Mice Lacking Selenoprotein P and Selenocysteine Lyase Exhibit Severe Neurological Dysfunction,Neurodegeneration, and Audiogenic Seizures

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.     

Disruption of the Selenocysteine Lyase-Mediated Selenium Recycling Pathway Leads to Metabolic Syndrome in Mice

Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins and is acquired either through diet or cellular recycling mechanisms. Selenocysteine lyase (Scly) is the enzyme that supplies Se for selenoprotein biosynthesis via decomposition of the amino acid selenocysteine (Sec). Knockout (KO) of Scly in a mouse affected hepatic glucose and lipid homeostasis. Mice lacking Scly and raised on an Se-adequate diet exhibit hyperinsulinemia, hyperleptinemia, glucose intolerance, and hepatic steatosis, with increased hepatic oxidative stress, but maintain selenoprotein levels and circulating Se status. Insulin challenge of Scly KO mice results in attenuated Akt phosphorylation but does not decrease phosphorylation levels of AMP kinase alpha (AMPKα). Upon dietary Se restriction, Scly KO animals develop several characteristics of metabolic syndrome, such as obesity, fatty liver, and hypercholesterolemia, with aggravated hyperleptinemia, hyperinsulinemia, and glucose intolerance. Hepatic glutathione peroxidase 1 (GPx1) and selenoprotein S (SelS) production and circulating selenoprotein P (Sepp1) levels are significantly diminished. Scly disruption increases the levels of insulin-signaling inhibitor PTP1B. Our results suggest a dependence of glucose and lipid homeostasis on Scly activity. These findings connect Se and energy metabolism and demonstrate for the first time a unique physiological role of Scly in an animal model.     

Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family

Selenium is an essential micronutrient that function through selenoproteins. Selenium deficiency results in lower concentrations of selenium and selenoproteins. The brain maintains it's selenium better than other tissues under low-selenium conditions. Recently, the selenium-containing protein selenoprotein P (Sepp) has been identified as a possible transporter of selenium. The targeted disruption of the selenoprotein P gene (Sepp1) results in decreased brain selenium concentration and neurological dysfunction, unless selenium intake is excessive However, the effect of selenoprotein P deficiency on the processes of memory formation and synaptic plasticity is unknown. In the present studies Sepp1(-/-) mice and wild type littermate controls (Sepp1(+/+)) fed a high-selenium diet (1 mg Se/kg) were used to characterize activity, motor coordination, and anxiety as well as hippocampus-dependent learning and memory. Normal associative learning, but disrupted spatial learning was observed in Sepp1(-/-) mice. In addition, severe alterations were observed in synaptic transmission, short-term plasticity and long-term potentiation in hippocampus area CA1 synapses of Sepp1(-/-) mice on a 1 mg Se/kg diet and Sepp1(+/+) mice fed a selenium-deficient (0 mg Se/kg) diet. Taken together, these data suggest that selenoprotein P is required for normal synaptic function, either through presence of the protein or delivery of required selenium to the CNS.     

Effects of selenium supplementation on diet-induced obesity in mice with a disruption of the selenocysteine lyase gene

BACKGROUNDThe 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.METHODSThree-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.RESULTSSe 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.CONCLUSIONThese 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.     

Selenoproteinless animals: selenophosphate synthetase SPS1 functions in a pathway unrelated to selenocysteine biosynthesis

Proteins containing the 21st amino acid, selenocysteine (Sec), have been described in all three domains of life, but the composition of selenoproteomes in organisms varies significantly. Here, we report that aquatic arthropods possess many selenoproteins also detected in other animals and unicellular eukaryotes, and that most of these proteins were either lost or replaced with cysteine-containing homologs in insects. As a result of this selective selenoproteome reduction, fruit flies and mosquitoes have three known selenoproteins, and the honeybee, Apis mellifera, a single detected candidate selenoprotein. Moreover, we identified the red flour beetle, Tribolium castaneum, and the silkworm, Bombyx mori, as the first animals that lack any Sec-containing proteins. These insects also lost the Sec biosynthesis and insertion machinery, but selenophosphate synthetase 1 (SPS1), an enzyme previously implicated in Sec biosynthesis, is present in all insects, including T. castaneum and B. mori. These data indicate that SPS1 functions in a pathway unrelated to selenoprotein synthesis. Since SPS1 evolved from a protein that utilizes selenium for Sec biosynthesis, an attractive possibility is that SPS1 may define a new pathway of selenium utilization in animals.     

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.     

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.     

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.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins, where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine. The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species. Available computational tools fail to correctly predict selenoproteins. Thus, we developed a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information, several programs were edited with PERL language to identify selenocysteine insertion sequence (SECIS) element, the coding potential of TGA codons, and cysteine-containing homologs of selenoprotein genes. Our results showed that 11365 genes were terminated with TGA codons, 918 of which contained SECIS elements. Similarity search revealed that 58genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons. Finally, 7genes were found to fully meet requirements for selenoproteins, although they have not been annotated as selenoproteins in NCBI databases. Deduced from their basic properties, the newly found selenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium. This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins,where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine.The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species.Available computational tools fail to correctly predict selenoproteins.Thus,we devel-oped a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information,several programs were edited with PERL language to identify selenocysteine insertion sequence(SECIS)element,the coding potential of TGA codons,and cys-teine-containing homologs of selenoprotein genes.Our results showed that 11365 genes were termi-nated with TGA codons,918 of which contained SECIS elements.Similarity search revealed that 58 genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons.Finally,7 genes were found to fully meet requirements for selenoproteins,although they have not been anno-tated as selenoproteins in NCBI databases.Deduced from their basic properties,the newly found se-lenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium.This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

Production of Selenoprotein P (Sepp1) by Hepatocytes Is Central to Selenium Homeostasis

Sepp1 is a widely expressed extracellular protein that in humans and mice contains 10 selenocysteine residues in its primary structure. Extra-hepatic tissues take up plasma Sepp1 for its selenium via apolipoprotein E receptor-2 (apoER2)-mediated endocytosis. The role of Sepp1 in the transport of selenium from liver, a rich source of the element, to peripheral tissues was studied using mice with selective deletion of Sepp1 in hepatocytes (Sepp1^c/c/alb-cre^+/− mice). Deletion of Sepp1 in hepatocytes lowered plasma Sepp1 concentration to 10% of that in Sepp1^c/c mice (controls) and increased urinary selenium excretion, decreasing whole-body and tissue selenium concentrations. Under selenium-deficient conditions, Sepp1^c/c/alb-cre^+/− mice accumulated selenium in the liver at the expense of extra-hepatic tissues, severely worsening clinical manifestations of dietary selenium deficiency. These findings are consistent with there being competition for metabolically available hepatocyte selenium between the synthesis of selenoproteins and the synthesis of selenium excretory metabolites. In addition, selenium deficiency down-regulated the mRNA of the most abundant hepatic selenoprotein, glutathione peroxidase-1 (Gpx1), to 15% of the selenium-replete value, while reducing Sepp1 mRNA, the most abundant hepatic selenoprotein mRNA, only to 61%. This strongly suggests that Sepp1 synthesis is favored in the liver over Gpx1 synthesis when selenium supply is limited, directing hepatocyte selenium to peripheral tissues in selenium deficiency. We conclude that production of Sepp1 by hepatocytes is central to selenium homeostasis in the organism because it promotes retention of selenium in the body and effects selenium distribution from the liver to extra-hepatic tissues, especially under selenium-deficient conditions.     

Selenoproteins and selenocysteine insertion system in the model plant cell system,Chlamydomonas reinhardtii

Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.     

Hepatic selenoprotein P (SePP) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice

SePP (selenoprotein P) is central for selenium transport and distribution. Targeted inactivation of the Sepp gene in mice leads to reduced selenium content in plasma, kidney, testis and brain. Accordingly, activities of selenoenzymes are reduced in Sepp(-/-) organs. Male Sepp(-/-) mice are infertile. Unlike selenium deficiency, Sepp deficiency leads to neurological impairment with ataxia and seizures. Hepatocyte-specific inactivation of selenoprotein biosynthesis reduces plasma and kidney selenium levels similarly to Sepp(-/-) mice, but does not result in neurological impairment, suggesting a physiological role of locally expressed SePP in the brain. In an attempt to define the role of liver-derived circulating SePP in contrast with locally expressed SePP, we generated Sepp(-/-) mice with transgenic expression of human SePP under control of a hepatocyte-specific transthyretin promoter. Secreted human SePP was immunologically detectable in serum from SEPP1-transgenic mice. Selenium content and selenoenzyme activities in serum, kidney, testis and brain of Sepp(-/-;SEPP1) (SEPP1-transgenic Sepp(-/-)) mice were increased compared with Sepp(-/-) controls. When a selenium-adequate diet (0.16-0.2 mg/kg of body weight) was fed to the mice, liver-specific expression of SEPP1 rescued the neurological defects of Sepp(-/-) mice and rendered Sepp(-/-) males fertile. When fed on a low-selenium diet (0.06 mg/kg of body weight), Sepp(-/-;SEPP1) mice survived 4 weeks longer than Sepp(-/-) mice, but ultimately developed the neurodegenerative phenotype. These results indicate that plasma SePP derived from hepatocytes is the main transport form of selenium supporting the kidney, testis and brain. Nevertheless, local Sepp expression is required to maintain selenium content in selenium-privileged tissues such as brain and testis during dietary selenium restriction.     

Deletion of selenoprotein P upregulates urinary selenium excretion and depresses whole-body selenium content

Deletion of the mouse selenoprotein P gene (Sepp1) lowers selenium concentrations in many tissues. We examined selenium homeostasis in Sepp1(-/-) and Sepp1(+/+) mice to assess the mechanism of this. The liver produces and exports selenoprotein P, which transports selenium to peripheral tissues, and urinary selenium metabolites, which regulate whole-body selenium. At intakes of selenium near the nutritional requirement, Sepp1(-/-) mice had whole-body selenium concentrations 72 to 75% of Sepp1(+/+) mice. Genotype did not affect dietary intake of selenium. Sepp1(-/-) mice excreted in their urine approximately 1.5 times more selenium in relation to their whole-body selenium than did Sepp1(+/+) mice. In addition, Sepp1(-/-) mice gavaged with (75)SeO(2-)(3) excreted 1.7 to 2.4 times as much of the (75)Se in the urine as did Sepp1(+/+) mice. These findings demonstrate that deletion of selenoprotein P raises urinary excretion of selenium. When urinary small-molecule (75)Se was injected intravenously into mice, over 90% of the (75)Se appeared in the urine within 24 h, regardless of selenium status. This shows that urinary selenium is dedicated to excretion and not to utilization by tissues. Our results indicate that deletion of selenoprotein P leads to increased urinary selenium excretion. We propose that the absence of selenoprotein P synthesis in the liver makes more selenium available for urinary metabolite synthesis, increasing loss of selenium from the organism and causing the decrease in whole-body selenium and some of the decreases observed in tissues of Sepp1(-/-) mice.     

Selenium and selenoproteins in the brain and brain diseases

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.     

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.     

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.     

硒蛋白的分子生物学研究进展

已有35种硒蛋白被分离和表征,但许多硒蛋白及其功能仍未完全阐明.硒半胱氨酸(Sec)作为参入蛋白质的第21种氨基酸,由硒蛋白mRNA上的UGA编码.在原核生物,Sec参入硒蛋白的复杂机制已经较为明确,需要四种基因产物（SELA、SELB、SELC和SELD）和一个存在于硒蛋白mRNA上的被称为Sec插入序列(SECIS)的茎环(stem loop)样二级结构.在真核生物,硒蛋白生物合成途径可能在SECIS的结构和位置、特异的延伸因子及其他RNA-RNA或RNA-蛋白质因子之间的相互作用等方面与原核生物不同.另外,哺乳动物硒蛋白mRNA上的UGA翻译为Sec的过程低效,特定位点的UGA密码子不同功能(终止密码和Sec密码)的调控可能是硒蛋白表达低效的关键.