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Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.

ABSTRACT: Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3'-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

SUBMITTER: Fujiwara N

PROVIDER: S-EPMC1220269 | biostudies-other | 1999 Jun

REPOSITORIES: biostudies-other

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Project description:Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1-4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

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Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.

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