Characterization of Alternative Cytosolic Forms and Cellular Targets of Mouse Mitochondrial Thioredoxin Reductase

Turanov, Anton A.; Su, Dan; Gladyshev, Vadim N.

doi:10.1074/jbc.m604326200

Cited by 78 publications

(68 citation statements)

References 43 publications

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“…TR1 and TR3 forms may be targeted by their N-terminal sequences to different cellular compartments or to distinct interacting partners. In some cases, an alternative form of TR1, normally a cytosolic protein, localizes to mitochondria, whereas the mitochondrial TR3 can be targeted to the cytosol when alternative first exon splicing skips over the sequences coding for the mitochondrial signal (34). There is also an intriguing isoform of TR1 that has a Grx domain that shows no activity in the Grx assays.…”

Section: Discussionmentioning

confidence: 99%

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Gerashchenko

Gladyshev

2010

Journal of Biological Chemistry

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Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.

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Section: Discussionmentioning

confidence: 99%

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Gerashchenko

Gladyshev

2010

Journal of Biological Chemistry

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“…The thioredoxin reductase was purified from E. coli (BL21(DE3), Novagen) transformed with the construct and induced to express the enzyme as previously described (50). Purity of the enzyme was confirmed by fractionation of the purified protein on a 10% SDS-polyacrylamide gel followed by Coomassie staining.…”

Section: Methodsmentioning

confidence: 99%

Paraquat Increases Cyanide-insensitive Respiration in Murine Lung Epithelial Cells by Activating an NAD(P)H:Paraquat Oxidoreductase

Gray¹,

Heck

Mishin

et al. 2007

Journal of Biological Chemistry

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Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10 -100 M) was found to cause a 2-4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial-but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 M), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.Exposure of humans and animals to toxic doses of paraquat (1,1Ј-dimethyl-4,4Ј-bipyridylium) is known to damage the lung leading to pulmonary edema and hypertension, acute respiratory distress syndrome, and progressive lung fibrosis (1). In target cells paraquat undergoes redox cycling which may contribute to its toxic actions. Several mammalian NADPH oxidases have been identified as potential inducers of paraquat redox cycling including cytochrome P450 reductase and nitric-oxide synthase (2, 3). These enzymes generate a reduced paraquat radical that can act as an electron donor (4) (see Reaction 1). Reacting rapidly with molecular oxygen, the paraquat radical recycles back to paraquat and in the process forms highly toxic oxidants including superoxide anion, hydrogen peroxide, hydroxyl radicals, and in the presence of nitric oxide, peroxynitrite (5-7). Cellular damage generated by these oxidants, including lipid peroxidation, may be important in paraquatinduced lung damage (8, 9).Redox cycling reactions are known to consume significant quantities of oxygen (10 -12). In cells, this has the potential to reduce levels of oxygen available for metabolic processes resulting in oxidative stress and toxicity (13). Previous studies have demonstrated that paraquat can stimulate oxygen uptake by microsomes from rat and rabbit liver and rabbit lung as...

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“…TR3 is localized in the mitochondria, where it is involved in reduction of mitochondrial thioredoxin (Trx2) and glutaredoxin 2 (Grx2). Similar to TR1, multiple TR3 isoforms have been described (352). Both TR1 and TR3 are present in all vertebrates, and knockout of either of them leads to embryonic lethality in mice (29,67,160).…”

Section: Thioredoxin Reductasesmentioning

confidence: 99%

Selenoproteins: Molecular Pathways and Physiological Roles

Labunskyy

Hatfield

Gladyshev³

2014

Physiological Reviews

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1,065

835

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Selenium is an essential micronutrient with important functions in human health and relevance to several pathophysiological conditions. The biological effects of selenium are largely mediated by selenium-containing proteins (selenoproteins) that are present in all three domains of life. Although selenoproteins represent diverse molecular pathways and biological functions, all these proteins contain at least one selenocysteine (Sec), a seleniumcontaining amino acid, and most serve oxidoreductase functions. Sec is cotranslationally inserted into nascent polypeptide chains in response to the UGA codon, whose normal function is to terminate translation. To decode UGA as Sec, organisms evolved the Sec insertion machinery that allows incorporation of this amino acid at specific UGA codons in a process requiring a cis-acting Sec insertion sequence (SECIS) element. Although the basic mechanisms of Sec synthesis and insertion into proteins in both prokaryotes and eukaryotes have been studied in great detail, the identity and functions of many selenoproteins remain largely unknown. In the last decade, there has been significant progress in characterizing selenoproteins and selenoproteomes and understanding their physiological functions. We discuss current knowledge about how these unique proteins perform their functions at the molecular level and highlight new insights into the roles that selenoproteins play in human health.

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Characterization of Alternative Cytosolic Forms and Cellular Targets of Mouse Mitochondrial Thioredoxin Reductase

Cited by 78 publications

References 43 publications

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Paraquat Increases Cyanide-insensitive Respiration in Murine Lung Epithelial Cells by Activating an NAD(P)H:Paraquat Oxidoreductase

Selenoproteins: Molecular Pathways and Physiological Roles

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