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Fifty years have passed since the discovery of the first selenoprotein by Rotruck and colleagues. In that time, the essential nature of selenium has come to light including the dependence of the brain on selenium to function properly. Animal models have shown that a lack of certain selenoproteins in the brain is detrimental for neuronal health, sometimes leading to neurodegeneration. There is also potential for selenoprotein-mediated redox balance to impact neuronal activity, including neurotransmission. Important insights on these topics have been gained over the past several years. This review briefly summarizes the known roles of specific selenoproteins in the brain while highlighting recent advancements regarding selenoproteins in neuronal function. Hypothetical models of selenoprotein function and emerging topics in the field are also provided.

The use of glucocorticoid medications is known to cause metabolic side effects such as overeating, excess weight gain, and insulin resistance. The hypothalamus, a central regulator of feeding behavior and energy expenditure, is highly responsive to glucocorticoids, and it has been proposed that it plays a role in glucocorticoid-induced metabolic defects. Glucocorticoids can alter the expression and activity of antioxidant enzymes and promote the accumulation of reactive oxygen species. Recent evidence indicates that selenium can counter the effects of glucocorticoids, and selenium is critical for proper hypothalamic function. This study sought to determine whether selenium is capable of protecting hypothalamic cells from dysfunction caused by glucocorticoid exposure. We treated mHypoE-44 mouse hypothalamic cells with corticosterone to study the effects on cellular physiology and the involvement of selenium. We found that corticosterone administration rendered cells more vulnerable to endoplasmic reticulum stress and the subsequent impairment of insulin signaling. Supplementing the cell culture media with additional selenium alleviated endoplasmic reticulum stress and promoted insulin signaling. These findings implicate a protective role of selenium against chronic glucocorticoid-induced hypothalamic dysfunction.

In this minireview, we provide a historical outline of the events that led to the identification and characterization of the deiodinases, the recognition that deiodination plays a major role in thyroid hormone action, and the cloning of the 3 deiodinase genes. The story starts in 1820, when it was first determined that elemental iodine was important for normal thyroid function. Almost 100 years later, it was found that the primary active principle of the gland, T4, contains iodine. Once radioactive iodine became available in the 1940s, it was demonstrated that the metabolism of T4 included deiodination, but at the time it was assumed to be merely a degradative process. However, this view was questioned after the discovery of T3 in 1952. We discuss in some detail the events of the next 20 years, which included some failures followed by the successful demonstration that deiodination is indeed essential to normal thyroid hormone action. Finally, we describe how the 3 deiodinases were identified and characterized and their genes cloned.

BACKGROUND The amino acid selenocysteine (Sec) is an integral part of selenoproteins, a class of proteins mostly involved in strong redox reactions. The enzyme Sec lyase (SCLY) decomposes Sec into selenide allowing for the recycling of the selenium (Se) atom via the selenoprotein synthesis machinery. We previously demonstrated that disruption of the Scly gene (Scly KO) in mice leads to the development of obesity and metabolic syndrome, with effects on glucose homeostasis, worsened by Se deficiency or a high-fat diet, and exacerbated in male mice. Our objective was to determine whether Se supplementation could ameliorate obesity and restore glucose homeostasis in the Scly KO mice. METHODS Three-weeks old male and female Scly KO mice were fed in separate experiments a diet containing 45 % kcal fat and either sodium selenite or a mixture of sodium selenite and selenomethionine (selenite/SeMet) at moderate (0.25 ppm) or high (0.5–1 ppm) levels for 9 weeks, and assessed for metabolic parameters, oxidative stress and expression of selenoproteins. RESULTS Se supplementation was unable to prevent obesity and elevated epididymal white adipose tissue weights in male Scly KO mice. Serum glutathione peroxidase activity in Scly KO mice was unchanged regardless of sex or dietary Se intake; however, supplementation with a mixture of selenite/SeMet improved oxidative stress biomarkers in the male Scly KO mice. CONCLUSION These results unveil sex- and selenocompound-specific regulation of energy metabolism after the loss of Scly, pointing to a role of this enzyme in the control of whole-body energy metabolism regardless of Se levels.

Sexual dimorphism, the condition in which males and females in a species differ beyond the morphology of sex organs, delineates critical aspects of the biology of higher eukaryotes, including selenium metabolism. While sex differences in selenium biology have been described by several laboratories, delineation of the effects of sex in selenium function and regulation of selenoprotein expression is still in its infancy. This review encompasses the available information on sex-dependent parameters of selenium metabolism, as well as the effects of selenium on sex hormones. Gaps in the current knowledge of selenium and sex are identified and discussed.

People with obesity are often dyslipidemic and prescribed statins to prevent cardiovascular events. A common side effect of statin use is myopathy. This could potentially be caused by the reduction of selenoproteins that curb oxidative stress, in turn, affecting creatine metabolism. We determined if statins regulate hepatic and muscular selenoprotein expression, oxidative stress and creatine metabolism. Mice lacking selenocysteine lyase (Scly KO), a selenium-provider enzyme for selenoprotein synthesis, were fed a high-fat, Se-supplemented diet and treated with simvastatin. Statin improved creatine metabolism in females and oxidative responses in both sexes. Male Scly KO mice were heavier than females after statin treatment. Hepatic selenoproteins were unaffected by statin and genotype in females. Statin upregulated muscular Gpx1 in females but not males, while Scly loss downregulated muscular Gpx1 in males and Selenon in females. Osgin1 was reduced in statin-treated Scly KO males after AmpliSeq analysis. These results refine our understanding of the sex-dependent role of selenium in statin responses.

Selenium is an essential dietary micronutrient. Ingested selenium is absorbed by the intestines and transported to the liver where it is mostly metabolized to selenocysteine (Sec). Sec is then incorporated into selenoproteins, including selenoprotein P (SELENOP), which is secreted into plasma and serves as a source of selenium to other tissues of the body. Herein, we provide an overview of the biology of selenium from its absorption and distribution to selenoprotein uptake and degradation, with a particular focus on the latter. Molecular mechanisms of selenoprotein degradation include the lysosome-mediated pathway for SELENOP and endoplasmic reticulum-mediated degradation of selenoproteins via ubiquitin-activated proteasomal pathways. Ubiquitin-activated pathways targeting full-length selenoproteins include the peroxisome proliferator-activated receptor gamma-dependent pathway and substrate-dependent ubiquitination. An alternate mechanism is utilized for truncated selenoproteins, in which cullin-RING E3 ubiquitin ligase 2 targets the defective proteins for ubiquitin-proteasomal degradation. Selenoproteins, particularly SELENOP, may have their Sec residues reutilized for new selenoprotein synthesis via Sec decomposition. This review will explore these aspects in selenium biology, providing insights to knowledge gaps that remain to be uncovered.

Selenoprotein P (SelenoP) functions as a plasma transporter of selenium (Se) from liver to other tissues via incorporation into multiple selenocysteine (Sec) residues. Selenocysteine lyase (Scly) is an intracellular enzyme that decomposes Sec into selenide, providing Se for the synthesis of new selenoproteins. Both SelenoP and Scly are mostly produced by the liver. Previous studies demonstrated that male mice lacking SelenoP (SelenoP KO) or Scly (Scly KO) had increased or decreased total hepatic Se, respectively. While SelenoP regulation by Se is well-studied, Scly regulation by Se has not been reported. We hypothesize that Scly is negatively regulated by Se levels, and that absence of SelenoP jeopardizes Scly-dependent Se recycling. Using in vitro and in vivo models, we unveiled a tissue-specific Se regulation of Scly gene expression. We also determined that SelenoP, a considered source of intracellular Se, affects Scly expression and activity in vitro but not in vivo, as in the absence of SelenoP, Scly levels and activity remain normal. We also showed that absence of SelenoP does not increase levels of transsulfuration pathway enzymes, which would result in available selenocompounds being decomposed by the actions of cystathionine γ-lyase (CGL or CTH) and cystathionine β-synthase (CBS). Instead, it affects levels of thioredoxin reductase 1 (Txnrd1), an enzyme that can reduce selenite to selenide to be used in selenoprotein production. This study evaluates a potential interplay between SelenoP and Scly, providing further insights into the regulation of selenium metabolism.

The hypothalamus is the central neural site governing food intake and energy expenditure. During the past 25 years, understanding of the hypothalamic cell types, hormones, and circuitry involved in the regulation of energy metabolism has dramatically increased. It is now well established that the adipocyte-derived hormone, leptin, acts upon two distinct groups of hypothalamic neurons that comprise opposing arms of the central melanocortin system. These two cell populations are anorexigenic neurons expressing proopiomelanocortin (POMC) and orexigenic neurons that express agouti-related peptide (AGRP). Several important studies have demonstrated that reactive oxygen species and endoplasmic reticulum stress significantly impact these hypothalamic neuronal populations that regulate global energy metabolism. Reactive oxygen species and redox homeostasis are influenced by selenoproteins, an essential class of proteins that incorporate selenium co-translationally in the form of the 21st amino acid, selenocysteine. Levels of these proteins are regulated by dietary selenium intake and they are widely expressed in the brain. Of additional relevance, selenium supplementation has been linked to metabolic alterations in both animal and human studies. Recent evidence also indicates that hypothalamic selenoproteins are significant modulators of energy metabolism in both neurons and tanycytes, a population of glial-like cells lining the floor of the 3rd ventricle within the hypothalamus. This review article will summarize current understanding of the regulatory influence of redox status on hypothalamic nutrient sensing and highlight recent work revealing the importance of selenoproteins in the hypothalamus.

Seafood is an important source of nutrients for fetal neurodevelopment. Most individuals are exposed to the toxic element mercury through seafood. Due to the neurotoxic effects of mercury, United States government agencies recommend no more than 340 g (12 oz) per week of seafood consumption during pregnancy. However, recent studies have shown that selenium, also abundant in seafood, can have protective effects against mercury toxicity. In this study, we analyzed mercury and selenium levels and selenoprotein mRNA, protein, and activity in placenta of a cohort of women in Hawaii in relation to maternal seafood consumption assessed with dietary surveys. Fish consumption resulted in differences in mercury levels in placenta and cord blood. When taken as a group, those who consumed no fish exhibited the lowest mercury levels in placenta and cord blood. However, there were numerous individuals who either had higher mercury with no fish consumption or lower mercury with high fish consumption, indicating a lack of correlation. Placental expression of selenoprotein mRNAs, proteins and enzyme activity was not statistically different in any region among the different dietary groups. While the absence of seafood consumption correlates with lower average placental and cord blood mercury levels, no strong correlations were seen between seafood consumption or its absence and the levels of either selenoproteins or selenoenzyme activity.

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.

Selenoproteins are a unique family of proteins, characterized by the co-translational incorporation of selenium as selenocysteine, which play key roles in antioxidant defense. Among selenoproteins, selenoprotein P (Sepp1) is particularly distinctive due to the fact that it contains multiple selenocysteine residues and has been postulated to act in selenium transport. Within the brain, Sepp1 delivers selenium to neurons by binding to the ApoER2 receptor. Upon feeding a selenium-deficient diet, mice lacking ApoER2 or Sepp1 develop severe neurological dysfunction and exhibit widespread brainstem neurodegeneration, indicating an important role for ApoER2-mediated Sepp1 uptake in normal brain function. Selenocysteine lyase (Scly) is an enzyme that plays an important role in selenium homeostasis, in that it catalyzes the decomposition of selenocysteine and allows selenium to be recycled for additional selenoprotein synthesis. We previously reported that constitutive deletion of Scly results in neurological deficits only when mice are challenged with a low selenium diet. To gain insight into the relationship between Sepp1 and Scly in selenium metabolism, we created novel transgenic mice constitutively lacking both genes (Scly(-/-)Sepp1(-/-)) and characterized the neurobehavioral phenotype. We report that deletion of Scly in conjunction with Sepp1 further aggravates the phenotype of Sepp1(-/-) mice, as these mice needed supraphysiological selenium supplementation to survive, and surviving mice exhibited impaired motor coordination, audiogenic seizures, and brainstem neurodegeneration. These findings provide the first in vivo evidence that Scly and Sepp1 work cooperatively to maintain selenoprotein function in the mammalian brain.

Selenium is an essential trace element linked to normal development and antioxidant defense mechanisms through its incorporation into selenoproteins via the amino acid, selenocysteine (Sec). Male mice lacking both the Se transporter, selenoprotein P (SELENOP), and selenocysteine lyase (Scly), which plays a role in intracellular Se utilization, require Se supplementation for viability and exhibit neuromotor deficits. Previously, we demonstrated that male SELENOP/Scly double knockout (DKO) mice suffer from loss of motor function and audiogenic seizures due to neurodegeneration, both of which are alleviated by prepubescent castration. The current study examined the neuromotor function of female DKO mice using the rotarod and open field test, as well as the effects of dietary Se restriction. Female DKO mice exhibited a milder form of neurological impairment than their male counterparts. This impairment is exacerbated by removal of Se supplementation during puberty. These results indicate there is a critical time frame in which Se supplementation is essential for neurodevelopment. These sex-specific differences may unveil new insights into dietary requirements for this essential nutrient in humans.

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all of the known selenoproteins are found in brain, where expression is facilitated by selenocysteine-laden selenoprotein P. The brain also expresses selenocysteine lyase, an enzyme that putatively salvages selenocysteine and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of selenocysteine lyase to selenoprotein P knockout mice for similarity of neurological impairments, and whether dietary selenium modulates these parameters. We report that selenocysteine lyase knockout mice do not display neurological dysfunction comparable to selenoprotein P knockout mice. Feeding a low-selenium diet to selenocysteine lyase knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of selenoprotein P is exacerbated in male versus female mice. These findings indicate that selenocysteine recycling via selenocysteine lyase becomes limiting under selenium deficiency, and suggest the presence of a complementary mechanism for processing selenocysteine. Our studies illuminate the interaction between selenoprotein P and selenocysteine lyase in the distribution and turnover of body and brain selenium, and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology.

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line ( approximately 5Kb average telomere length) and SY5Y neuroblastoma cell line (approximately 4.2Kb average telomere length), we observed a significant reduction (-0.6 to -1.1 Kb; Por= 0.01) in telomere length as compared to control cells. This reduction in telomere length was independent of affects on telomerase, since both telomerase activity levels and Tert mRNA expression levels were not altered by knock-down of SBP2 expression. Furthermore, telomeres were particularly sensitive to S1 nuclease digestion following SBP2 knock-down, indicating an increased frequency of oxidative damage-induced lesions in the telomeric DNA in these cells. Together, these observations imply that selenoproteins may help protect telomeric reserve in mammalian cells.

The incidence of breast cancer among women is high and increasing. This study investigated the inhibitory effect of an extract from bamboo Phyllostachys edulis on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in female Sprague-Dawley rats. The rats were fed with bamboo extract (BEX) supplemented diet or control diet, and treated with DMBA after 3 weeks of the dietary regime. The incidence of mammary tumors was monitored by palpation for the next 11 weeks. At the end of the experiment, blood samples were collected for total antioxidant capacities (TAC) assay and liver samples for phase II enzyme activity assays. The TAC values, total contents of phenolics and flavonoids of BEX were also measured. The results showed that BEX delayed the onset of mammary tumor by 1 week, decreased the tumor incidence by 44% and tumor multiplicity by 67%, and increased the total sulfotransferases (SULT) activity by 63%. BEX showed high levels of TAC, total phenolic and total flavonoids. However, the serum TAC values were not affected by BEX supplementation. In summary, the results indicate that BEX possesses a potent anti-breast cancer effect, and the upregulation of SULT activity, therefore estrogen metabolism may be the underlying mechanism.

The long-sought pathway by which selenocysteyl-tRNA[Ser]Sec is synthesized in eukaryotes has been revealed. Seryl-tRNA[Ser]Sec is O-phosphorylated and SecS, a pyridoxal phosphate-dependent protein, catalyzes the reaction of O-phosphoseryl-tRNA[Ser]Sec with monoselenophosphate to give selenocysteyl-tRNA[Ser]Sec . 1 H- 77 Se HMQC-TOCSY NMR spectroscopy has been developed to detect the selenium-containing amino acids present in selenized yeast after protease XIV digestion. An archived selenized yeast sample is found to contain the novel amino acid S-(methylseleno)cysteine in addition to selenomethionine. Arsenite and selenite react with GSH to form (GS) 2 AsSe−. The structure of this compound has been determined by EXAFS, 77 Se NMR and Raman spectroscopic and chromatographic studies. Its formation under biological conditions has been demonstrated.

Mitochondria are the major reactive oxygen species (ROS)--generating sites in mammalian cells. Blockade of complexes in the electron transport chain (ETC) increases the leakage of single electrons to O(2) and therefore increases ROS levels. Complexes I and III have been reported to be the major ROS-generating sites in mitochondria. In this study, using mouse hippocampal HT22 cells as in vitro model, we monitored the change of intracellular ROS level in response to the blockade of ETC at different complex, and measured changes of gene expression of antioxidant enzymes and phase II enzymes, also evaluated potential protective effect of selenium (Se) supplementation to the cells under this oxidative stress. In summary, our results showed that complex I was the major ROS-generating site in HT22 cells. Complex I blockade upregulated the mRNA levels of glutamylcysteine synthetase heavy and light chains, glutathione-S-transferases omega1 and alpha 2, hemoxygenase 1, thioredoxin reductase 1, and selenoprotein H. Unexpectedly, the expression of the enzymes that directly scavenge ROS decreased, including superoxide dismutases 1 and 2, glutathione peroxidase 1, and catalase. Se supplementation increased glutathione levels and glutathione peroxidase activity, indicating a potential protective role in oxidative stress caused by ETC blockade.

Aims: Selenocysteine lyase (Scly) mediates selenocysteine decomposition. It was previously demonstrated that, upon adequate caloric intake (12% kcal fat) and selenium deficiency, disruption of Scly in mice leads to development of metabolic syndrome. In this study, we investigate the effect of a high-fat (45% kcal) selenium-adequate diet in Scly knockout (KO) mice on development of metabolic syndrome. Involvement of selenoproteins in energy metabolism after Scly disruption was also examined in vitro in the murine hepatoma cell line, Hepa1-6, following palmitate treatment. Results: Scly KO mice were more susceptible to diet-induced obesity than their wild-type counterparts after feeding a high-fat selenium-adequate diet. Scly KO mice had aggravated hyperinsulinemia, hypercholesterolemia, glucose, and insulin intolerance, but unchanged inflammatory cytokines and expression of most selenoproteins, except increased serum selenoprotein P (Sepp1). Scly KO mice also exhibited enhanced hepatic levels of pyruvate and enzymes involved in the regulation of pyruvate cycling, such as pyruvate carboxylase (Pcx) and pyruvate dehydrogenase (Pdh). However, in vitro silencing of Scly in Hepa1-6 cells led to diminished Sepp1 expression, and concomitant palmitate treatment decreased Pdh expression. Innovation: The role of selenium in lipid metabolism is recognized, but specific selenium-dependent mechanisms leading to obesity are unclear. This study uncovers that Scly has a remarkable effect on obesity and metabolic syndrome development triggered by high-fat exposure, independent of the expression of most selenoproteins. Conclusion: Diet-induced obesity in Scly KO mice is aggravated, with effects on pyruvate levels and consequent activation of energy metabolism independent of selenoprotein levels. Antioxid. Redox Signal. 23, 761–774.

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688–695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA [Ser]Sec , but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA [Ser]Sec kinase ( pstk ). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA [Ser]Sec and, in addition, had a requirement for ATP and Mg 2+ . Proteins with homology to mammalian PSTK occur in Drosophila , Caenorhabditis elegans , Methanopyrus kandleri , and Methanococcus jannaschii , suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation.

Over the past three decades, selenium has been intensively investigated as an antioxidant trace element. It is widely distributed throughout the body, but is particularly well maintained in the brain, even upon prolonged dietary selenium deficiency. Changes in selenium concentration in blood and brain have been reported in Alzheimer's disease and brain tumors. The functions of selenium are believed to be carried out by selenoproteins, in which selenium is specifically incorporated as the amino acid, selenocysteine. Several selenoproteins are expressed in brain, but many questions remain about their roles in neuronal function. Glutathione peroxidase has been localized in glial cells, and its expression is increased surrounding the damaged area in Parkinson's disease and occlusive cerebrovascular disease, consistent with its protective role against oxidative damage. Selenoprotein P has been reported to possess antioxidant activities and the ability to promote neuronal cell survival. Recent studies in cell culture and gene knockout models support a function for selenoprotein P in delivery of selenium to the brain. mRNAs for other selenoproteins, including selenoprotein W, thioredoxin reductases, 15-kDa selenoprotein and type 2 iodothyronine deiodinase, are also detected in the brain. Future research directions will surely unravel the important functions of this class of proteins in the brain.

Background : Insects appear to have diverged from both higher and lower organisms in their defense mechanisms against oxidative damage. They do not encode glutathione peroxidases or glutathione reductases, and their thioredoxin reductases exhibit distinct properties from those of higher and lower species. Nonetheless, appropriate balance of antioxidants and pro-oxidants, and protection from damaging reactive oxygen species are clearly crucial in insects for viability, normal functioning of signalling pathways and morphogenesis, and have been implicated in studies on longevity in flies and other organisms. We have used RNAi in D. melanogaster embryos and in Schneider S2 cells to inhibit expression of these proteins. We report that inhibition of either dselH or dselK expression significantly reduces viability in embryos. We further show that dselH silencing decreases total anti-oxidant capacity in embryos and Schneider cells, and increases lipid peroxidation in cells. Conversely, transient expression of dselH in the cell line decreases lipid peroxidation, and reverses the toxic effects of a glutathione-depleting drug. The latter correlates with sparing of glutathione levels.

The goal of this review is to place the exciting advances that have occurred in our understanding of the molecular biology of the types 1, 2, and 3 (D1, D2, and D3, respectively) iodothyronine deiodinases into a biochemical and physiological context. We review new data regarding the mechanism of selenoprotein synthesis, the molecular and cellular biological properties of the individual deiodinases, including gene structure, mRNA and protein characteristics, tissue distribution, subcellular localization and topology, enzymatic properties, structure-activity relationships, and regulation of synthesis, inactivation, and degradation. These provide the background for a discussion of their role in thyroid physiology in humans and other vertebrates, including evidence that D2 plays a significant role in human plasma T3 production. We discuss the pathological role of D3 overexpression causing “consumptive hypothyroidism” as well as our current understanding of the pathophysiology of iodothyronine deiodination during illness and amiodarone therapy. Finally, we review the new insights from analysis of mice with targeted disruption of the Dio2 gene and overexpression of D2 in the myocardium. (Endocrine Reviews 23: 38–89, 2002)

In neurons, calcium (Ca(2+) ) channels regulate a wide variety of functions ranging from synaptic transmission to gene expression. They also induce neuroplastic changes that alter gene expression following psychostimulant administration. Ca(2+) channel blockers have been considered as potential therapeutic agents for the treatment of methamphetamine (METH) dependence because of their ability to reduce drug craving among METH users. Here, we studied the effects of METH exposure on voltage-gated Ca(2+) channels using SH-SY5Y cells as a model of dopaminergic neurons. We found that METH has different short- and long-term effects. A short-term effect involves immediate ( 5 min) direct inhibition of Ca(2+) ion movements through Ca(2+) channels. Longer exposure to METH (20 min or 48 h) selectively up-regulates the expression of only the CACNA1C gene, thus increasing the number of L-type Ca(2+) channels. This up-regulation of CACNA1C is associated with the expression of the cAMP-responsive element-binding protein (CREB), a known regulator of CACNA1C gene expression, and the MYC gene, which encodes a transcription factor that putatively binds to a site proximal to the CACNA1C gene transcription initiation site. The short-term inhibition of Ca(2+) ion movement and later, the up-regulation of Ca(2+) channel gene expression together suggest the operation of cAMP-responsive element-binding protein- and C-MYC-mediated mechanisms to compensate for Ca(2+) channel inhibition by METH. Increased Ca(2+) current density and subsequent increased intracellular Ca(2+) may contribute to the neurodegeneration accompanying chronic METH abuse. Methamphetamine (METH) exposure has both short- and long-term effects. Acutely, methamphetamine directly inhibits voltage-gated calcium channels. Chronically, neurons compensate by up-regulating the L-type Ca(2+) channel gene, CACNA1C. This compensatory mechanism is mediated by transcription factors C-MYC and CREB, in which CREB is linked to the dopamine D1 receptor signaling pathway. These findings suggest Ca(2+) -mediated neurotoxicity owing to over-expression of calcium channels.

Selenoproteins are a distinct class of proteins that are characterized by the co-translational incorporation of selenium (Se) in the form of the 21st amino acid selenocysteine. Selenoproteins provide a key defense against oxidative stress, as many of these proteins participate in oxidation-reduction reactions neutralizing reactive oxygen species, where selenocysteine residues act as catalytic sites. Many selenoproteins are highly expressed in the brain, and mouse knockout studies have determined that several are required for normal brain development. In parallel with these laboratory studies, recent reports of rare human cases with mutations in genes involved in selenoprotein biosynthesis have described individuals with an assortment of neurological problems that mirror those detailed in knockout mice. These deficits include impairments in cognition and motor function, seizures, hearing loss, and altered thyroid metabolism. Additionally, due to the fact that oxidative stress is a key feature of neurodegenerative disease, there is considerable interest in the therapeutic potential of selenium supplementation for human neurological disorders. Studies performed in cell culture and rodent models have demonstrated that selenium administration attenuates oxidative stress, prevents neurodegeneration, and counters cell signaling mechanisms known to be dysregulated in certain disease states. However, there is currently no definitive evidence in support of selenium supplementation to prevent and/or treat common neurological conditions in the general population. It appears likely that, in humans, supplementation with selenium may only benefit certain subpopulations, such as those that are either selenium-deficient or possess genetic variants that affect selenium metabolism.

Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery.

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.

Background Selenocysteine incorporation has been reported to be inefficient in all systems studied, including Escherichia coli, baculovirus-insect cell systems, rabbit reticulocyte in vitro translation systems, transiently transfected mammalian cells, and intact animals. Nonetheless, full-length selenoproteins containing up to 17 selenocysteine residues are produced in animals, indicating that the efficiency observed in manipulated systems might not accurately reflect the true efficiency of this process in nature. Results To begin to address this apparent discrepancy, we have examined the polysome profiles of endogenously expressed selenoprotein mRNAs in a mammalian cell line, and compared them with nonselenoprotein mRNAs. We report that three selenoprotein mRNAs, type 1 deiodinase, glutathione peroxidase and selenoprotein P, are under-loaded with ribosomes, based on their predicted open reading frame sizes. The average numbers of ribosomes per mRNA correspond to the sizes predicted by termination at the UGA selenocysteine codons. Appropriate loading on the type 1 deiodinase mRNA is seen following substitution of a cysteine codon for the selenocysteine codon, indicating that the UGA codon confers a translational penalty on the mRNA. Surprisingly, ribosomal loading is also increased by the expression of eukaryotic release factors eRF1 and eRF3. Conclusions These results suggest that the presence of a selenocysteine codon confers a translational penalty on selenoprotein mRNAs, and that increased levels of release factors may alter the kinetics of termination.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3′-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3′-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3'-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.

Selenium, an essential trace element, is a component of prokaryotic and eukaryotic antioxidant proteins. A candidate selenoprotein homologous to glutathione peroxidase was deduced from the sequence of molluscum contagiosum, a poxvirus that causes persistent skin neoplasms in children and acquired immunodeficiency syndrome (AIDS) patients. Selenium was incorporated into this protein during biosynthesis, and a characteristic stem-loop structure near the end of the messenger RNA was required for alternative selenocysteine decoding of a potential UGA stop codon within the open reading frame. The selenoprotein protected human keratinocytes against cytotoxic effects of ultraviolet irradiation and hydrogen peroxide, providing a mechanism for a virus to defend itself against environmental stress.

Cotranslational insertion of selenocysteine occurs in both prokaryotic and eukaryotic organisms. For the most part, what is known about this process in eukaryotes has been derived from extensive analysis of the RNA requirements for selenocysteine incorporation. Studies which have defined the selenocysteine insertion sequence (SECIS) element, identified its critical sequence and structural motifs, and established constraints on its spatial relationship with the UGA selenocysteine codon, have given mechanistic insights into this critical process.

Selenium is an essential trace element that is co-translationally incorporated into selenoproteins in the form of the 21st amino acid, selenocysteine. This class of proteins largely functions in oxidation-reduction reactions and is critically involved in maintaining proper redox balance essential to health. Selenoprotein M (SelM) is a thioredoxin-like endoplasmic reticulum-resident protein that is highly expressed in the brain and possesses neuroprotective properties. In this study, we first assessed the regional pattern of SelM expression in the mouse brain to provide insights into the potential functional implications of this protein in physiology and behavior. Next, we generated transgenic mice with a targeted deletion of the SelM gene and subjected them to a battery of neurobehavioral tests to evaluate motor coordination, locomotion, and cognitive function in comparison with wild-type controls. Finally, these mice were tested for several measures of metabolic function and body composition. Our results show that SelM knock-out (KO) mice display no deficits in measures of motor coordination and cognitive function but exhibit increased weight gain, elevated white adipose tissue deposition, and diminished hypothalamic leptin sensitivity. These findings suggest that SelM plays an important role in the regulation of body weight and energy metabolism. Background: Selenoprotein M (SelM) is highly expressed in the brain and postulated to have neuroprotective properties. Results: SelM expression is present in high levels in hypothalamic nuclei involved in energy metabolism, and SelM KO mice exhibit increased adiposity without apparent cognitive deficits. Conclusion: SelM protects against obesity. Significance: Increased understanding of the genes that protect against obesity may yield improved treatments and prevention strategies.

HIV infection and methamphetamine (Meth) abuse both may lead to oxidative stress. This study used HIV-1 transgenic (HIV-1Tg) rats to investigate the independent and combined effects of HIV viral protein expression and low dose repeated Meth exposure on the glutathione (GSH)-centered antioxidant system and oxidative stress in the brain. Total GSH content, gene expression and/or enzymatic activities of glutamylcysteine synthetase (GCS), gamma-glutamyltransferase (GGT), glutathione reductase (GR), glutathione peroxidase (GPx), glutaredoxin (Glrx), and glutathione-s-transferase (GST) were measured. The protein expression of cystine transporter (xCT) and oxidative stress marker 4-hydroxynonenal (HNE) were also analyzed. Brain regions studied include thalamus, frontal and remainder cortex, striatum, cerebellum and hippocampus. HIV-1Tg rats and Meth exposure showed highly regional specific responses. In the F344 rats, the thalamus had the highest baseline GSH concentration and potentially higher GSH recycle rate. HIV-1Tg rats showed strong transcriptional responses to GSH depletion in the thalamus. Both HIV-1Tg and Meth resulted in decreased GR activity in thalamus, and decreased Glrx activity in frontal cortex. However, the increased GR and Glrx activities synergized with increased GSH concentration, which might have partially prevented Meth-induced oxidative stress in striatum. Interactive effects between Meth and HIV-1Tg were observed in thalamus on the activities of GCS and GGT, and in thalamus and frontal cortex on Glrx activity and xCT protein expression. Findings suggest that HIV viral protein and low dose repeated Meth exposure have separate and combined effects on the brain’s antioxidant capacity and the oxidative stress response that are regional specific.

Selenium is an essential micronutrient in mammals, but is also recognized as toxic in excess. It is a non-metal with properties that are intermediate between the chalcogen elements sulfur and tellurium. Selenium exerts its biological functions through selenoproteins. Selenoproteins contain selenium in the form of the 21st amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur-containing side chain replaced by a Se-containing side chain. Sec is encoded by the codon UGA, which is one of three termination codons for mRNA translation in non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by specialized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome. Most of the selenoprotein genes were discovered by bioinformatics approaches, searching for SECIS elements downstream of in-frame UGA codons. Sec has been described as having stronger nucleophilic and electrophilic properties than cysteine, and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins, whose functions are known, are involved in redox systems and signaling pathways. However, several selenoproteins are not well characterized in terms of their function. The selenium field has grown dramatically in the last few decades, and research on selenium biology is providing extensive new information regarding its importance for human health.

The regulation of translation frequently involves protein-RNA interactions. An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine. Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS element) and a unique selenocysteyl-tRNA, are required for this process. In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding. whether eukaryotes use a similar mechanism is currently the subject of intense investigation.

Selenoprotein H is a redox-sensing DNA binding protein that upregulates genes involved in antioxidant responses. Given the known links between oxidative stress and heavy metals, we investigated the potential for regulation of selenoprotein H by metals. In silico analysis of the selenoprotein H genes from nine species reveals multiple predicted metal response elements (MREs). To validate MRE function, we investigated the effects of zinc or cadmium addition and metal-responsive transcription factor 1 (MTF-1) knockout on selenoprotein H mRNA levels. Chromatin immunoprecipitation was used to directly assess physical binding of the transcription factor to MREs in the human and mouse selenoprotein H genes. The results reported herein show that selenoprotein H is a newly identified target for MTF-1. Further, whereas nearly all prior studies of MREs focused on those located in promoters, we demonstrate binding of MTF-1 to MREs located downstream of the transcription start sites in the human and murine selenoprotein H genes. Finally, we identified MREs in downstream sequences in 15 additional MTF-1 regulated genes lacking promoter MREs, and demonstrated MTF-1 binding in three of these genes. This regulation via sequences downstream of promoters highlights a new direction for identifying previously unrecognized target genes for MTF-1.