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.