Vadim N. Gladyshev

How selenium has altered our understanding of the genetic code.

Selenium is an essential micronutrient in the diet of many life forms, including humans and other mammals. Significant health benefits have been attributed to this element. It is rapidly becoming recognized as one of the more promising cancer chemopreventive agents (19), and there are strong indications that it has a role in reducing viral expression (4), in preventing heart disease and other cardiovascular and muscle disorders (23), and in delaying the progression of AIDS in human immunodeficiency virus-infected patients (3). Additional evidence suggests that selenium may have a role in mammalian development (51), in immune function (70), in male reproduction (30), and in slowing the aging process (70). Despite the many potential health benefits of selenium, the means by which this element promotes better health are only just beginning to be elucidated (31, 91). There are about 20 known selenium-containing proteins in mammals (33), and it would seem very likely that several of these are mediators of health benefits of dietary selenium. Therefore, it is critical to understand how selenium is inserted into protein and the identities and functions of the resulting protein products. Selenium is present in naturally occurring selenium-containing proteins in two basic forms. It can be inserted posttranslationally as a dissociable cofactor (32). This rare form of protein-associated selenium has been found only in several bacterial molybdenum-containing enzymes and will not be discussed further in this review. Selenium is also cotranslationally inserted into protein as the amino acid selenocysteine (Sec). Such occurrence of this element in protein is widespread in all major domains of life and is responsible for the majority of biological effects of selenium. The elucidation of how Sec is incorporated into protein has progressed at a rapid pace in the last decade and has revealed some surprising results. In fact, unraveling this mystery has altered our understanding of the genetic code, as the code has now been expanded to include Sec as the 21st naturally occurring amino acid. When the code was deciphered in the mid-1960s (48, 79), 20 amino acids were assigned to 61 of the possible 64 codons within the triplet code and 3 codons were found to function as terminators for protein synthesis. Each of the 64 code words was therefore assigned a function, and there did not appear to be room for additional amino acids. Although it was recognized in the mid-1960s that one code word, AUG, had a dual role of initiating protein synthesis and inserting methionine at internal protein positions, the possibility that a second codon also had two functions was not considered at that time. We now know that UGA serves as both a termination codon and a Sec codon. The means by which UGA serves as a Sec codon and how Sec is biosynthesized and incorporated into protein have been examined in considerable detail with eubacteria (reviewed in reference 7) and with mammals (this review). While the fundamental mechanism of Sec insertion in these organisms appears to be similar, recent studies suggest that mammals evolved additional components that allow incorporation of multiple Secs into a single protein and provide stringent regulation of Sec biosynthesis. The present review discusses our current knowledge of these features in mammals. It should be noted that selenium can also be incorporated nonspecifically into protein (42). The nonspecific occurrence of this element in protein arises when selenium replaces sulfur in the biosynthesis of cysteine or methionine and the resulting selenoamino acid (Sec or selenomethionine) is inserted in place of the natural amino acid. Such misincorporation of selenium into protein may be toxic; this subject has been reviewed elsewhere (42).

Redox Regulation of Cell Signaling by Selenocysteine in Mammalian Thioredoxin Reductases

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the ‘queen’, who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat’s exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Selenoproteins: Molecular Pathways and Physiological Roles

Selenium is an essential micronutrient with important functions in human health and relevance to several pathophysiological conditions. The biological effects of selenium are largely mediated by selenium-containing proteins (selenoproteins) that are present in all three domains of life. Although selenoproteins represent diverse molecular pathways and biological functions, all these proteins contain at least one selenocysteine (Sec), a selenium-containing amino acid, and most serve oxidoreductase functions. Sec is cotranslationally inserted into nascent polypeptide chains in response to the UGA codon, whose normal function is to terminate translation. To decode UGA as Sec, organisms evolved the Sec insertion machinery that allows incorporation of this amino acid at specific UGA codons in a process requiring a cis-acting Sec insertion sequence (SECIS) element. Although the basic mechanisms of Sec synthesis and insertion into proteins in both prokaryotes and eukaryotes have been studied in great detail, the identity and functions of many selenoproteins remain largely unknown. In the last decade, there has been significant progress in characterizing selenoproteins and selenoproteomes and understanding their physiological functions. We discuss current knowledge about how these unique proteins perform their functions at the molecular level and highlight new insights into the roles that selenoproteins play in human health.

Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.

Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype and Tumorigenicity of Lung Carcinoma Cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

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.

New Mammalian Selenocysteine-containing Proteins Identified with an Algorithm That Searches for Selenocysteine Insertion Sequence Elements

Mammalian selenium-containing proteins identified thus far contain selenium in the form of a selenocysteine residue encoded by UGA. These proteins lack common amino acid sequence motifs, but 3′-untranslated regions of selenoprotein genes contain a common stem-loop structure, selenocysteine insertion sequence (SECIS) element, that is necessary for decoding UGA as selenocysteine rather than a stop signal. We describe here a computer program, SECISearch, that identifies mammalian selenoprotein genes by recognizing SECIS elements on the basis of their primary and secondary structures and free energy requirements. When SECISearch was applied to search human dbEST, two new mammalian selenoproteins, designated SelT and SelR, were identified. We determined their cDNA sequences and expressed them in a monkey cell line as fusion proteins with a green fluorescent protein. Incorporation of selenium into new proteins was confirmed by metabolic labeling with 75Se, and expression of SelT was additionally documented in immunoblot assays. SelT and SelR did not have homology to previously characterized proteins, but their putative homologs were detected in various organisms. SelR homologs were present in every organism characterized by complete genome sequencing. The data suggest applicability of SECISearch for identification of new selenoprotein genes in nucleotide data bases.

Selenium and selenocysteine: roles in cancer, health, and development

The many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace, elucidating its many roles in health, development, and in cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions.