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

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

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

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

Quantifying Turnover Dynamics of Selenoproteome by Isotopic Perturbation.

Selenium, as an essential trace element of life, is closely related to human health and is required to produce selenoproteins, a family of important functional proteins in many living organisms. All selenoproteins contain a special amino acid, selenocysteine, which often serves as their active-site residue, and the expression and activity of selenoproteins are fine-tuned. However, the turnover dynamics of selenoproteome has never been systematically investigated, especially in a site-specific manner for selenocysteines. In the current work, we developed a chemical proteomic strategy named "SElenoprotein Turnover Rate by Isotope Perturbation (SETRIP)" to quantitatively monitor the turnover dynamics of selenoproteins at the proteomic level. The kinetic rates and half-lives of nine selenoproteins were accurately measured by combining Na274SeO3 metabolic labeling with pulse-chase chemoproteomics. The half-lives of selenoproteins were measured to range from 6 to 32 h with the housekeeping selenoprotein glutathione peroxidases (GPX4) showing a faster turnover rate, implying that the hierarchy regulation also exists in the turnover of selenoproteins in addition to expression and activity. Our study generated a global portrait of dynamic changes in the selenoproteome and provided important clues to study the roles of selenium in biology.

Chemoproteomic interrogation of selenocysteine by low-pH isoTOP-ABPP.

Selenoproteins comprise a small group of selenocysteine (Sec) containing proteins, often involved in redox homeostasis. While Sec is functionally similar to cysteine (Cys), with both acting as protein-centered nucleophiles, chemoproteomic strategies employing electrophilic probes have often failed to rigorously identify Sec residues, due to their relatively low abundance with respect to Cys across a proteome. To improve the enrichment and detection of selenoproteins, herein we describe a chemoproteomic strategy that relies on the unique properties of Sec as compared to Cys, such as reduced pKa and the unique isotopic distribution of selenium. Low pH electrophilic probe labeling of mouse proteomes reduces Cys reactivity, resulting in increased identification of most soluble selenoproteins. This quantitative chemoproteomic platform provides a method to reliably measure changes in selenoprotein abundance across growth conditions as well as quantify inhibition by selenoprotein specific inhibitors, such as Auranofin.

Application of alpha-methyl selenocysteine as a tool for the study of selenoproteins.

Selenocysteine (Sec) is the 21st proteogenic amino acid and it is now widely accepted that Sec is involved in redox biochemistry when incorporated in proteins. However, many of the chemical mechanisms for Sec bioactivity remain unknown. Herein, we describe a derivative of Sec, alpha-methyl Sec ((αMe)Sec), that is a useful chemical tool to study selenoenzyme mechanisms. (αMe)Sec is identical to Sec except the Cα-H is replaced with a Cα-methyl group, which prevents this derivative from undergoing oxygen-mediated ß-syn elimination to dehydroalanine, which is a common problem with Sec-containing peptides and proteins. Thus, since (αMe)Sec-containing peptides and proteins cannot lose the side-chain selenium atom when oxidized, mechanistic studies can be performed that are not always possible with Sec. In this chapter, we provide detailed methods for the incorporation of (αMe)Sec into peptides using solid phase peptide synthesis and subsequent incorporation into mammalian thioredoxin reductase using protein semisynthesis. We then provide two examples of how (αMe)Sec has been used as a chemical tool to study selenoenzyme mechanism. Finally, we discuss future applications where we envision (αMe)Sec will be useful.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

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.

Therapeutic Potential and Main Methods of Obtaining Selenium Nanoparticles.

This review presents the latest data on the importance of selenium nanoparticles in human health, their use in medicine, and the main known methods of their production by various methods. In recent years, a multifaceted study of nanoscale complexes in medicine, including selenium nanoparticles, has become very important in view of a number of positive features that make it possible to create new drugs based on them or significantly improve the properties of existing drugs. It is known that selenium is an essential trace element that is part of key antioxidant enzymes. In mammals, there are 25 selenoproteins, in which selenium is a key component of the active site. The important role of selenium in human health has been repeatedly proven by several hundred works in the past few decades; in recent years, the study of selenium nanocomplexes has become the focus of researchers. A large amount of accumulated data requires generalization and systematization in order to improve understanding of the key mechanisms and prospects for the use of selenium nanoparticles in medicine, which is the purpose of this review.

Roles for Selenoprotein I and Ethanolamine Phospholipid Synthesis in T Cell Activation.

The selenoprotein family includes 25 members, many of which are antioxidant or redox regulating enzymes. A unique member of this family is Selenoprotein I (SELENOI), which does not catalyze redox reactions, but instead is an ethanolamine phosphotransferase (Ept). In fact, the characteristic selenocysteine residue that defines selenoproteins lies far outside of the catalytic domain of SELENOI. Furthermore, data using recombinant SELENOI lacking the selenocysteine residue have suggested that the selenocysteine amino acid is not directly involved in the Ept reaction. SELENOI is involved in two different pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, which are constituents of cellular membranes. Ethanolamine phospholipid synthesis has emerged as an important process for metabolic reprogramming that occurs in pluripotent stem cells and proliferating tumor cells, and this review discusses roles for upregulation of SELENOI during T cell activation, proliferation, and differentiation. SELENOI deficiency lowers but does not completely diminish de novo synthesis of PE and plasmenyl PE during T cell activation. Interestingly, metabolic reprogramming in activated SELENOI deficient T cells is impaired and this reduces proliferative capacity while favoring tolerogenic to pathogenic phenotypes that arise from differentiation. The implications of these findings are discussed related to vaccine responses, autoimmunity, and cell-based therapeutic approaches.

Toxicology and pharmacology of synthetic organoselenium compounds: an update.

Here, we addressed the pharmacology and toxicology of synthetic organoselenium compounds and some naturally occurring organoselenium amino acids. The use of selenium as a tool in organic synthesis and as a pharmacological agent goes back to the middle of the nineteenth and the beginning of the twentieth centuries. The rediscovery of ebselen and its investigation in clinical trials have motivated the search for new organoselenium molecules with pharmacological properties. Although ebselen and diselenides have some overlapping pharmacological properties, their molecular targets are not identical. However, they have similar anti-inflammatory and antioxidant activities, possibly, via activation of transcription factors, regulating the expression of antioxidant genes. In short, our knowledge about the pharmacological properties of simple organoselenium compounds is still elusive. However, contrary to our early expectations that they could imitate selenoproteins, organoselenium compounds seem to have non-specific modulatory activation of antioxidant pathways and specific inhibitory effects in some thiol-containing proteins. The thiol-oxidizing properties of organoselenium compounds are considered the molecular basis of their chronic toxicity; however, the acute use of organoselenium compounds as inhibitors of specific thiol-containing enzymes can be of therapeutic significance. In summary, the outcomes of the clinical trials of ebselen as a mimetic of lithium or as an inhibitor of SARS-CoV-2 proteases will be important to the field of organoselenium synthesis. The development of computational techniques that could predict rational modifications in the structure of organoselenium compounds to increase their specificity is required to construct a library of thiol-modifying agents with selectivity toward specific target proteins.

Dissecting the Potential of Selenoproteins Extracted from Selenium-Enriched Rice on Physiological, Biochemical and Anti-Ageing Effects In Vivo.

Ageing is an irreversible phenomenon and the processes which can delay it are under consideration for a long time by the scientific community. Selenium is an important candidate for it, but the impact of selenoprotein on nutritional changes and ageing has not been reported well. In this regard, antioxidant activities and free radical scavenging effect of selenoproteins extracted from selenium-rich rice were studied. Mice were administered a subcutaneous abdominal injection of D-galactose to induce the ageing model and fed with different selenoprotein dosage diet. Deviations among biochemical activities (total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA)) in liver and serum of the mice were assessed. The degree of liver injury, antioxidant genes and protein relative expression were estimated. The protein content, selenium content, hydroxyl scavenging and DPPH radicals were accessed in selenoprotein components. The selenoprotein constituent had protein and selenium contents in different components as water-soluble proteins > alkali-soluble proteins > salt-soluble proteins > ethanol-soluble proteins. The enzymatic activity (total antioxidant capacity, GSH-Px and SOD) in liver and serum of mice was significantly enhanced in selenoprotein diet groups. D-Galactose-induced liver injury was significantly reduced by selenoprotein diet of 25 µg/(kg day). Real-time qPCR and Western blot disclosed the enhanced relative expression of antioxidant genes (SOD2, GPX1, TrxR2 and Nrf2) and HO-1 protein in the positive control (Vc) and selenoprotein diet groups. In conclusion, selenoprotein treatment was found to have a positive influence on liver hepatocytes and biochemical features in mice. It might be used as a potential diet in scavenging oxidative injury and supporting enzymatic antioxidant system.

Effects of soft electrophiles on selenium physiology.

This review examines the effects of neurotoxic electrophiles on selenium (Se) metabolism. Selenium-dependent enzymes depend on the unique and elite functions of selenocysteine (Sec), the 21st proteinogenic amino acid, to perform their biochemical roles. Humans possess 25 selenoprotein genes, ~ half of which are enzymes (selenoenzymes) required for preventing, controlling, or reversing oxidative damage, while others participate in regulating calcium metabolism, thyroid hormone status, protein folding, cytoskeletal structure, Sec synthesis and Se transport. While selenoproteins are expressed in tissue dependent distributions and levels in all cells of all vertebrates, they are particularly important in brain development, health, and functions. As the most potent intracellular nucleophile, Sec is subject to binding by mercury (Hg) and other electron poor soft neurotoxic electrophiles. Epidemiological and environmental studies of the effects of exposures to methyl-Hg (CH3Hg+), elemental Hg (Hg°), and/or other metallic/organic neurotoxic soft electrophiles need to consider the concomitant effects of all members of this class of toxicants in relation to the Se status of their study populations. The contributions of individual electrophiles' discrete and cooperative rates of Se sequestration need to be evaluated in relation to tissue Se reserves of the exposed populations to identify sensitive subgroups which may be at accentuated risk due to poor Se status. Additional study is required to examine possibilities of inherited, acquired, or degenerative neurological disorders of Se homeostasis that may influence vulnerability to soft electrophile exposures. Investigations of soft electrophile toxicity will be enhanced by considering the concomitant effects of combined exposures on tissue Se-availability in relation to pathological consequences during fetal development or in relation to etiologies of neurological disorders and neurodegenerative diseases. Since selenoenzymes are molecular "targets" of soft electrophiles, concomitant evaluation of aggregate exposures to these toxicants in relation to dietary Se intakes will assist regulatory agencies in their goals of improving and protecting public health.

Strategies for the development of selenium-based anticancer drugs.

Many experimental models demonstrated that inorganic and organic selenium (Se) compounds may have an anticancer activity. However, large clinical studies failed to demonstrate that Se supplementations may prevent the outcome of cancers. Moreover, there are few randomized trials in cancer patients and there is not yet any Se compound recognized as anticancer drug. There is still a need to develop new Se compounds with new strategies. For that, it may be necessary to consider that Se compounds may have a dual role, either as anti-oxidant or as pro-oxidant. Experimental studies demonstrated that it is as pro-oxidant that Se compounds have anticancer effects, even though cancer cells have a pro-oxidant status. The oxidative status differs according to the type of cancer, the stage of the disease and to other parameters. We propose to adapt the doses of the Se compounds to markers of the oxidative stress, but also to markers of angiogenesis, which is strongly related with the oxidative status. A dual role of Se on angiogenesis has also been noted, either as pro-angiogenesis or as anti-angiogenesis. The objective for the development of new Se compounds, having a great selectivity on cancer cells, could be to try to normalize these oxidative and angiogenic markers in cancer patients, with an individual adaptation of doses.

Biochemical characterization of the selenoproteome in Gallus gallus via bioinformatics analysis: structure-function relationships and interactions of binding molecules.

Knowledge about mammalian selenoproteins is increasing. However, the selenoproteome of birds remains considerably less understood, especially concerning its biochemical characterization, structure-function relationships and the interactions of binding molecules. In this work, the SECIS elements, subcellular localization, protein domains and interactions of binding molecules of the selenoproteome in Gallus gallus were analyzed using bioinformatics tools. We carried out comprehensive analyses of the structure-function relationships and interactions of the binding molecules of selenoproteins, to provide biochemical characterization of the selenoproteome in Gallus gallus. Our data provided a wealth of information on the biochemical functions of bird selenoproteins. Members of the selenoproteome were found to be involved in various biological processes in chickens, such as in antioxidants, maintenance of the redox balance, Se transport, and interactions with metals. Six membrane-bound selenoproteins (SelI, SelK, SelS, SelT, DIO1 and DIO3) played important roles in maintaining the membrane integrity. Chicken selenoproteins were classified according to their ligand binding sites as zinc-containing matrix metalloselenoproteins (Sep15, MsrB1, SelW and SelM), POP-containing selenoproteins (GPx1-4), FAD-interacting selenoproteins (TrxR1-3), secretory transport selenoproteins (GPx3 and SelPa) and other selenoproteins. The results of our study provided new evidence for the unknown biological functions of the selenoproteome in birds. Future research is required to confirm the novel biochemical functions of bird selenoproteins.

Selenium and redox signaling.

Selenium compounds that contain selenol functions or can be metabolized to selenols are toxic via superoxide and H2O2 generation, when ingested at dosages beyond requirement. At supra-nutritional dosages various forms of programmed cell death are observed. At physiological intakes, selenium exerts its function as constituent of selenoproteins, which overwhelmingly are oxidoreductases. Out of those, the glutathione peroxidases counteract hydroperoxide-stimulated signaling cascades comprising inflammation triggered by cytokines or lipid mediators, insulin signaling and different forms of programmed cell death. Similar events are exerted by peroxiredoxins, which functionally depend on the selenoproteins of the thioredoxin reductase family. The thiol peroxidases of both families can, however, also act as sensors for hydroperoxides, thereby initiating signaling cascades. Although the interaction of selenoproteins with signaling events has been established by genetic techniques, the in vivo relevance of these findings is still hard to delineate for several reasons: The biosynthesis of individual selenoproteins responds differently to variations of selenium intakes; selenium is preferentially delivered to privileged tissues via inter-organ trafficking and receptor-mediated uptake, and only half of the selenoproteins known by sequence have been functionally characterized. The fragmentary insights do not allow any uncritical use of selenium for optimizing human health.

Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950.

Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g) were found in yeast cells after 24-hour culture conducted in control (YPD) medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours) the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium) of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

Membrane-bound selenoproteins.

SIGNIFICANCE: Selenoproteins employ selenium to supplement the chemistry available through the common 20 amino acids. These powerful enzymes are affiliated with redox biology, often in connection with the detection, management, and signaling of oxidative stress. Among them, membrane-bound selenoproteins play prominent roles in signaling pathways, Ca(2+) regulation, membrane complexes integrity, and biosynthesis of lipophilic molecules. RECENT ADVANCES: The number of selenoproteins whose physiological roles, protein partners, expression, evolution, and biosynthesis are characterized is steadily increasing, thus offering a more nuanced view of this specialized family. This review focuses on human membrane selenoproteins, particularly the five least characterized ones: selenoproteins I, K, N, S, and T. CRITICAL ISSUES: Membrane-bound selenoproteins are the least understood, as it is challenging to provide the membrane-like environment required for their biochemical and biophysical characterization. Hence, their studies rely mostly on biological rather than structural and biochemical assays. Another aspect that has not received much attention is the particular role that their membrane association plays in their physiological function. FUTURE DIRECTIONS: Findings cited in this review show that it is possible to infer the structure and the membrane-binding mode of these lesser-studied selenoproteins and design experiments to examine the role of the rare amino acid selenocysteine.

Selenium as a feed supplement for heat-stressed poultry: a review.

Heat stress is associated with compromised performance and productivity in poultry due to declines in feed intake, nutrient utilization, growth rate, egg production and quality, and feed efficiency. Emerging evidences have shown that acute heat exposure results in increased production of free radicals and causes oxidative damage to lipids, proteins, and DNA. Additionally, heat stress can influence immune response by changing the expression of cytokines and by making the immune cells more susceptible to oxidative stress. Selenium, as a part of specific selenoproteins, can help to maintain antioxidant defenses, thereby preventing damages to tissues. An optimum response with supplementation of selenium in diet has been found to improve feed intake, body weight gain, feed efficiency, egg production and quality, and antioxidant status in heat-stressed poultry. Selenium compounds are also known to improve immune responses by altering the production of certain cytokines secreted by cells of the immune system and by enhancing the resistance of the immune cells to oxidative stress. It was reported that selenium supplementation had inhibitory effects on tumor necrosis factor alpha levels in heat-stressed broiler chicks, but the details are not completely elucidated. In the present review, the effect of selenium on production performance, nutrient utilization, antioxidative status, and immune responses of heat-stressed poultry is summarized.

Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex.

Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.