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Selenium (Se) is an essential trace element for life. Seleno-methylselenocysteine (SeMCys) can serve as a Se supplement with anticarcinogenic activity and can improve cognitive deficits. We engineered Escherichia coli for microbial production of SeMCys. The genes involved in the synthesis of SeMCys were divided into three modules–the selenocysteine (SeCys) synthesis, methyl donor synthesis and SMT modules–and expressed in plasmids with different copy numbers. The higher copy number of the SeCys synthesis module facilitated SeMCys production. The major routes for SeCys degradation were then modified. Deletion of the cysteine desulfurase gene csdA or sufS improved SeMCys production the most, and the strain that knocked out both genes doubled SeMCys production. The addition of serine in the mid-logarithmic growth phase significantly improved SeMCys synthesis. When the serine synthetic pathway was enhanced, SeMCys production increased by 12.5 %. Fed-batch culture for sodium selenite supplementation in the early stationary phase improved SeMCys production to 3.715 mg/L. This is the first report of the metabolic engineering of E. coli for the production of SeMCys and provide information on Se metabolism. • Higher copy number of the SeCys synthesis module facilitated SeMCys production. • Cysteine desulfurase CsdA and SufS are involved in the degradation of SeCys. • The enhanced Ser synthetic pathway increased SeMCys synthesis. [ABSTRACT FROM AUTHOR]

Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β‐ME. Furthermore, the FDA‐approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin‐1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress. [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) is an essential amino acid that distinguishes itself from cysteine by a selenium atom in place of a sulfur atom. This single change imparts distinct chemical properties to Sec which are crucial for selenoprotein (Sec-containing protein) function. These properties include a lower pKa, enhanced nucleophilicity, and reversible oxidation. However, studying Sec incorporation in proteins is a complex process. While we find Sec in all domains of life, each domain has distinct translation mechanisms. These mechanisms are unique to canonical translation and are composed of Sec-specific enzymes and an mRNA hairpin to drive recoding of the UGA stop codon with Sec. In this review, we highlight the obstacles that arise when investigating Sec insertion, and the role that Sec has in proteins. We discuss the strategic methods implemented in this field to address these challenges. Though the Sec translation system is complex, a remarkable amount of information has been obtained and specialized tools have been developed. Continued studies in this area will provide a deeper understanding on the role of Sec in the context of proteins, and the necessity that we have for maintaining this complex translation machinery to make selenoproteins. [ABSTRACT FROM AUTHOR]

Selenocysteine methyltransferase (SMT) is a key enzyme involved in the Se metabolism pathway, and it is responsible for the catalysis of Se-methylselenocysteine (SeMSC) compound formation. Previous studies showed that selenium treatment activated SMT expression and promoted the accumulation of glucosinolates (GSLs) and sulforaphane, but the roles and functional mechanisms of SMT in mediating GSLs and sulforaphane synthesis remain unclear. In this study, we identified the BoSMT gene in broccoli and uncovered its roles in mediating GSLs biosynthesis. Transgenic assays revealed that BoSMT is involved in SeMSC biosynthesis in broccoli. More importantly, the contents of GSLs and sulforaphane were significantly increased in the BoSMT-overexpressing broccoli lines but decreased in the knockdown lines, suggesting that BoSMT played a positive role in regulating GSLs and sulforaphane synthesis. Further evidence indicated that BoSMT-mediated overaccumulation of GSLs and sulforaphane might be due to the increase in the endogenous SeMSC content. Compared with the mock (water) treatment, selenite-induced significantly increases of the SeMSC content in the BoSMT-knockdown plants partially compensated the phenotype of GSLs and sulforaphane loss. Compared with the mock treatment, exogenous SeMSC treatment significantly increased the contents of GSL and sulforaphane and activated GSL synthesis-related gene expression, suggesting that SeMSC acted as a positive regulator for GSL and sulforaphane production. Our findings provided novel insights into selenium-mediated GSLs and sulforaphane accumulation. The genetic manipulation of BoSMT might be a useful strategy for improving the dietary nutritional values of broccoli. [ABSTRACT FROM AUTHOR]

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15. [ABSTRACT FROM AUTHOR]

Selenium is an essential trace element in our diet, crucial for the composition of human selenoproteins, which include 25 genes such as glutathione peroxidases and thioredoxin reductases. The regulation of the selenoproteome primarily hinges on the bioavailability of selenium, either from dietary sources or cell culture media. This selenium-dependent control follows a specific hierarchy, with "housekeeping" selenoproteins maintaining constant expression while "stress-regulated" counterparts respond to selenium level fluctuations. This study investigates the variability in fetal bovine serum (FBS) selenium concentrations among commercial batches and its effects on the expression of specific stress-related cellular selenoproteins. Despite the limitations of our study, which exclusively used HEK293 cells and focused on a subset of selenoproteins, our findings highlight the substantial impact of serum selenium levels on selenoprotein expression, particularly for GPX1 and GPX4. The luciferase reporter assay emerged as a sensitive and precise method for evaluating selenium levels in cell culture environments. While not exhaustive, this analysis provides valuable insights into selenium-mediated selenoprotein regulation, emphasizing the importance of serum composition in cellular responses and offering guidance for researchers in the selenoprotein field. [ABSTRACT FROM AUTHOR]

In this study, the transcriptome analysis was practiced to identify potential genes of probiotic Bacillus subtilis BSN313 involved in selenium (Se) enrichment metabolism. The transcriptomic variation of the strain was deliberated in presence of three different sodium selenite concentrations (0, 3, and 20 μg/mL). The samples were taken at 1 and 13 h subsequent to inoculation of selenite and gene expression profiles in Se metabolism were analyzed through RNA sequencing. The gene expression levels of the pre log phase were lower than the stationary phase. It is because, the bacteria has maximum grown with high concentration of Se (enriched with organic Se), at stationary phase. Bacterial culture containing 3 μg/mL concentration of inorganic Se (sodium selenite) has shown highest gene expression as compared to no or high concentration of Se. This concentration (3 μg/mL) of sodium selenite (as Se) in the medium promoted the upregulation of thioredoxin reductase expression, whereas its higher Se concentration inhibited the formation of selenomethionine (SeMet). The result of 5 L bioreactor fermentation showed that SeMet was also detected in the fermentation supernatant as the growth entered in the late stationary phase and reached up to 857.3 ng/mL. The overall intracellular SeMet enriched content in BSN313 was extended up to 23.4 μg/g dry cell weight. The other two selenoamino acids (Se‐AAs), methyl‐selenocysteine, and selenocysteine were hardly detected in medium supernatant. From this study, it was concluded that SeMet was the highest content of organic Se byproduct biosynthesized by B. subtilis BSN313 strain in Se‐enriched medium during stationary phase. Thus, B. subtilis BSN313 can be considered a commercial probiotic strain that can be used in the food and pharmaceutical industries. This is because it can meet the commercial demand for Se‐AAs (SeMet) in both industries. [ABSTRACT FROM AUTHOR]

The antioxidant properties of 21 proteinogenic amino acids (AAs) and 3,4-dioxophenylanine (DOPA) have been studied in implicit water using density functional theory (DFT). All the calculations have been performed according to three oxidation mechanisms: (1) hydrogen-atom transfer (HAT); (2) single electron transfer followed by proton transfer (SET-PT); and (3) sequential proton-loss electron transfer (SPLET). As a result, five AAs with the highest antioxidant capacity have been established: DOPA, selenocysteine (Sec), tyrosine (Tyr), cysteine (Cys), and tryptophan (Trp). Also, global reactivity in terms of hardness/softness has been evaluated, as well as Fukui indices of local reactivity. Trp has been determined as the most reactive molecule, whereas selenium atom of Sec has been established as the most reactive atom. All the findings are in agreement with the recent literature on both experimental and theoretical studies of amino acids antioxidant activity. However, to the best of my knowledge, the calculations for one electron redox reactions of zwitterionic amino acids in implicit water have been performed for the first time. [ABSTRACT FROM AUTHOR]

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells. [ABSTRACT FROM AUTHOR]

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss–cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The targeting of human thioredoxin reductase is widely recognized to be crucially involved in the anticancer properties of several metallodrugs, including Au(I) complexes. In this study, the mechanism of reaction between a set of five N-heterocyclic carbene Au(I) complexes and models of the active Sec residue in human thioredoxin reductase was investigated by means of density functional theory approaches. The study was specifically addressed to the kinetics and thermodynamics of the tiled process by aiming at elucidating and explaining the differential inhibitory potency in this set of analogous Au(I) bis-carbene complexes. While the calculated free energy profile showed a substantially similar reactivity, we found that the binding of these Au(I) bis-carbene at the active CysSec dyad in the TrxR enzyme could be subjected to steric and orientational restraints, underlining both the approach of the bis-carbene scaffold and the attack of the selenol group at the metal center. A new and detailed mechanistic insight to the anticancer activity of these Au(I) organometallic complexes was thus provided by consolidating the TrxR targeting paradigm. [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) was discovered as the 21st genetically encoded amino acid. In nature, site-directed incorporation of Sec into proteins requires specialized biosynthesis and recoding machinery that evolved distinctly in bacteria compared to archaea and eukaryotes. Many organisms, including higher plants and most fungi, lack the Sec-decoding trait. We review the discovery of Sec and its role in redox enzymes that are essential to human health and important targets in disease. We highlight recent genetic code expansion efforts to engineer site-directed incorporation of Sec in bacteria and yeast. We also review methods to produce selenoproteins with 21 or more amino acids and approaches to delivering recombinant selenoproteins to mammalian cells as new applications for selenoproteins in synthetic biology. [ABSTRACT FROM AUTHOR]

Selenocysteine selenenic acids (Sec–SeOHs) and selenocysteine selenenyl iodides (Sec–SeIs) have long been recognized as crucial intermediates in the catalytic cycle of glutathione peroxidase (GPx) and iodothyronine deiodinase (Dio), respectively. However, the observation of these reactive species remained elusive until our recent study, where we successfully stabilized Sec–SeOHs and Sec–SeIs using a protective molecular cradle. Here, we report the first demonstration of the chemical transformation from a Sec–SeI to a Sec–SeOH through alkaline hydrolysis. A stable Sec–SeI derived from a selenocysteine methyl ester was synthesized using the protective cradle, and its structure was determined by crystallographic analysis. The alkaline hydrolysis of the Sec–SeI at −50 °C yielded the corresponding Sec–SeOH in an 89% NMR yield, the formation of which was further confirmed by its reaction with dimedone. The facile and nearly quantitative conversion of the Sec–SeI to the Sec–SeOH not only validates the potential involvement of this process in the catalytic mechanism of Dio, but also highlights its utility as a method for producing a Sec–SeOH. [ABSTRACT FROM AUTHOR]

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e‐SE) platform for the efficient site‐selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e‐SE is highlighted through late‐stage C‐terminal modification of the FDA‐approved cancer drug leuprolide and assembly of a library of anti‐HER2 affibody conjugates bearing complex cargoes. Following assembly by e‐SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated. [ABSTRACT FROM AUTHOR]

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e‐SE) platform for the efficient site‐selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e‐SE is highlighted through late‐stage C‐terminal modification of the FDA‐approved cancer drug leuprolide and assembly of a library of anti‐HER2 affibody conjugates bearing complex cargoes. Following assembly by e‐SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated. [ABSTRACT FROM AUTHOR]

The development of straightforward synthetic and characterization methods for selenopeptides is essential to discovering hierarchical structured functional materials. Here, the synthesis of a series of homo-selenopeptides 1-4, having the benzyl (Bzl) group-protected selenocysteine [(Bzl)SeCH2CH(NH2)COOH] monomer units in the sequence, is reported. The homo-selenopeptides 1-4 are characterized by 1D (1H, 13C, and 77Se) and 2D (1H-1H COSY, 1H-1H NOESY, and 1H-1H TOCSY) NMR spectroscopy, and ESI-MS spectrometry. The triselenopeptide 1 shows a propensity for self-assembly into β-sheet amyloid-like fibril structure in acetonitrile (ACN) solution at room temperature. This has systematically been analyzed and established through the spectroscopic techniques; FT-IR, CD, and ThT-based fluorescence spectroscopy for secondary bonding analyses and microscopic techniques; SEM and TEM for the amyloid-like fibril structure in ACN solution. The amide I band vibrational stretching frequencies observed in the range 1600-1700 cm−1 confirm that all peptides in the homologous series have a strong propensity to form amyloid-like fibril structures. Synthesis of a series of homo-selenopeptides 1-4 through the solid-phase peptide synthesis (SPPS), using Fmoc protected Sec(Bzl)-OH as a source of amino acid, has been described. Spectroscopic and morphological imaging studies revealed that the homo-selenopeptides have a high propensity to get self-organized into amyloid-like fibrillar structures. [ABSTRACT FROM AUTHOR]

Background: Seleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and cognitive deficits. In a previous study, a SeMCys producing strain of Bacillus subtilis GBACB was generated by releasing feedback inhibition by overexpression of cysteine-insensitive serine O-acetyltransferase, enhancing the synthesis of S-adenosylmethionine as methyl donor by overexpression of S-adenosylmethionine synthetase, and expressing heterologous selenocysteine methyltransferase. In this study, we aimed to improve GBACB SeMCys production by synthesizing methylmethionine as a donor to methylate selenocysteine and by inhibiting the precursor degradation pathway. Results: First, the performance of three methionine S-methyltransferases that provide methylmethionine as a methyl donor for SeMCys production was determined. Integration of the NmMmt gene into GBACB improved SeMCys production from 20.7 to 687.4 μg/L. Next, the major routes for the degradation of selenocysteine, which is the precursor of SeMCys, were revealed by comparing selenocysteine hyper-accumulating and non-producing strains at the transcriptional level. The iscSB knockout strain doubled SeMCys production. Moreover, deleting sdaA, which is responsible for the degradation of serine as a precursor of selenocysteine, enhanced SeMCys production to 4120.3 μg/L. Finally, the culture conditions in the flasks were optimized. The strain was tolerant to higher selenite content in the liquid medium and the titer of SeMCys reached 7.5 mg/L. Conclusions: The significance of methylmethionine as a methyl donor for SeMCys production in B. subtilis is reported, and enhanced precursor supply facilitates SeMCys synthesis. The results represent the highest SeMCys production to date and provide insight into Se metabolism. [ABSTRACT FROM AUTHOR]

Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec498 residue of the enzyme, but the neighboring Cys497 and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec498. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of N-acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec498 and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis. [ABSTRACT FROM AUTHOR]

The global understanding of selenium (Se) in plant biology mainly comes from the fields of medicine and animal science, while the research on Se in plant biology in the field of plant science lags behind. This paper summarized the physiological functions of Se in plants. These studies indicate that Se can promote plant seed development and growth and plant photosynthesis, increase plant economic yield and quality, and enhance plant antioxidant capacity and resistance to stress. However, its effects have a "dual" character, and its concentration or dosage range is very narrow. At appropriate concentrations, Se has an important impact on the physiological processes of plants and is a beneficial element for many plants to maintain health and good growth and development. [ABSTRACT FROM AUTHOR]

Investigation of the induction of antioxidant activity (AOA) by selenium compounds (Na2SeO3, diacetophenonyl selenide (DAPS-25), L-selenocystine) in various organisms is of interest as a way of protecting cell membranes from oxidative stress. A comparative analysis of the antioxidant activity of 23 amino acids was performed by coulometric titration with electrogenerated bromine and iodine. The activity decreases in the order: cystine > tryptophan > selenocystine > tyrosine > 3,3'-dimethyl-L-selenocystine > methionine. Only amino acids with sulfhydryl and selenol groups as more active reductants can interact with the electrogenerated iodine: cysteine > selenocysteine > threo-3-methyl-L-selenocysteine. Probably the correction of the antioxidant status at the amino acids level is based on the sulfhydryl and selenol groups in radicals. In case they are not enough, cystine, selenocystin, tryptophan, tyrosine, and methionine will act as scavenger-reductants. It was found that selenium compounds dose-dependently induce the total antioxidant activity of the Aspergillus niger mycelium and affect indicators of the antioxidant status (amino acid composition and catalase activity), which in turn stimulates the biomass accumulations. DAPS-25 and sodium selenite treatment at high doses (0.025 mg Se/L) caused the greatest effect on the total AOA induction (3.4–5.5 times). Lower concentrations (0.0025–0.00025 mg Se/L) had a lesser effect (25.8–41.7%). Activity in samples with L-selenocystin increased by 1.6–43.3%. It is noted that the iodine antioxidant activity in the mycelium was generally lower than the bromine one. [ABSTRACT FROM AUTHOR]

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 highaffinity ApoER2 in a Sec lyase–independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP. [ABSTRACT FROM AUTHOR]

Selenocysteine is a catalytic residue at the active site of all selenoenzymes in bacteria and mammals, and it is incorporated into the polypeptide backbone by a co-translational process that relies on the recoding of a UGA termination codon into a serine/selenocysteine codon. The best-characterized selenoproteins from mammalian species and bacteria are discussed with emphasis on their biological function and catalytic mechanisms. A total of 25 genes coding for selenoproteins have been identified in the genome of mammals. Unlike the selenoenzymes of anaerobic bacteria, most mammalian selenoenzymes work as antioxidants and as redox regulators of cell metabolism and functions. Selenoprotein P contains several selenocysteine residues and serves as a selenocysteine reservoir for other selenoproteins in mammals. Although extensively studied, glutathione peroxidases are incompletely understood in terms of local and time-dependent distribution, and regulatory functions. Selenoenzymes take advantage of the nucleophilic reactivity of the selenolate form of selenocysteine. It is used with peroxides and their by-products such as disulfides and sulfoxides, but also with iodine in iodinated phenolic substrates. This results in the formation of Se-X bonds (X = O, S, N, or I) from which a selenenylsulfide intermediate is invariably produced. The initial selenolate group is then recycled by thiol addition. In bacterial glycine reductase and D-proline reductase, an unusual catalytic rupture of selenium–carbon bonds is observed. The exchange of selenium for sulfur in selenoproteins, and information obtained from model reactions, suggest that a generic advantage of selenium compared with sulfur relies on faster kinetics and better reversibility of its oxidation reactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selenium is an essential micronutrient and has been shown to have an antioxidant effect. This can prevent different diseases such as coronary illness or cancer. Its deficiency generates neuromuscular disorders, which is why a recommended daily intake for humans of 55 μg/day has been proposed but it is not always covered. Selenium is integrated as selenoaminoacids to glutathione peroxidases, which are enzymes involved in redox reactions. Some yeast are capable of introducing inorganic selenium into the polypeptide chains of ribosome to later transform it into selenoaminoacids. In some cases, selenized yeasts are used as starter cultures in fermented foods processing. These yeasts are considered bioavailable selenium supplements and currently food science has developed strategies to include them within innovation processes of enriched products. Due to the importance of selenium in human health, the objective of this review is to present a state of the art of selenized foods, going through the importance of selenium in human health and the biochemical mechanism of biotransformation of inorganic selenium to organic species by yeasts. [ABSTRACT FROM AUTHOR]

AuI‐carbene and PtIV−AuI‐carbene prodrugs display low to sub‐μM activity against several cancer cell lines and overcome cisplatin (cisPt) resistance. Linking a cisPt‐derived PtIV(phenylbutyrate) complex to a AuI‐phenylimidazolylidene complex 2, yielded the most potent prodrug. While in vivo tests against Lewis Lung Carcinoma showed that the prodrug PtIV(phenylbutyrate)‐AuI‐carbene (7) and the 1 : 1 : 1 co‐administration of cisPt: phenylbutyrate:2 efficiently inhibited tumor growth (≈95 %), much better than 2 (75 %) or cisPt (84 %), 7 exhibited only 5 % body weight loss compared to 14 % for 2, 20 % for cisPt and >30 % for the co‐administration. 7 was much more efficient than 2 at inhibiting TrxR activity in the isolated enzyme, in cells and in the tumor, even though it was much less efficient than 2 at binding to selenocysteine peptides modeling the active site of TrxR. Organ distribution and laser‐ablation (LA)‐ICP‐TOFMS imaging suggest that 7 arrives intact at the tumor and is activated there. [ABSTRACT FROM AUTHOR]

AuI‐carbene and PtIV−AuI‐carbene prodrugs display low to sub‐μM activity against several cancer cell lines and overcome cisplatin (cisPt) resistance. Linking a cisPt‐derived PtIV(phenylbutyrate) complex to a AuI‐phenylimidazolylidene complex 2, yielded the most potent prodrug. While in vivo tests against Lewis Lung Carcinoma showed that the prodrug PtIV(phenylbutyrate)‐AuI‐carbene (7) and the 1 : 1 : 1 co‐administration of cisPt: phenylbutyrate:2 efficiently inhibited tumor growth (≈95 %), much better than 2 (75 %) or cisPt (84 %), 7 exhibited only 5 % body weight loss compared to 14 % for 2, 20 % for cisPt and >30 % for the co‐administration. 7 was much more efficient than 2 at inhibiting TrxR activity in the isolated enzyme, in cells and in the tumor, even though it was much less efficient than 2 at binding to selenocysteine peptides modeling the active site of TrxR. Organ distribution and laser‐ablation (LA)‐ICP‐TOFMS imaging suggest that 7 arrives intact at the tumor and is activated there. [ABSTRACT FROM AUTHOR]

Ferroptosis, characterized by excessive iron accumulation and lipid peroxidation, is a novel form of iron-dependent cell death, which is morphologically, genetically, and biochemically distinct from other known cell death types, such as apoptosis, necrosis, and autophagy. Emerging evidence shows that glutathione peroxidase 4 (GPX4), a critical core regulator of ferroptosis, plays an essential role in protecting cells from ferroptosis by removing the product of iron-dependent lipid peroxidation. The fast-growing studies on ferroptosis in cancer have boosted a perspective on its use in cancer therapeutics. In addition, significant progress has been made in researching and developing tumor therapeutic drugs targeting GPX4 based on ferroptosis, especially in acquired drug resistance. Selenium modulates GPX4-mediated ferroptosis, and its existing form, selenocysteine (Sec), is the active center of GPX4. This review explored the structure and function of GPX4, with the overarching goal of revealing its mechanism and potential application in tumor therapy through regulating ferroptosis. A deeper understanding of the mechanism and application of GPX4-mediated ferroptosis in cancer therapy will provide new strategies for the research and development of antitumor drugs. [ABSTRACT FROM AUTHOR]

The level of selenocysteine (Sec) in the human body is closely related to a variety of pathophysiological states, so it is important to study its fluorescence sensing mechanism for designing efficient fluorescent probes. Herein, we used time-dependent density functional theory to investigate the fluorescence sensing mechanism of phenanthroimidazole derivates A4 and B4 for the detection of Sec, which are proposed to be designed based on excited state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) mechanisms. The calculation results show that the fluorescence quenching mechanism of A4 and B4 is due to the photo-induced electron transfer (PET) process with the sulfonate group acts as the electron acceptor. Subsequently, A4 and B4 react with Sec, the sulfonate group is substituted by hydroxyl groups, PET is turned off, and significant fluorescence enhancement of the formed A3 and B3 is observed. The theoretical results suggest that the fluorescence enhancement mechanism of B3 is not based on ICT mechanism, and the charge transfer phenomenon was not observed by calculating the frontier molecular orbitals, and proved to be a local excitation mode. The reason for the fluorescence enhancement of A3 based on ESIPT is also explained by the calculated potential energy curves. [ABSTRACT FROM AUTHOR]

The effect of deprotonation on the structure and stability of the 21st proteinogenic amino acid selenocysteine, generated as bare deprotonated species by electrospray ionization, has been investigated by infrared multiple photon dissociation (IRMPD) spectroscopy over an extended frequency range (700-3600 cm−1), encompassing both the fingerprint and X–H (X = C, N, O) stretching ranges. IRMPD spectra, interpreted by anharmonic DFT calculations, provide evidence of a thermally averaged ion population of two types of low-lying canonical conformers deprotonated at the selenol (Se–H) group and involved in different H-bonding motifs. The broadened and diffuse band structures observed in the H-stretching range are well interpreted by Born-Oppenheimer molecular dynamics computations that provide a valuable description of the flexible backbone arrangements of Sec and of the proton sharing dynamics in the Se–HO and Se–HN moieties. Other prototropic isomers, deprotonated at the carboxylic group or with a zwitterionic structure, should not be significantly populated, according to their higher free energy and calculated IR spectra inconsistent with experimental evidence. [Display omitted] • The structure of isolated deprotonated amino acid selenocysteine was unveiled. • The deprotonation site on the selenol group was assessed. • IR ion spectroscopy was used over an extended frequency range (700–3600 cm⁻1). • Anharmonic IR calculations confirmed the presence of several conformers. • BOMD computations accurately interpret diffuse IR band structures. [ABSTRACT FROM AUTHOR]

A potential target of precision nutrition in cancer therapeutics is the micronutrient selenium (Se). Se is metabolized and incorporated as the amino acid selenocysteine (Sec) into 25 human selenoproteins, including glutathione peroxidases (GPXs) and thioredoxin reductases (TXNRDs), among others. Both the processes of Se and Sec metabolism for the production of selenoproteins and the action of selenoproteins are utilized by cancer cells from solid tumors as a protective mechanism against oxidative damage and to resist ferroptosis, an iron-dependent cell death mechanism. Protection against ferroptosis in cancer cells requires sustained production of the selenoprotein GPX4, which involves increasing the uptake of Se, potentially activating Se metabolic pathways such as the trans-selenation pathway and the TXNRD1-dependent decomposition of inorganic selenocompounds to sustain GPX4 synthesis. Additionally, endoplasmic reticulum-resident selenoproteins also affect apoptotic responses in the presence of selenocompounds. Selenoproteins may also help cancer cells adapting against increased oxidative damage and the challenges of a modified nutrient metabolism that result from the Warburg switch. Finally, cancer cells may also rewire the selenoprotein hierarchy and use Se-related machinery to prioritize selenoproteins that are essential to the adaptations against ferroptosis and oxidative damage. In this review, we discuss both the evidence and the gaps in knowledge on how cancer cells from solid tumors use Se, Sec, selenoproteins, and the Se-related machinery to promote their survival particularly via resistance to ferroptosis. [ABSTRACT FROM AUTHOR]

Human β-defensin 3, HBD-3, is a 45-residue antimicrobial and immunomodulatory peptide that plays multiple roles in the host defense system. In addition to interacting with cell membranes, HBD-3 is also a ligand for melanocortin receptors, cytokine receptors and voltage-gated potassium channels. Structural and functional studies of HBD-3 have been hampered by inefficient synthetic and recombinant expression methods. Herein, we report an optimized Fmoc solid-phase synthesis of this peptide using an orthogonal disulfide bonds formation strategy. Our results suggest that utilization of an optimized resin, coupling reagents and pseudoproline dipeptide building blocks decrease chain aggregation and largely improve the amount of the target peptide in the final crude material, making the synthesis more efficient. We also present an alternative synthesis of HBD-3 in which a replacement of a native disulfide bridge with a diselenide bond improved the oxidative folding. Our work enables further biological and pharmacological characterization of HBD-3, hence advancing our understanding of its therapeutic potential. [ABSTRACT FROM AUTHOR]

Cyanobacteria are prokaryotic Gram-negative organisms prevalent in nearly all habitats. A detailed proteomics study of Cyanobacteria has not been conducted despite extensive study of their genome sequences. Therefore, we conducted a proteome-wide analysis of the Cyanobacteria proteome and found Calothrix desertica as the largest (680331.825 kDa) and Candidatus synechococcus spongiarum as the smallest (42726.77 kDa) proteome of the cyanobacterial kingdom. A Cyanobacterial proteome encodes 312.018 amino acids per protein, with a molecular weight of 182173.1324 kDa per proteome. The isoelectric point (pI) of the Cyanobacterial proteome ranges from 2.13 to 13.32. It was found that the Cyanobacterial proteome encodes a greater number of acidic-pI proteins, and their average pI is 6.437. The proteins with higher pI are likely to contain repetitive amino acids. A virtual 2D map of Cyanobacterial proteome showed a bimodal distribution of molecular weight and pI. Several proteins within the Cyanobacterial proteome were found to encode Selenocysteine (Sec) amino acid, while Pyrrolysine amino acids were not detected. The study can enable us to generate a high-resolution cell map to monitor proteomic dynamics. Through this computational analysis, we can gain a better understanding of the bias in codon usage by analyzing the amino acid composition of the Cyanobacterial proteome. [ABSTRACT FROM AUTHOR]

Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNASec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2R543Q-mutant brains. [ABSTRACT FROM AUTHOR]

Methionine (Met) can be oxidized to methionine sulfoxide (MetO), which exist as R- and S -diastereomers. Present in all three domains of life, methionine sulfoxide reductases (MSR) are the enzymes that reduce MetO back to Met. Most characterized among them are MSRA and MSRB, which are strictly stereospecific for the S - and R -diastereomers of MetO, respectively. While the majority of MSRs use a catalytic Cys to reduce their substrates, some employ selenocysteine. This is the case of mammalian MSRB1, which was initially discovered as selenoprotein SELR or SELX and later was found to exhibit an MSRB activity. Genomic analyses demonstrated its occurrence in most animal lineages, and biochemical and structural analyses uncovered its catalytic mechanism. The use of transgenic mice and mammalian cell culture revealed its physiological importance in the protection against oxidative stress, maintenance of neuronal cells, cognition, cancer cell proliferation, and the immune response. Coincident with the discovery of Met oxidizing MICAL enzymes, recent findings of MSRB1 regulating the innate immunity response through reversible stereospecific Met- R -oxidation of cytoskeletal actin opened up new avenues for biological importance of MSRB1 and its role in disease. In this review, we discuss the current state of research on MSRB1, compare it with other animal Msrs, and offer a perspective on further understanding of biological functions of this selenoprotein. [Display omitted] • Met residues can be oxidized to Met sulfoxide and reduced by methionine sulfoxide reductases (MSR). • The selenoprotein MSRB1 is present in animals and its expression regulated by dietary selenium. • MSRB1 protects against oxidative stress but few targets are known. • MSRB1 regulates actin polymerization in a cycle of Met oxidation/reduction with MICALs enzymes. • CaMKII and CaM oxidation and reduction by MSRB1 may protect cardiac and nervous cells. [ABSTRACT FROM AUTHOR]

Granulocyte colony stimulating factor (G‐CSF) is a cytokine used to treat neutropenia. Different glycosylated and non‐glycosylated variants of G‐CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O‐glycoforms of G‐CSF, thereby enabling the preparation of selectively and homogeneously post‐translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N‐terminal segment with a synthetic C‐terminal part, transiently equipped with a side‐chain‐linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G‐CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays. [ABSTRACT FROM AUTHOR]

The synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3′untranslated region (3′UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than m7G-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase 1 (GPx1) is an important cellular antioxidant enzyme that is found in the cytoplasm and mitochondria of mammalian cells. Like most selenoenzymes, it has a single redox-sensitive selenocysteine amino acid that is important for the enzymatic reduction of hydrogen peroxide and soluble lipid hydroperoxides. Glutathione provides the source of reducing equivalents for its function. As an antioxidant enzyme, GPx1 modulates the balance between necessary and harmful levels of reactive oxygen species. In this review, we discuss how selenium availability and modifiers of selenocysteine incorporation alter GPx1 expression to promote disease states. We review the role of GPx1 in cardiovascular and metabolic health, provide examples of how GPx1 modulates stroke and provides neuroprotection, and consider how GPx1 may contribute to cancer risk. Overall, GPx1 is protective against the development and progression of many chronic diseases; however, there are some situations in which increased expression of GPx1 may promote cellular dysfunction and disease owing to its removal of essential reactive oxygen species. [Display omitted] • GPx1 is a selenoenzyme that reduces hydrogen peroxide and lipid hydroperoxides. • Selenium availability and translational mechanisms regulate the expression of GPx1. • GPx1 modulates the balance between necessary and harmful levels of H 2 O 2. • Removal of essential reactive oxygen species can promote dysfunction and disease. • Overall, GPx1 protects against the progression and development of many diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selenium (Se) is an important trace element that mainly occurs in the form of selenocysteine in selected proteins. In prokaryotes, Se is also required for the synthesis of selenouridine and Se-containing cofactor. A large number of selenoprotein families have been identified in diverse prokaryotic organisms, most of which are thought to be involved in various redox reactions. In the last decade or two, computational prediction of selenoprotein genes and comparative genomics of Se metabolic pathways and selenoproteomes have arisen, providing new insights into the metabolism and function of Se and their evolutionary trends in bacteria and archaea. This review aims to offer an overview of recent advances in bioinformatics analysis of Se utilization in prokaryotes. We describe current computational strategies for the identification of selenoprotein genes and generate the most comprehensive list of prokaryotic selenoproteins reported to date. Furthermore, we highlight the latest research progress in comparative genomics and metagenomics of Se utilization in prokaryotes, which demonstrates the divergent and dynamic evolutionary patterns of different Se metabolic pathways, selenoprotein families, and selenoproteomes in sequenced organisms and environmental samples. Overall, bioinformatics analyses of Se utilization, function, and evolution may contribute to a systematic understanding of how this micronutrient is used in nature. [ABSTRACT FROM AUTHOR]

Follicular ovarian cysts (FOCs) are characterized by follicles in the ovaries that are >20 mm in diameter and persist for >10 days without the corpus luteum, leading to anovulation, dysregulation of folliculogenesis and subfertility in humans and livestock species. Despite their clinical significance, the precise impact of FOCs on oocyte reserve, maturation, and quality still needs to be explored. While FOCs are observed in both human and livestock populations, they are notably prevalent in livestock species. Consequently, livestock species serve as valuable models for investigating the molecular intricacies of FOCs. Thus, in this study, using goat FOCs, we performed integrated proteomic, metabolomic and functional analyses to demonstrate that oocyte maturation is hampered due to increased reactive oxygen species (ROS) in FOCs follicular fluid (FF) via downregulation of glutathione peroxidase (GPX1), a critical antioxidant seleno enzyme required to negate oxidative stress. Notably, GPX1 reduction was positively correlated with the FF's decline of free selenium and selenocysteine metabolic enzymes, O-phosphoryl-tRNA (Sec) selenium transferase (SEPSECS) and selenocysteine lyase (SCLY) levels. Adding GPX1, selenocysteine, or selenium to the culture media rescued the oocyte maturation abnormalities caused by FOCs FF by down-regulating the ROS. Additionally, we demonstrate that substituting GPX1 regulator, Insulin-like growth factor-I (IGF-1) in the in vitro maturation media improved the oocyte maturation in the cystic FF by down-regulating the ROS activity via suppressing Non-sense-mediated decay (NMD) of GPX1. In contrast, inhibition of IGF-1R and the target of rapamycin complex 1 (mTORC1) hampered the oocyte maturation via NMD up-regulation. These findings imply that the GPX1 regulation via selenocysteine metabolism and the IGF-1-mediated NMD may be critical for the redox homeostasis of FF. We propose that GPX1 enhancers hold promise as therapeutics for enhancing the competence of FOCs oocytes. However, further in vivo studies are necessary to validate these findings observed in vitro. • Elevated reactive oxygen species (ROS) levels in the follicular ovarian cystic microenvironment (FOC) lead to oocyte death. • In the FOC follicular fluid, there is a notable reduction in the GPX1 antioxidant enzyme levels, linked to diminished selenium metabolism. • Supplementing culture media with recombinant GPX1, selenocysteine, or selenium enhanced FOC oocyte maturation by lowering ROS levels. • We propose that GPX1 enhancers exhibit potential as therapeutic agents for improving the competency of FOCs oocytes. [ABSTRACT FROM AUTHOR]

The trace element selenium (Se) is essential to the physiology of most organisms on the planet. The most well documented of Se's biological forms are selenoproteins, where selenocysteine often serves as the catalytic center for crucial redox processes. Se is also found in several other classes of biological molecules, including nucleic acids, sugars, and modified amino acids, although its role in the function of these metabolites is less understood. Despite its prevalence, only a small number of Se-specific biosynthetic pathways have been discovered. Around half of these were first characterized in the past three years, suggesting that the selenometabolome may be more diverse than previously appreciated. Here, we review the recent advances in our understanding of this intriguing biochemical space, and discuss prospects for future discovery efforts. [ABSTRACT FROM AUTHOR]

The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3. [ABSTRACT FROM AUTHOR]

We succeeded in expressing selenocysteine β-lyase (SCL) from a lactic acid bacterium, Leuconostoc mesenteroides LK-151 (Lm-SCL), in the soluble fractions of Escherichia coli Rosetta (DE3) using a novel expression vector of pET21malb constructed by ourselves that has both maltose binding protein (MBP)- and 6 × His-tag. Lm-SCL acted on l-selenocysteine, l-cysteine, and l-cysteine sulfinic acid but showed a high preference for l-selenocysteine. The kcat and kcat/Km values of Lm-SCL were determined to be 108 (min−1) and 42.0 (min−1・mM−1), respectively, and this was enough catalytic efficiency to suggest that Lm-SCL might also be involved in supplying elemental selenium from l-selenocysteine to selenoproteins like other SCLs. The optimum temperature and optimum pH of Lm-SCL were determined to be 37 °C and pH 6.5, respectively. Lm-SCL was stable at 37–45 °C and pH 6.5–7.5. Lm-SCL was completely inhibited by the addition of hydroxylamine, semicarbazide, and iodoacetic acid. The enzyme activity of Lm-SCL was decreased in the presence of various metal ions, especially Cu2+. The quaternary structure of Lm-SCL is a homodimer with a subunit molecular mass of 47.5 kDa. The similarity of the primary structure of Lm-SCL to other SCLs from Citrobacter freundii, Escherichia coli, humans, or mouse was calculated to be 47.0, 48.0, 12.5, or 24.0%, respectively. Unlike Ec-SCL, our mutational and molecular docking simulation studies revealed that C362 of Lm-SCL might also catalyze the deselenation of l-selenocysteine in addition to the desulfuration of l-cysteine. [ABSTRACT FROM AUTHOR]

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol‐disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one‐pot chemical synthesis of the thioredoxin‐like domain of SELENOF, highlighted by the use of Fmoc‐protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)‐like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non‐native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER. [ABSTRACT FROM AUTHOR]

Ferrocene (Fc) conjugated selenopeptides were synthesized using a new modified solid‐phase peptide synthesis (SPSS) protocol and their self‐assembled nanostructure formation into spheres, nanofibers, and nanotubes studied. Four selenopeptide conjugates have been successfully prepared: two containing both selenocysteine (Sec) and Fc, the other two have either one of these units. These compounds were well‐characterized with various spectroscopic techniques, including 1D and 2D NMR. The importance of the Fc unit, aromatic moieties, and weak intermolecular interactions on the self‐assembled process was revealed by systematic morphological studies. [ABSTRACT FROM AUTHOR]

β-Seleno-α-amino acids, known as selenocysteine (Sec) derivatives, have emerged as important targets because of their role in chemical biology, not only as part of selenoproteins with important redox properties, but also because of their activity as antivirals or metabolites effective in inhibiting carcinogenesis. In addition, there is demand for this type of compounds due to their use in native chemical ligation to construct large peptides. Therefore, this review summarizes the various synthetic methods that have been published to construct Sec derivatives. Most of them involve the generation of the C–Se bond by nucleophilic substitution reactions, but other reactions such as radical or multicomponent strategies are also reported. Of particular importance is the Se -Michael addition of Se -nucleophiles to chiral bicyclic dehydroalanines, in which the stereogenic center is generated under complete stereocontrol. 1 Introduction 2 Previously Reviewed Synthesis of Sec 3 Retrosynthesis of Sec Derivatives 4 Sec Derivatives by Nucleophilic Substitutions 5 Sec Derivatives by Radical Processes 6 Sec Derivatives by 1,4-Conjugate Additions 7 Conclusion [ABSTRACT FROM AUTHOR]

As an essential trace element, selenium (Se) plays a tremendous role in the functioning of the human organism being used for the biosynthesis of selenoproteins (proteins containing one or several selenocysteine residues). The functions of human selenoproteins in vivo are extremely diverse. Many selenoproteins have an antioxidant activity and, hence, play a key role in cell antioxidant defense and maintenance of redox homeostasis, which accounts for their involvement in diverse biological processes, such as signal transduction, proliferation, cell transformation and aging, ferroptosis, immune system functioning, etc. One of the critical functions of selenoenzymes is participation in the synthesis of thyroid hormones regulating basal metabolism in all body tissues. Over the last decades, optimization of population Se intake for prevention of diseases related to Se deficiency or excess has been recognized as a pressing issue in modern healthcare worldwide. [ABSTRACT FROM AUTHOR]