Selenocysteine-containing proteins in mammals

Since the recent discovery of selenocysteine as the 21st amino acid in protein, the field of selenium biology has rapidly expanded. Twelve mammalian selenoproteins have been characterized to date and each contains selenocysteine that is incorporated in response to specific UGA code words. These selenoproteins have different cellular functions, but in those selenoproteins for which the function is known, selenocysteine is located at the active center. The presence of selenocysteine at critical sites in naturally occurring selenoproteins provides an explanation for the important role of selenium in human health and development. This review describes known mammalian selenoproteins and discusses recent developments and future directions in the selenium field.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.     

Translation regulation of mammalian selenoproteins

Background

Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21^st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins.

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.     

The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but directs selenocysteine incorporation.

Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.     

Selenocysteine: the 21st amino acid

Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in-frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co-translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.     

The genomics of selenium: Its past,present and future

In the last two decades, genomic analyses have enriched the study of the biology of selenium in many ways. These include the identification of selenoproteins in prokaryotic and eukaryotic genomes, the discovery of genetic variants that mediate humans and other vertebrates' adaptations to their selenium nutritional histories, and the association of specific genotypes with common and rare human selenium disorders. We briefly review these computational, evolutionary and association studies and their contribution to the genomics of selenium, selenocysteine and selenoproteins in the 200th anniversary of the discovery of this trace element.     

Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins

In the genetic code, UGA serves as either a signal for termination or a codon for selenocysteine (Sec). Sec rarely occurs in protein and is different from other amino acids in that much of the biosynthetic machinery governing its incorporation into protein is unique to this amino acid. Sec-containing proteins have diverse functions and lack a common amino acid motif or consensus sequence. Sec has previously been considered to be a relic of the primordial genetic code that was counter-selected by the presence of oxygen in the atmosphere. In the present report, it is proposed that Sec was added to the already existing genetic code and its use has accumulated during evolution of eukaryotes culminating in vertebrates. The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions. Further understanding of the evolution of selenoproteins as well as biological properties and biomedical applications of the trace element selenium requires identification and functional characterization of all mammalian selenoproteins.     

Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.     

The selenium to selenoprotein pathway in eukaryotes: More molecular partners than anticipated

The amino acid selenocysteine (Sec) is the major biological form of the trace element selenium. Sec is co-translationally incorporated in selenoproteins. There are 25 selenoprotein genes in humans, and Sec was found in the active site of those that have been attributed a function. This review will discuss how selenocysteine is synthesized and incorporated into selenoproteins in eukaryotes. Sec biosynthesis from serine on the tRNA^Sec requires four enzymes. Incorporation of Sec in response to an in-frame UGA codon, otherwise signaling termination of translation, is achieved by a complex recoding machinery to inform the ribosomes not to stop at this position on the mRNA. A number of the molecular partners acting in this machinery have been identified but their detailed mechanism of action has not been deciphered yet. Here we provide an overview of the literature in the field. Particularly striking is the higher than originally envisaged number of factors necessary to synthesize Sec and selenoproteins. Clearly, selenoprotein synthesis is an exciting and very active field of research.     

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.     

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA^[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.     

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.     

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.     

The microbial selenoproteome of the Sargasso Sea

Background  

Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project.     

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.     

Selenoproteins

Selenium is an essential micronutrient for man and animals. The role of selenium has been attributed largely to its presence in selenoproteins as the 21st amino acid, selenocysteine (Sec, U). Sec is encoded by TGA in DNA. A unique mechanism is used to decode the UGA codon in mRNA to co-translationally incorporate Sec into the growing polypeptide because there is no free pool of Sec. In the human genome, 25 genes for selenoproteins have been identified. Selenoproteins such as glutathione peroxidases, thioredoxin reductases, and iodothyronine deiodinases are involved in redox reactions, and Sec is an active-site residue essential for catalytic activity. Selenoproteins have biological functions in oxidoreductions, redox signaling, antioxidant defense, thyroid hormone metabolism, and immune responses. They thus possess a strong correlation with human diseases such as cancer, Keshan disease, virus infections, male infertility, and abnormalities in immune responses and thyroid hormone function.     

Selenoprotein synthesis: UGA does not end the story

It is well established that the beneficial effects of the trace element selenium are mediated by its major biological product, the amino acid selenocysteine, present in the active site of selenoproteins. These fulfill different functions, as varied as oxidation-reduction of metabolites in bacteria, reduction of reactive oxygen species, control of the redox status of the cell or thyroid hormone maturation. This review will focus on the singularities of the selenocysteine biosynthesis pathway and its unique incorporation mechanism into eukaryal selenoproteins. Selenocysteine biosynthesis from serine is achieved on tRNA(Sec) and requires four proteins. As this amino acid is encoded by an in-frame UGA codon, otherwise signaling termination of translation, ribosomes must be told not to stop at this position in the mRNA. Several molecular partners acting in cis or in trans have been identified, but their knowledge has not enabled yet to firmly establish the molecular events underlying this mechanism. Data suggest that other, so far uncharacterized factors might exist. In this survey, we attempted to compile all the data available in the literature and to describe the latest developments in the field.     

Molecular biology of selenium with implications for its metabolism.

Selenium has a highly specific metabolism centered around its incorporation as selenocysteine into selenoproteins. An outline of this metabolism has emerged from recent molecular biological and biochemical studies of bacteria and animals. A unique tRNA, designated tRNA[Ser]Sec, is charged with L-serine, which is then converted through at least two steps to selenocysteine. With the aid of a unique translation factor, the selenocysteinyl-tRNA[Ser]Sec recognizes specific UGA codons in mRNA to insert selenocysteine into the primary structure of selenoproteins. Turnover of selenoproteins presumably liberates selenocysteine which is toxic in its free form. Selenocysteine beta-lyase catabolizes free selenocysteine and makes its selenium available for reuse. Proteins contain almost all the selenium in animals. Of the known selenoproteins, the glutathione peroxidases contain the most selenium. Cellular and plasma glutathione peroxidases are products of different genes but have 44% identity of amino acid sequence. There is evidence for other proteins of this family. Selenoprotein P is an unrelated protein with multiple selenocysteines in its primary structure. It contains most of the selenium in rat plasma. Studies of the regulation of cellular glutathione peroxidase by selenium have yielded conflicting results, but there is a strong suggestion that mRNA levels of the rodent liver glutathione peroxidase decrease in selenium deficiency. This could be a mechanism for directing selenium to the synthesis of other selenoproteins. Although present knowledge allows construction of an outline of selenium metabolism, several steps have not been characterized and little is known about mechanisms of its regulation.