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.

Impairment in selenocysteine synthesis as a candidate mechanism of inducible coagulopathy in COVID-19 patients.

Coagulopathy has recently been recognized as a recurring complication of COVID-19, most typically associated with critical illness. There are epidemiological, mechanistic and transcriptomic evidence that link Selenium with SARS-CoV-2's intracellular latency. Taking into consideration the vital role of selenoproteins in maintaining an adequate immune response, endothelial homeostasis and a non-prothrombotic platelet activation status, we propose that impairment in selenocysteine synthesis, via perturbations in the aforementioned physiological functions, potentially constitutes a mechanism of coagulopathy in COVID 19 patients other than those developed in critical illness.

Trypanosomatid selenophosphate synthetase structure, function and interaction with selenocysteine lyase.

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

Initial steps in selenocysteine biosynthesis: The interaction between selenocysteine lyase and selenophosphate synthetase.

The selenocysteine (Sec) incorporation is a co-translational event taking place at an in-frame UGA-codon and dependent on an organized molecular machinery. Selenium delivery requires mainly two enzymes, the selenocysteine lyase (CsdB) is essential for Sec recycling and conversion to selenide, further used by the selenophosphate synthetase (SelD), responsible for the conversion of selenide in selenophosphate. Therefore, understanding the catalytic mechanism involved in selenium compounds delivery, such as the interaction between SelD and CsdB (EcCsdB.EcSelD), is fundamental for the further comprehension of the selenocysteine synthesis pathway and its control. In Escherichia coli, EcCsdB.EcSelD interaction must occur to prevent cell death from the release of the toxic intermediate selenide. Here, we demonstrate and characterize the in vitro EcSelD.EcCsdB interaction by biophysical methods. The EcSelD.EcCsdB interaction occurs with a stoichiometry of 1:1 in presence of selenocysteine and at a low-nanomolar affinity (~1.8 nM). The data is in agreement with the small angle X-ray scattering model fitted using available structures. Moreover, yeast-2-hybrid assays supported the macromolecular interaction in the cellular environment. This is the first report that demonstrates the interaction between EcCsdB and EcSelD supporting the hypothesis that EcSelD.EcCsdB interaction is necessary to sequester the selenide during the selenocysteine incorporation pathway in Bacteria.

Serine-enriched minimal medium enhances conversion of selenium into selenocysteine by Streptococcus thermophilus.

Selenium is included in selenoprotein sequences, which participate in enzymatic processes necessary to preserve optimal health. Some lactic acid bacteria carry out the biotransformation of inorganic selenium in their metabolism. The complete biochemical mechanism of selenium biotransformation is still unknown; however, it is known that both the selenocysteine synthesis process and its subsequent incorporation into selenoproteins include serine as part of the action of seryl-RNAt synthetase. Therefore, the aim of this work was to determine the effect of serine during the biotransformation of selenium and the subsequence growth of Streptococcus thermophilus in a minimal medium. Two culture media were prepared, one enriched with the minimum inhibitory concentration of selenite (as Na2SeO3) and the other as a mixture of the minimum inhibitory concentration of selenite and serine. The absorbed selenium concentration was measured by inductively coupled plasma, and the selenocysteine identification was performed by reverse-phase HPLC. In the second culture medium, decreases in both times, the adaptation and the logarithmic phase, were observed. According to the results, it was possible to establish that the presence of serine allowed the biotransformation of selenite into selenocysteine by Strep. thermophilus.

Biological implications of selenium and its role in trypanosomiasis treatment.

Selenium (Se) is an essential trace element for several organisms and is present in proteins as selenocysteine (Sec or U), an amino acid that is chemically distinct from serine and cysteine by a single atom (Se instead of O or S, respectively). Sec is incorporated into selenoproteins at an in-frame UGA codon specified by an mRNA stem-loop structure called the selenocysteine incorporating sequence (SECIS) presented in selenoprotein mRNA and specific selenocysteine synthesis and incorporation machinery. Selenoproteins are presented in all domains but are not found in all organisms. Although several functions have been attributed to this class, the majority of the proteins are involved in oxidative stress defense. Here, we discuss the kinetoplastid selenocysteine pathway and how selenium supplementation is able to alter the infection course of trypanosomatids in detail. These organisms possess the canonical elements required for selenoprotein production such as phosphoseryl tRNA kinase (PSTK), selenocysteine synthase (SepSecS), selenophosphase synthase (SelD or SPS), and elongation factor EFSec (SelB), whereas other important factors presented in mammal cells, such as SECIS binding protein 2 (SBP) and SecP 43, are absent. The selenoproteome of trypanosomatids is small, as is the selenoproteome of others parasites, which is in contrast to the large number of selenoproteins found in bacteria, aquatic organisms and higher eukaryotes. Trypanosoma and Leishmania are sensitive to auranofin, a potent selenoprotein inhibitor; however, the probable drug mechanism is not related to selenoproteins in kinetoplastids. Selenium supplementation decreases the parasitemia of various Trypanosome infections and reduces important parameters associated with diseases such as anemia and parasite-induced organ damage. New experiments are necessary to determine how selenium acts, but evidence suggests that immune response modulation and increased host defense against oxidative stress contribute to control of the parasite infection.

Gene expression of endoplasmic reticulum resident selenoproteins correlates with apoptosis in various muscles of se-deficient chicks.

Dietary selenium (Se) deficiency causes muscular dystrophy in various species, but the molecular mechanism remains unclear. Our objectives were to investigate: 1) if dietary Se deficiency induced different amounts of oxidative stress, lipid peroxidation, and cell apoptosis in 3 skeletal muscles; and 2) if the distribution and expression of 4 endoplasmic reticulum (ER) resident selenoprotein genes (Sepn1, Selk, Sels, and Selt) were related to oxidative damages in these muscles. Two groups of day-old layer chicks (n = 60/group) were fed a corn-soy basal diet (33 µg Se/kg; produced in the Se-deficient area of Heilongjiang, China) or the diet supplemented with Se (as sodium selenite) at 0.15 mg/kg for 55 d. Dietary Se deficiency resulted in accelerated (P < 0.05) cell apoptosis that was associated with decreased glutathione peroxidase activity and elevated lipid peroxidation in these muscles. All these responses were stronger in the pectoral muscle than in the thigh and wing muscles (P < 0.05). Relative distribution of the 4 ER resident selenoprotein gene mRNA amounts and their responses to dietary Se deficiency were consistent with the resultant oxidative stress and cell apoptosis in the 3 muscles. Expression of Sepn1, Sels, and Selt in these muscles was correlated with (r > 0.72; P < 0.05) that of Sepsecs encoding a key enzyme for biosynthesis of selenocysteine (selenocysteinyl-tRNA synthase). In conclusion, the pectoral muscle demonstrated unique expression patterns of the ER resident selenoprotein genes and GPx activity, along with elevated susceptibility to oxidative cell death, compared with the other skeletal muscles. These features might help explain why it is a primary target of Se deficiency diseases in chicks.

Synthesis and decoding of selenocysteine and human health.

Selenocysteine, the 21st amino acid, has been found in 25 human selenoproteins and selenoenzymes important for fundamental cellular processes ranging from selenium homeostasis maintenance to the regulation of the overall metabolic rate. In all organisms that contain selenocysteine, both the synthesis of selenocysteine and its incorporation into a selenoprotein requires an elaborate synthetic and translational apparatus, which does not resemble the canonical enzymatic system employed for the 20 standard amino acids. In humans, three synthetic enzymes, a specialized elongation factor, an accessory protein factor, two catabolic enzymes, a tRNA, and a stem-loop structure in the selenoprotein mRNA are critical for ensuring that only selenocysteine is attached to selenocysteine tRNA and that only selenocysteine is inserted into the nascent polypeptide in response to a context-dependent UGA codon. The abnormal selenium homeostasis and mutations in selenoprotein genes have been causatively linked to a variety of human diseases, which, in turn, sparked a renewed interest in utilizing selenium as the dietary supplement to either prevent or remedy pathologic conditions. In contrast, the importance of the components of the selenocysteine-synthetic machinery for human health is less clear. Emerging evidence suggests that enzymes responsible for selenocysteine formation and decoding the selenocysteine UGA codon, which by extension are critical for synthesis of the entire selenoproteome, are essential for the development and health of the human organism.

[Expanding genetic code: amino acids 21 and 22--selenocysteine and pyrrolysine].

The discovery of two nonstandard amino acids, selenocysteine and pyrrolysine, in the genetic code is discussed. These findings have expanded our understanding of the genetic code, since the repertoire of amino acids in the genetic code was supplemented by two novel ones, in addition of the standard 20 amino acids. Current views on specific mechanisms of selenocysteine insertion in forming selenoproteins are considered, as well as the results of studies of new translational components involved in biosynthesis and incorporation of selenocysteine at different stages of translation. Similarity in the strategies of decoding UGA and UAG as codons for respectively selenocysteine and pyrrolysine is discussed. The review also presents evidence on the medical and biological role of selenium and selenoproteins containing selenocysteine as the main biological form of selenium.

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.

[Biochemical selenocysteine synthesis and the phylogenic study].

Selenium (Se) is an essential trace element. Se is found as selenocysteine (Sec) in Se-proteins. Sec is the 21(st) amino acid, because Sec has its tRNA, the codon UGA and those components in its translational machinery. Sec UGA codon shares with major stop codon UGA. We purified Sec synthesizing enzymes, such as seryl-tRNA synthetase (SerRS), Sec synthetase (SecS) and selenophosphate synthetase (SePS). I described the procedures to prepare Sec tRNA, SerRS, SecS, SePS and [(75)Se]H(2)Se in detail. We clarified that SecS composed of two proteins, SecSalpha and SecSbeta. Sec synthesizing and incorporating systems present in Monela, Animalia and Protoctista but not in Plantae and Fungi. We showed that protozoa had Sec tRNA on which Sec was synthesized from Ser-tRNA by bovine and protozoa SecS. Some worms, such as Caenorhabditis elegans and Fasiola gigantica, also had Sec tRNA on which Sec was synthesized by bovine liver SecS or C. elegans enzymes. We showed recognition sites of mammalian Sec tRNA by SecS. The identity units of Sec tRNA are 9 bp aminoacyl- and 6 bp D-stems. This recognition is not the base-specific manner but the length-specific manner. From comparison of the phylogeny trees of Sec synthesizing system and translation system, we concluded that the evolution of Sec synthesizing system is older than that of the translation system.

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.

Biosynthesis of selenocysteine on its tRNA in eukaryotes.

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase.

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selDHI) in an E. coli DeltaselD strain. Gene selDHI gave rise to a selenium-containing gene product and also supported - via its activity - the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGASec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif.

The path of unspecific incorporation of selenium in Escherichia coli.

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating 75Se only and specifically into selenoproteins.

Selenocysteine.

Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon. Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome. Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine. A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA. Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria. The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed. Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification. Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine. The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5' deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed. In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine. This substitution caused complete loss of glycine reductase selenoprotein A activity.