Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA[Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.Selenium is an essential micronutrient in the diet of mammals and numerous other life forms (see reference 26 for a review). Many health benefits have been attributed to this element, including a role in the prevention of cancer (10) and heart disease and other cardiovascular and muscle disorders (11), in delaying the aging process (33) and the onset of AIDS in human immunodeficiency virus-positive patients (1), in male reproduction (17), in mammalian development (5), in immune function (33), and as an antiviral agent (2). Selenium is incorporated into protein in the form of selenocysteine (Sec), and Sec has its own tRNA (designated Sec tRNA[Ser]Sec ) and its own code word, UGA (26). Sec is indeed the 21st naturally occurring amino acid in the genetic code. Most certainly, the health benefits of selenium are due in large part to its presence in protein (26).Sec tRNA [Ser]Sec is the only known tRNA that governs the expression of an entire class of proteins, the selenoproteins (26). This provides a unique opportunity to study the expression of selenoproteins by manipulating the levels and characteristics of Sec tRNA [Ser]Sec . For example, the levels of numerous selenoproteins were reduced in a protein-and tissue-specific manner in transgenic mice carrying mutant Sec tRNA [Ser]Sec transgenes lacking the highly modified base isopentenyladenosine in its anticodon (37). Glutathione peroxidase 1 (GPx1) and thioredoxin reductases 1 (TR1) and 3 (TR3) were the most and least affected selenoproteins, respectively, and selenoprotein expre...

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.

Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b 561 (Cyt b 561 ) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b 561 , immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b 561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b 561 . In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.

Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiaeΔfre1Δfre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the Δfre1Δfre2 mutant cells in iron‐deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+‐EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site‐directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate‐dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins.

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization of protein and interprotein disulfide bonds and localized this function to its thiol domain. TGR targets include proteins that form structural components of the sperm, including glutathione peroxidase GPx4/PHGPx. Together, TGR and GPx4 can serve as a novel disulfide bond formation system. Both enzymes contain a catalytic selenocysteine consistent with the role of selenium in male reproduction.

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Thioredoxin reductase 1 (TR1) is a key component in the thioredoxin system, one of major redox systems in mammals that links NADPH and thiol-dependent processes. Mammalian TR1 genes are known to be regulated by alternative splicing. In this report, comparative genomic analyses were used to identify and characterize species-specific and common alternative forms of mammalian TR1 genes. Six human TR1 isoforms were identified that were derived from a large number of transcripts and differed in their N-terminal sequences. One isoform resulted from exons located 30-70 kb upstream of the previously identified core TR1 promoter and was composed of a basic TR1 module fused to a glutaredoxin (Grx) domain that contained an unusual active site CTRC sequence. This TR1 form occurred in humans, dogs, and chimpanzees but was inactivated in mice and rats. The CTRC motif in the human enzyme made the N-terminal domain inactive in the Grx assays tested. However, when mutated to CPYC, an active site present in most Grxs, the Grx domain was active. In addition, the presence of the Grx domain interfered with the TR1 activity, distinguishing this enzyme from other proteins with Grx and TR fusions. The data suggest that the fusion of the basic TR1 module and variable N-terminal sequences links the pyridine nucleotide thiol/disulfide oxidoreductase pathway to specific cellular redox functions and may control spatial and temporal expression of TR1 transcripts. Our data also suggest that various N-terminal extensions in mammalian TRs are often expressed in testes.