The Selenium Transport Protein,Selenoprotein P,Requires Coding Sequence Determinants to Promote Efficient Selenocysteine Incorporation

Selenoproteins are an essential and unique group of proteins in which selenocysteine (Sec) is incorporated in response to a stop codon (UGA). Reprograming of UGA for Sec insertion in eukaryotes requires a cis-acting stem–loop structure in the 3′ untranslated region of selenoprotein mRNA and several trans-acting factors. Together these factors are sufficient for Sec incorporation in vitro, but the process is highly inefficient. An additional challenge is the synthesis of selenoprotein P (SELENOP), which uniquely contains multiple UGA codons. Full-length SELENOP expression requires processive Sec incorporation, the mechanism for which is not understood. In this study, we identify core coding region sequence determinants within the SELENOP mRNA that govern SELENOP synthesis. Using ⁷⁵Se labeling in cells, we determined that the N-terminal coding sequence (upstream of the second UGA) and C-terminal coding sequence context are two independent determinants for efficient synthesis of full-length SELENOP. In addition, the distance between the first UGA and the consensus signal peptide is also critical for efficiency.     

Mice Lacking Selenoprotein P and Selenocysteine Lyase Exhibit Severe Neurological Dysfunction,Neurodegeneration, and Audiogenic Seizures

Selenoproteins are a unique family of proteins, characterized by the co-translational incorporation of selenium as selenocysteine, which play key roles in antioxidant defense. Among selenoproteins, selenoprotein P (Sepp1) is particularly distinctive due to the fact that it contains multiple selenocysteine residues and has been postulated to act in selenium transport. Within the brain, Sepp1 delivers selenium to neurons by binding to the ApoER2 receptor. Upon feeding a selenium-deficient diet, mice lacking ApoER2 or Sepp1 develop severe neurological dysfunction and exhibit widespread brainstem neurodegeneration, indicating an important role for ApoER2-mediated Sepp1 uptake in normal brain function. Selenocysteine lyase (Scly) is an enzyme that plays an important role in selenium homeostasis, in that it catalyzes the decomposition of selenocysteine and allows selenium to be recycled for additional selenoprotein synthesis. We previously reported that constitutive deletion of Scly results in neurological deficits only when mice are challenged with a low selenium diet. To gain insight into the relationship between Sepp1 and Scly in selenium metabolism, we created novel transgenic mice constitutively lacking both genes (Scly^−/−Sepp1^−/−) and characterized the neurobehavioral phenotype. We report that deletion of Scly in conjunction with Sepp1 further aggravates the phenotype of Sepp1^−/− mice, as these mice needed supraphysiological selenium supplementation to survive, and surviving mice exhibited impaired motor coordination, audiogenic seizures, and brainstem neurodegeneration. These findings provide the first in vivo evidence that Scly and Sepp1 work cooperatively to maintain selenoprotein function in the mammalian brain.     

Molecular mechanism of selenoprotein P synthesis

Background

Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

Isoform-specific Binding of Selenoprotein P to the β-Propeller Domain of Apolipoprotein E Receptor 2 Mediates Selenium Supply

Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery.     

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.     

Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine

Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7–15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys.     

Selenoproteins—What unique properties can arise with selenocysteine in place of cysteine?

The defining entity of a selenoprotein is the inclusion of at least one selenocysteine (Sec) residue in its sequence. Sec, the 21st naturally occurring genetically encoded amino acid, differs from its significantly more common structural analog cysteine (Cys) by the identity of a single atom: Sec contains selenium instead of the sulfur found in Cys. Selenium clearly has unique chemical properties that differ from sulfur, but more striking are perhaps the similarities between the two elements. Selenium was discovered by Jöns Jacob Berzelius, a renowned Swedish scientist instrumental in establishing the institution that would become Karolinska Institutet. Written at the occasion of the bicentennial anniversary of Karolinska Institutet, this mini review focuses on the unique selenium-derived properties that may potentially arise in a protein upon the inclusion of Sec in place of Cys. With 25 human genes encoding selenoproteins and in total several thousand selenoproteins yet described in nature, it seems likely that the presence of that single selenium atom of Sec should convey some specific feature, thereby explaining the existence of selenoproteins in spite of demanding and energetically costly Sec-specific synthesis machineries. Nonetheless, most, if not all, of the currently known selenoproteins are also found as Cys-containing non-selenoprotein orthologues in other organisms, wherefore any potentially unique properties of selenoproteins are yet a matter of debate. The pK_a of free Sec (approximately 5.2) being significantly lower than that of free Cys (approximately 8.5) has often been proposed as one of the unique features of Sec. However, as discussed herein, this pK_a difference between Sec and Cys can hardly provide an evolutionary pressure for maintenance of selenoproteins. Moreover, the typically 10- to 100-fold lower enzymatic efficiencies of Sec-to-Cys mutants of selenoprotein oxidoreductases, are also weak arguments for the overall existence of selenoproteins. Here, it is however emphasized that the inherent high nucleophilicity of Sec and thereby its higher chemical reaction rate with electrophiles, as compared to Cys, seems to be a truly unique property of Sec that cannot easily be mimicked by the basicity of Cys, even within the microenvironment of a protein. The chemical rate enhancement obtained with Sec can have other consequences than those arising from a low redox potential of some Cys-dependent proteins, typically aiming at maintaining redox equilibria. Another unique aspect of Sec compared to Cys seems to be its efficient potency to support one-electron transfer reactions, which, however, has not yet been unequivocally shown as a Sec-dependent step during the natural catalysis of any known selenoprotein enzyme.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Partitioning between recoding and termination at a stop codon–selenocysteine insertion sequence

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA^Sec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.     

Adjustments,extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.     

Selenoprotein synthesis and regulation in Archaea

Background

The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism.

Effects of selenium supplementation on diet-induced obesity in mice with a disruption of the selenocysteine lyase gene

BACKGROUNDThe amino acid selenocysteine (Sec) is an integral part of selenoproteins, a class of proteins mostly involved in strong redox reactions. The enzyme Sec lyase (SCLY) decomposes Sec into selenide allowing for the recycling of the selenium (Se) atom via the selenoprotein synthesis machinery. We previously demonstrated that disruption of the Scly gene (Scly KO) in mice leads to the development of obesity and metabolic syndrome, with effects on glucose homeostasis, worsened by Se deficiency or a high-fat diet, and exacerbated in male mice. Our objective was to determine whether Se supplementation could ameliorate obesity and restore glucose homeostasis in the Scly KO mice.METHODSThree-weeks old male and female Scly KO mice were fed in separate experiments a diet containing 45 % kcal fat and either sodium selenite or a mixture of sodium selenite and selenomethionine (selenite/SeMet) at moderate (0.25 ppm) or high (0.5–1 ppm) levels for 9 weeks, and assessed for metabolic parameters, oxidative stress and expression of selenoproteins.RESULTSSe supplementation was unable to prevent obesity and elevated epididymal white adipose tissue weights in male Scly KO mice. Serum glutathione peroxidase activity in Scly KO mice was unchanged regardless of sex or dietary Se intake; however, supplementation with a mixture of selenite/SeMet improved oxidative stress biomarkers in the male Scly KO mice.CONCLUSIONThese results unveil sex- and selenocompound-specific regulation of energy metabolism after the loss of Scly, pointing to a role of this enzyme in the control of whole-body energy metabolism regardless of Se levels.     

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.     

The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA^Sec) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA^Sec binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA^Sec binding and accommodation into the ribosomal A-site.     

Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 Å resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (α₂)₂ homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process.     

Selenocysteine insertion sequence (SECIS)-binding protein 2 alters conformational dynamics of residues involved in tRNA accommodation in 80 S ribosomes

Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.     

A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.     

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.