The selenophosphate synthetase family: A review.

Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.

Facultative protein selenation regulates redox sensitivity, adipose tissue thermogenesis, and obesity.

Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well.

Processive Recoding and Metazoan Evolution of Selenoprotein P: Up to 132 UGAs in Molluscs.

Selenoproteins typically contain a single selenocysteine, the 21st amino acid, encoded by a context-redefined UGA. However, human selenoprotein P (SelenoP) has a redox-functioning selenocysteine in its N-terminal domain and nine selenium transporter-functioning selenocysteines in its C-terminal domain. Here we show that diverse SelenoP genes are present across metazoa with highly variable numbers of Sec-UGAs, ranging from a single UGA in certain insects, to 9 in common spider, and up to 132 in bivalve molluscs. SelenoP genes were shaped by a dynamic evolutionary process linked to selenium usage. Gene evolution featured modular expansions of an ancestral multi-Sec domain, which led to particularly Sec-rich SelenoP proteins in many aquatic organisms. We focused on molluscs, and chose Pacific oyster Magallana gigas as experimental model. We show that oyster SelenoP mRNA with 46 UGAs is translated full-length in vivo. Ribosome profiling indicates that selenocysteine specification occurs with ∼5% efficiency at UGA1 and approaches 100% efficiency at distal 3' UGAs. We report genetic elements relevant to its expression, including a leader open reading frame and an RNA structure overlapping the initiation codon that modulates ribosome progression in a selenium-dependent manner. Unlike their mammalian counterparts, the two SECIS elements in oyster SelenoP (3'UTR recoding elements) do not show functional differentiation in vitro. Oysters can increase their tissue selenium level up to 50-fold upon supplementation, which also results in extensive changes in selenoprotein expression.

Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements.

Dynamic redefinition of the 10 UGAs in human and mouse selenoprotein P (Sepp1) mRNAs to specify selenocysteine instead of termination involves two 3' UTR structural elements (SECIS) and is regulated by selenium availability. In addition to the previously known human Sepp1 mRNA poly(A) addition site just 3' of SECIS 2, two further sites were identified with one resulting in 10-25% of the mRNA lacking SECIS 2. To address function, mutant mice were generated with either SECIS 1 or SECIS 2 deleted or with the first UGA substituted with a serine codon. They were fed on either high or selenium-deficient diets. The mutants had very different effects on the proportions of shorter and longer product Sepp1 protein isoforms isolated from plasma, and on viability. Spatially and functionally distinctive effects of the two SECIS elements on UGA decoding were inferred. We also bioinformatically identify two selenoprotein S mRNAs with different 5' sequences predicted to yield products with different N-termini. These results provide insights into SECIS function and mRNA processing in selenoprotein isoform diversity.

Identification of a selenium-dependent glutathione peroxidase in the blood-sucking insect Rhodnius prolixus.

The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrogenase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus.

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.

Composition and evolution of the vertebrate and mammalian selenoproteomes.

BACKGROUND: Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian (25 proteins) selenoproteomes, and describe how they evolved along lineages through gene duplication (20 events), gene loss (10 events) and replacement of Sec with cysteine (12 events). We show that an intronless selenophosphate synthetase 2 gene evolved in early mammals and replaced functionally the original multiexon gene in placental mammals, whereas both genes remain in marsupials. Mammalian thioredoxin reductase 1 and thioredoxin-glutathione reductase evolved from an ancestral glutaredoxin-domain containing enzyme, still present in fish. Selenoprotein V and GPx6 evolved specifically in placental mammals from duplications of SelW and GPx3, respectively, and GPx6 lost Sec several times independently. Bony fishes were characterized by duplications of several selenoprotein families (GPx1, GPx3, GPx4, Dio3, MsrB1, SelJ, SelO, SelT, SelU1, and SelW2). Finally, we report identification of new isoforms for several selenoproteins and describe unusually conserved selenoprotein pseudogenes. CONCLUSIONS/SIGNIFICANCE: This analysis represents the first comprehensive survey of the vertebrate and mammal selenoproteomes, and depicts their evolution along lineages. It also provides a wealth of information on these selenoproteins and their forms.