Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA[Ser]Sec as substrates to generate selenocysteyl-tRNA[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.

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 [ S elenocysteine (Sec) has its own code word, UGA, and its own tRNA, and therefore is viewed as the 21st amino acid in the genetic code (reviewed in refs. 1-4). Although UGA usually codes for the termination of protein synthesis, it also specifies Sec if specific requirements are met. The presence of a stemloop structure downstream of UGA, called a Sec insertion sequence (SECIS) element, is the critical component in selenoprotein mRNAs that dictates UGA to code for Sec (reviewed in ref. 5). In mammals, the SECIS element occurs in the 3Ј untranslated region of selenoprotein mRNAs. A specific elongation factor, EFsec, specifically recognizes selenocysteyltRNA [Ser]Sec (6, 7), and a SECIS element binding protein, SBP2, binds specifically to the SECIS element (8), directing the insertion of Sec into protein in response to UGA.It has been known for several years that the biosynthesis of Sec occurs on its tRNA in both bacteria (9) and mammals (10) after the tRNA is initially aminoacylated with serine by seryl-tRNA synthetase. In Escherichia coli, the pathway for the biosynthesis of Sec has been completely established (reviewed in ref. 1). After the aminoacylation of bacterial tRNA [Ser]Sec with serine, the hydroxyl group is removed from the seryl moiety to yield an aminoacrylyl intermediate, and this step is catalyzed by a pyridoxal phosphate-dependent Sec synthase. The aminoacrylyl intermediate serves as the acceptor for the activated selenium donor, monoselenophosphate, which is synthesized from selenite and ATP in the presence of selenophosphate synthetase (reviewed in ref. 1). Once selenium is donated to the intermediate, the biosynthesis of Sec on tRNA [Ser]Sec is complete.In eukaryotes, however, the biosynthesis of Sec has not been established, but several components have been identified over the years that play a role in this process. For example, in 1970, a minor seryl-tRNA was reported to form phosphoseryl-tRNA by a kinase activity from rooster liver (11), and a minor seryl-tRNA from bovine, rabbit, and chicken livers was reported to specifically decode the nonsense codon UGA (12). Although it was subsequently shown that both the minor seryl-tRNA that formed phosphoseryl-tRNA and the one that decoded UGA were the same tRNA (13), it was not known at the time these components were ...

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

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Numerous studies characterizing the function of glutathione peroxidase 4 (GPx4) have demonstrated that this selenoenzyme is protective against oxidative stress. Herein, we characterized the function of this protein by targeting GPx4 downregulation using RNA interference. Partial knockdown of GPx4 levels resulted in growth retardation and morphological changes. Surprisingly, GPx4 knockdown cells showed virtually unchanged levels of intracellular ROS, yet highly increased levels of oxidized lipid by-products. GPx1, another glutathione peroxidase and a major cellular peroxide scavenging enzyme, did not rescue GPx4-deficient cells and did not reduce lipid peroxide levels. The data established an essential role of GPx4 in protecting cells against lipid hydroperoxide damage, yet a limited role as a general antioxidant enzyme. As oxidized lipid hydroperoxides are a characteristic of neurodegenerative diseases, we analyzed brain tissues of mice suffering from a model of Alzheimer's disease and found that oxidized lipid by-products were enriched, and expression of both GPx4 and guanine-rich sequence-binding factor, which is known to control GPx4 synthesis, was downregulated. Brain tissue from an Alzheimer's diseased human also manifested enhanced levels of one of the oxidized lipid by-products, 4-hydroxynonenal. These data suggest a role of GPx4 in neurodegenerative diseases through its function in removal of lipid hydroperoxides.

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA ½SerSec and its insertion was dependent on the Sec insertion sequence element in the 3′ UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA ½SerSec to generate Cys-tRNA ½SerSec . Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinerybased mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.de novo synthesis | new biosynthetic pathway | selenium deficiency

Although selenophosphate synthetase 1 (SPS1/SelD) is an essential gene in Drosophila, its function has not been determined. To elucidate its intracellular role, we targeted the removal of SPS1/SelD mRNA in Drosophila SL2 cells using RNA interference technology that led to the formation of vacuole-like globular structures. Surprisingly, these structures were identified as megamitochondria, and only depolarized mitochondria developed into megamitochondria. The mRNA levels of l(2)01810 and glutamine synthetase 1 (GS1) were increased by SPS1/SelD knockdown. Blocking the expression of GS1 and l(2)01810 completely inhibited the formation of megamitochondria induced by loss of SPS1/SelD activity and decreased the intracellular levels of glutamine to those of control cells suggesting that the elevated level of glutamine is responsible for megamitochondrial formation. Overexpression of GS1 and l(2)01810 had a synergistic effect on the induction of megamitochondrial formation and on the synthesis of glutamine suggesting that l(2)01810 is involved in glutamine synthesis presumably by activating GS1. Our results indicate that, in Drosophila, SPS1/SelD regulates the intracellular glutamine by inhibiting GS1 and l(2)01810 expression and that elevated levels of glutamine lead to a nutritional stress that provides a signal for megamitochondrial formation.Selenium is an essential trace element in the diet of humans and many other life forms. It provides many health benefits such as roles in preventing cancer and heart disease, serving as an antiviral agent, stimulating the immune system, reactive oxygen species (ROS) 4 scavenging, and male reproduction (1-6). Many of the benefits of selenium are most likely due to the presence of this element in selenoproteins as the amino acid selenocysteine (Sec) (7-9). Sec is the 21st amino acid in the genetic code (10 -12) and is incorporated into selenoproteins in response to UGA Sec codons (13,14). The active donor of selenium in Sec biosynthesis is monoselenophosphate (15), which is synthesized from selenite and ATP by an enzyme designated as selenophosphate synthetase (SPS) (16). There are two isoforms of SPS in higher eukaryotes, SPS1/SelD and SPS2, whereas only one type of SPS (SelD) exists in lower eukaryotes and eubacteria (17). The sequences of SPS1/SelD and SPS2 are highly conserved. For example, the amino acid sequence homology between human SPS1/SelD and SPS2 is 72% and that between Drosophila SPS1/SelD and SPS2 is ϳ45%. One of the major differences between SPS1/SelD and SPS2 is that SPS1/ SelD has an arginine at the position corresponding to Sec in SPS2 (18,19).Initially, both SPS1/SelD and SPS2 were thought to be involved in selenophosphate synthesis. However, it was subsequently shown that only SPS2 catalyzes selenophosphate synthesis. In in vitro experiments, SPS2 synthesized selenophosphate from selenide and ATP, but SPS1/SelD did not have this activity (20). Knockdown of SPS2 in NIH3T3 cells led to the loss of selenoprotein biosynthesis, whereas the inhibition of SPS1/ SelD...