroxidase activities (P450 [30] , glutathione peroxidase [47] , thioredoxine reductase [57] ...) and in the nitroxide (aminoxyl radical) moiety that supports the ESR signature and that cannot be shielded from reduction by bulky substituents (a strategy proposed for "stable" nitroxides)…”

Section: Resultsmentioning

confidence: 99%

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Bézière

Hardy

Poulhès

et al. 2014

Free Radical Biology and Medicine

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Reactive oxygen species (ROS) are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, superoxide radical is of special interest as the origin of several damaging species such as H 2 O 2 , hydroxyl radical, or peroxynitrite (ONOO -). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In the present manuscript, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original set up combining a stoppedflow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.3

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The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Cited by 61 publications

References 69 publications

Cross-Linking of Thioredoxin Reductase by the Sulfur Mustard Analogue Mechlorethamine (Methylbis(2-chloroethyl)amine) in Human Lung Epithelial Cells and Rat Lung: Selective Inhibition of Disulfide Reduction but Not Redox Cycling

Cross-Linking of Thioredoxin Reductase by the Sulfur Mustard Analogue Mechlorethamine (Methylbis(2-chloroethyl)amine) in Human Lung Epithelial Cells and Rat Lung: Selective Inhibition of Disulfide Reduction but Not Redox Cycling

Crystal structure of the human thioredoxin reductase–thioredoxin complex

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

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