Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.

A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium.

The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.

Molecular Mechanisms of the Cytotoxic Effect of Recombinant Selenoprotein SELENOM on Human Glioblastoma Cells.

Currently, selenobiology is an actively developing area, primarily due to the study of the role of the trace element selenium and its organic and inorganic compounds in the regulation of vital processes occurring in the cell. In particular, the study of the functions of selenium nanoparticles has gained great popularity in recent years. However, a weak point in this area of biology is the study of the functions of selenoproteins, of which 25 have been identified in mammals to date. First of all, this is due to the difficulties in obtaining native forms of selenoproteins in preparative quantities, due to the fact that the amino acid selenocysteine is encoded by one of the three stop codons of the TGA universal genetic code. A complex system for recognizing a given codon as a selenocysteine codon has a number of features in pro- and eukaryotes. The selenoprotein SELENOM is one of the least studied mammalian selenoproteins. In this work, for the first time, studies of the molecular mechanisms of regulation of the cytotoxic effect of this protein on human glioblastoma cells were carried out. The cytotoxicity of cancer cells in our experiments was already observed when cells were exposed to 50 µg of SELENOM and increased in proportion to the increase in protein concentration. Apoptosis of human glioblastoma cells was accompanied by an increase in mRNA expression of a number of pro-apoptotic genes, an increase in endoplasmic reticulum stress, and activation of the UPR IRE1α signaling pathway. The results obtained also demonstrate a dose-dependent depletion of the Ca2+ pool under the action of SELENOM, which proves the important role of this protein in the regulation of calcium homeostasis in the cell.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.

In vivo probing of SECIS-dependent selenocysteine translation in Archaea.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure-function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial ß-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Selenium and breast cancer - An update of clinical and epidemiological data.

There is an urgent need for new and improved therapeutic strategies in breast cancer, which is the most common malignancy affecting women in the United States and worldwide. Selenium (Se) is an essential trace element of the human diet and plays a critical role in many aspects of human health. Clinical and epidemiological studies summarized here clearly demonstrate that Se status correlates with breast cancer survival. As a result, one way to curb breast cancer mortality would be via Se supplementation, especially in patients with severely deplete Se status. Se manifests its biological activity through incorporation into selenoproteins as selenocysteine. However, a better understanding of tissue-specific mechanisms and roles for selenoproteins in general is required. Additionally, many human selenoproteins harbor single nucleotide polymorphisms, which impact protein expression and activity and have been associated with cancer susceptibility or impacting survival. Increasing evidence indicates that these genetic variations impinge on the interactions between Se and breast cancer. This highlights the importance of integrating the Se status with genetic factors to fully define the benefit of Se in breast cancer. While Se supplementation would clearly benefit a subset of patients, this requires first the identification of at-risk patients and warrants validation through intervention trials.

Molecular characterization and expression analysis of selenoprotein W gene in rainbow trout (Oncorhynchus mykiss) with dietary selenium levels.

Selenium (Se) plays an essential role in the growth of fish and performs its physiological functions mainly through incorporation into selenoproteins. Our previous studies suggested that the selenoprotein W gene (selenow) is sensitive to changes in dietary Se in rainbow trout. However, the molecular characterization and tissue expression pattern of selenow are still unknown. Here, we revealed the molecular characterization, the tissue expression pattern of rainbow trout selenow and analyzed its response to dietary Se. The open reading frame (ORF) of the selenow gene was composed of 393 base pairs (bp) and encodes a 130-amino-acid protein. The 3' untranslated region (UTR) was 372 bp with a selenocysteine insertion sequence (SECIS) element. Remarkably, the rainbow trout selenow gene sequence was longer than those reported for mammals and most other fish. A ß1-α1-ß2-ß3-ß4-α2 pattern made up the secondary structure of SELENOW. Furthermore, multiple sequence alignment revealed that rainbow trout SELENOW showed a high level of identity with SELENOW from Salmo salar. In addition, the selenow gene was ubiquitously distributed in 13 tissues with various abundances and was predominantly expressed in muscle and brain. Interestingly, dietary Se significantly increased selenow mRNA expression in muscle. Our results highlight the vital role of selenow in rainbow trout muscle response to dietary Se levels and provide a theoretical basis for studies of selenow.

The selenocysteine toolbox: A guide to studying the 21st amino acid.

Selenocysteine (Sec), the 21st genetically encoded amino acid, is structurally similar to cysteine (Cys) but with a sulfur to selenium replacement. This small change confers Sec with related chemical properties to Cys but often with enhanced reactivity. In organisms, Sec is present in selenoproteins taking on various roles such as cellular maintenance, immune response, hormone regulation, and oxidative stress. The detailed reactions of Sec in these functions remains unclear and has been a difficult question to answer. This is related to the low natural expression of selenoproteins and their complicated biosynthesis pathway. As a result, the focus in selenoprotein research has been on the expansion of tools and techniques to promote research in this area. Over the past two decades there has been immense progress in the development of selenoprotein expression systems, Sec-detection methods, and genomic databases. In this review we have compiled these tools systematically, highlighting their strengths and clarifying the limitations, as a resource for future selenoprotein research.

Selenoproteins and the senescence-associated epitranscriptome.

Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.

Selenocysteine Machinery Primarily Supports TXNRD1 and GPX4 Functions and Together They Are Functionally Linked with SCD and PRDX6.

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD.

The selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation.

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3' UTR. Aside from the SECIS, selenoprotein mRNA 3' UTRs are not conserved between different selenoproteins within a species. In contrast, the 3'-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3'-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3' UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.

The Possible Mechanism of Physiological Adaptation to the Low-Se Diet and Its Health Risk in the Traditional Endemic Areas of Keshan Diseases.

Selenium is an essential trace element for humans and animals. As with oxygen and sulfur, etc., it belongs to the sixth main group of the periodic table of elements. Therefore, the corresponding amino acids, such as selenocysteine (Sec), serine (Ser), and cysteine (Cys), have similar spatial structure, physical, and chemical properties. In this review, we focus on the neglected but key role of serine in a possible mechanism of the physiological adaptation to Se-deficiency in human beings with an adequate intake of dietary protein: the insertion of Cys in place of Sec during the translation of selenoproteins dependent on the Sec insertion sequence element in the 3'UTR of mRNA at the UGA codon through a novel serine-dependent pathway for the de novo synthesis of the Cys-tRNA[Ser]Sec, similar to Sec-tRNA[Ser]Sec. We also discuss the important roles of serine in the metabolism of selenium directly or indirectly via GSH, and the maintenance of selenium homostasis regulated through the methylation modification of Sec-tRNA[Ser]Sec at the position 34U by SAM. Finally, we propose a hypothesis to explain why Keshan disease has gradually disappeared in China and predict the potential health risk of the human body in the physiological adaptation state of low selenium based on the results of animal experiments.

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine.

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure-activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Pathogenic Variants in Selenoproteins and Selenocysteine Biosynthesis Machinery.

Selenium is incorporated into selenoproteins as the 21st amino acid selenocysteine (Sec). There are 25 selenoproteins encoded in the human genome, and their synthesis requires a dedicated machinery. Most selenoproteins are oxidoreductases with important functions in human health. A number of disorders have been associated with deficiency of selenoproteins, caused by mutations in selenoprotein genes or Sec machinery genes. We discuss mutations that are known to cause disease in humans and report their allele frequencies in the general population. The occurrence of protein-truncating variants in the same genes is also presented. We provide an overview of pathogenic variants in selenoproteins genes from a population genomics perspective.

The hepatic transcriptome of the turkey poult (Meleagris gallopavo) is minimally altered by high inorganic dietary selenium.

There is interest in supplementing animals and humans with selenium (Se) above Se-adequate levels, but the only good biomarker for toxicity is tissue Se. We targeted liver because turkeys fed 5 µg Se/g have hepatic Se concentrations 6-fold above Se-adequate (0.4 µg Se/g) levels without effects on growth or health. Our objectives were (i) to identify transcript biomarkers for high Se status, which in turn would (ii) suggest proteins and pathways used by animals to adapt to high Se. Turkey poults were fed 0, 0.025, 0.4, 0.75 and 1.0 µg Se/g diet in experiment 1, and fed 0.4, 2.0 and 5.0 µg Se/g in experiment 2, as selenite, and the full liver transcriptome determined by RNA-Seq. The major effect of Se-deficiency was to down-regulate expression of a subset of selenoprotein transcripts, with little significant effect on general transcript expression. In response to high Se intake (2 and 5 µg Se/g) relative to Se-adequate turkeys, there were only a limited number of significant differentially expressed transcripts, all with only relatively small fold-changes. No transcript showed a consistent pattern of altered expression in response to high Se intakes across the 1, 2 and 5 µg Se/g treatments, and there were no associated metabolic pathways and biological functions that were significant and consistently found with high Se supplementation. Gene set enrichment analysis also found no gene sets that were consistently altered by high-Se and supernutritional-Se. A comparison of differentially expressed transcript sets with high Se transcript sets identified in mice provided high Se (~3 µg Se/g) also failed to identify common differentially expressed transcript sets between these two species. Collectively, this study indicates that turkeys do not alter gene expression in the liver as a homeostatic mechanism to adapt to high Se.

SBP2 deficiency in adipose tissue macrophages drives insulin resistance in obesity.

Proinflammatory activation and accumulation of adipose tissue macrophages (ATMs) are associated with increased risk of insulin resistance in obesity. Here, we described the previously unidentified role of selenocysteine insertion sequence-binding protein 2 (SBP2) in maintaining insulin sensitivity in obesity. SBP2 was suppressed in ATMs of diet-induced obese mice and was correlated with adipose tissue inflammation. Loss of SBP2 initiated metabolic activation of ATMs, inducing intracellular reactive oxygen species content and inflammasome, which subsequently promoted IL-1ß-associated local proliferation and infiltration of proinflammatory macrophages. ATM-specific knockdown of SBP2 in obese mice promoted insulin resistance by increasing fat tissue inflammation and expansion. Reexpression of SBP2 improved insulin sensitivity. Last, an herbal formula that specifically induced SBP2 expression in ATMs can experimentally improve insulin sensitivity. Clinical observation revealed that it improved hyperglycemia in patients with diabetes. This study identified SBP2 in ATMs as a potential target in rescuing insulin resistance in obesity.

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.

Four selenoprotein P genes exist in salmonids: Analysis of their origin and expression following Se supplementation and bacterial infection.

The following research was conducted to elucidate the evolution and expression of salmonid selenoprotein P (SelP), a selenoprotein that is unique in having multiple selenocysteine (Sec) residues, following supranutritional selenium supplementation and infection in rainbow trout. We show that in salmonids SelP is present as four paralogues and that the diversification of SelP genes during vertebrate evolution relates to whole genome duplication events. With 17 and 16 selenocysteine residues for rainbow trout (Oncorhynchus mykiss)/Atlantic salmon (Salmo salar) SelPa1 and SelPa2 proteins respectively and 1 or 2 (trout or salmon) and 4 or 3 (trout or salmon) selenocysteine residues for salmonid SelPb1 and SelPb2 proteins respectively, this is the highest number of (predicted) multiple selenocysteine containing SelP proteins reported for any vertebrate species to date. To investigate the effects of selenium form on SelP expression we added different concentrations (1 nM- 10 µM) of organic or inorganic selenium to a trout cell line (RTG-2 cells) and analysed changes in mRNA abundance. We next studied the impact of supplementation on the potential modulation of these transcripts by PAMPs and proinflammatory cytokines in RTG-2 and RTS-11 cells. These experiments revealed that selenium type influenced the responses, and that SelP gene subfunctionalisation was apparent. To get an insight into the expression patterns in vivo we conducted a feeding trial with 2 diets differing in selenium content and 5 weeks later challenged the trout with a bacterial pathogen (Aeromonas salmonicida). Four tissues were analysed for SelP paralogue expression. The results show a significant induction of SelPa1 in gills and intestine following infection in selenium supplemented fish and for SelPa2 in gills. SelPb1 was significantly reduced in head kidney of both diet groups following infection, whilst SelPb2 was significantly upregulated in skin of both diet groups post infection. Overall these findings reveal differential expression profiles for the SelPa/SelPb paralogues in trout, influenced by selenium supply, cell type/tissue and stimulant. The increase of multiple Sec containing SelP proteins in salmonids could indicate an enhanced requirement for selenium in this lineage.