A functional link between housekeeping selenoproteins and phase II enzymes

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.     

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.     

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.     

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.     

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.     

Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development

Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.     

A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.     

Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism

Selenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA^[Ser]Sec. Targeted removal of the tRNA^[Ser]Sec gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism.     

Selenoproteins mediate T cell immunity through an antioxidant mechanism

Selenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

High Error Rates in Selenocysteine Insertion in Mammalian Cells Treated with the Antibiotic Doxycycline,Chloramphenicol, or Geneticin

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.     

Regulation of selenoprotein mRNA expression by hormones and retinoic acid in bovine mammary cells

Selenium is essential for maintaining many body functions through the actions of selenoproteins. To find factors regulating selenoprotein biosynthesis in the bovine mammary cell line MAC-T, the effects of supplementation with selenite and also with retinoic acid, insulin, hydrocortisone and prolactin on the mRNA expression of a number of selenoproteins were investigated. It was found that MAC-T cells express glutathione peroxidase (GPx) 1 and 4, thioredoxin reductase 1 and selenoprotein P, but not GPx 3, which is interesting considering that GPx 3 is one of the only few selenoproteins detected in milk so far. Addition of selenite to the cell culture resulted in a large increase in GPx 1 expression and an increase in selenoprotein P expression, which is similar to the findings made in other systems investigated. Increased mRNA levels of GPx 1 were also observed in cells treated with insulin and hydrocortisone or with retinoic acid. The expression of thioredoxin reductase 1 was increased in cells treated with retinoic acid, whereas that of selenoprotein P was decreased in cells exposed to insulin. The results indicate that several hormones, selenium, and retinoic acid regulate the biosynthesis of various selenoproteins differently in the bovine mammary cell. The possible implications of the findings for processes related to milk formation and mammary carcinogenesis will need additional investigation. Further study of the detailed mechanisms involved is also necessary.     

Lactation defect in a widely used MMTV-Cre transgenic line of mice

Background

MMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines.

Methodology/Principal Findings

To explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development.

Conclusions/Significance

The identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase.     

Composition and evolution of the vertebrate and mammalian selenoproteomes

Background

Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24.

Methodology/Principal Findings

We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian (25 proteins) selenoproteomes, and describe how they evolved along lineages through gene duplication (20 events), gene loss (10 events) and replacement of Sec with cysteine (12 events). We show that an intronless selenophosphate synthetase 2 gene evolved in early mammals and replaced functionally the original multiexon gene in placental mammals, whereas both genes remain in marsupials. Mammalian thioredoxin reductase 1 and thioredoxin-glutathione reductase evolved from an ancestral glutaredoxin-domain containing enzyme, still present in fish. Selenoprotein V and GPx6 evolved specifically in placental mammals from duplications of SelW and GPx3, respectively, and GPx6 lost Sec several times independently. Bony fishes were characterized by duplications of several selenoprotein families (GPx1, GPx3, GPx4, Dio3, MsrB1, SelJ, SelO, SelT, SelU1, and SelW2). Finally, we report identification of new isoforms for several selenoproteins and describe unusually conserved selenoprotein pseudogenes.

Conclusions/Significance

This analysis represents the first comprehensive survey of the vertebrate and mammal selenoproteomes, and depicts their evolution along lineages. It also provides a wealth of information on these selenoproteins and their forms.