Orphan SelD proteins and selenium-dependent molybdenum hydroxylases

Bacterial and Archaeal cells use selenium structurally in selenouridine-modified tRNAs, in proteins translated with selenocysteine, and in the selenium-dependent molybdenum hydroxylases (SDMH). The first two uses both require the selenophosphate synthetase gene, selD. Examining over 500 complete prokaryotic genomes finds selD in exactly two species lacking both the selenocysteine and selenouridine systems, Enterococcus faecalis and Haloarcula marismortui. Surrounding these orphan selD genes, forming bidirectional best hits between species, and detectable by Partial Phylogenetic Profiling vs. selD, are several candidate molybdenum hydroxylase subunits and accessory proteins. We propose that certain accessory proteins, and orphan selD itself, are markers through which new selenium-dependent molybdenum hydroxylases can be found

Exposure to Silver Nanoparticles Inhibits Selenoprotein Synthesis and the Activity of Thioredoxin Reductase

BACKGROUND: Silver nanoparticles (AgNPs) and silver (Ag)-based materials are increasingly being incorporated into consumer products, and although humans have been exposed to colloidal Ag in many forms for decades, this rise in the use of Ag materials has spurred interest into their toxicology. Recent reports have shown that exposure to AgNPs or Ag ions leads to oxidative stress, endoplasmic reticulum stress, and reduced cell proliferation. Previous studies have shown that Ag accumulates in tissues as silver sulfides (Ag2S) and silver selenide (Ag2Se). OBJECTIVES: In this study we investigated whether exposure of cells in culture to AgNPs or Ag ions at subtoxic doses would alter the effective metabolism of selenium, that is, the incorporation of selenium into selenoproteins. METHODS: For these studies we used a keratinocyte cell model (HaCat) and a lung cell model (A549). We also tested (in vitro, both cellular and chemical) whether Ag ions could inhibit the activity of a key selenoenzyme, thioredoxin reductase (TrxR). RESULTS: We found that exposure to AgNPs or far lower levels of Ag ions led to a dose-dependent inhibition of selenium metabolism in both cell models. The synthesis of protein was not altered under these conditions. Exposure to nanomolar levels of Ag ions effectively blocked selenium metabolism, suggesting that Ag ion leaching was likely the mechanism underlying observed changes during AgNP exposure. Exposure likewise inhibited TrxR activity in cultured cells, and Ag ions were potent inhibitors of purified rat TrxR isoform 1 (cytosolic) (TrxR1) enzyme. CONCLUSIONS: Exposure to AgNPs leads to the inhibition of selenoprotein synthesis and inhibition of TrxR1. Further, we propose these two sites of action comprise the likely mechanism underlying increases in oxidative stress, increases endoplasmic reticulum stress, and reduced cell proliferation during exposure to Ag

Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the D-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions

Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO2 and TiO2 Nanoparticles

Immunomodulation by nanoparticles, especially as related to the biochemical properties of these unique materials, has scarcely been explored. In an in vitro model of human immunity, we demonstrate two catalytic nanoparticles, TiO2 (oxidant) and CeO2 (antioxidant), have nearly opposite effects on human dendritic cells and T helper (T-H) cells. For example, whereas TiO2 nanoparticles potentiated DC maturation that led towards T(H)1-biased responses, treatment with antioxidant CeO2 nanoparticles induced APCs to secrete the anti-inflammatory cytokine, IL-10, and induce a T(H)2-dominated T cell profile. In subsequent studies, we demonstrate these results are likely explained by the disparate capacities of the nanoparticles to modulate ROS, since TiO2, but not CeO2 NPs, induced inflammatory responses through an ROS/inflammasome/IL-1 beta pathway. This novel capacity of metallic NPs to regulate innate and adaptive immunity in profoundly different directions via their ability to modulate dendritic cell function has strong implications for human health since unintentional exposure to these materials is common in modern societies

Impact of Trivalent Arsenicals on Selenoprotein Synthesis

BACKGROUND: Exposure to arsenic has been associated with development of skin, lung, bladder, liver, and kidney cancer. Recent evidence suggests that an increase in oxidative stress in cells treated with arsenicals represents the molecular mechanism behind arsenic-induced carcinogenesis. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in defense against reactive oxygen species. A mutual sparing effect between arsenic and selenium has been shown in animal studies when both metalloids are present in high concentrations. OBJECTIVES: To determine whether changes in selenoprotein synthesis may be an underlying mechanism behind arsenic-induced carcinogenesis, we analyzed the new synthesis of selenoproteins within cells after exposure to inorganic or methylated arsenicals using a human keratinocyte cell model. RESULTS: Addition of arsenite to culture medium blocked new synthesis of selenoproteins when selenium was present in the form of selenite, and appeared to stimulate the use of serum-derived selenium. Monomethylarsonous acid (MMA(III)) treatment of cells, in contrast, did not block all new synthesis of selenoproteins but did result in an increase in cytosolic thioredoxin reductase (TrxR1) at both the mRNA and protein levels. MMA(III) also reduced the new synthesis of cellular glutatione peroxidase (cGpx) and other smaller selenoproteins. Dimethylarsinous acid (DMA(III)) stimulated selenoprotein synthesis by an as yet unknown mechanism. CONCLUSIONS: These results suggest that arsenite and MMA(III) are key metabolites that trigger higher levels of TrxR1, and both lead to a reduction in the expression of cGpx. Together these effects certainly could lead to carcinogenesis given the knowledge that many cancers have higher levels of TrxR, and reduced Gpx levels will reduce the cell’s ability to defend against reactive oxygen species. Based on these results, the impact of the trivalent arsenicals arsenite and MMA(III) on selenoprotein synthesis may indeed represent a potential molecular mechanism for the higher rates of cancer observed in populations exposed to high levels of arsenic

Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO2 and TiO2 Nanoparticles

Immunomodulation by nanoparticles, especially as related to the biochemical properties of these unique materials, has scarcely been explored. In an in vitro model of human immunity, we demonstrate two catalytic nanoparticles, TiO2 (oxidant) and CeO2 (antioxidant), have nearly opposite effects on human dendritic cells and T helper (T-H) cells. For example, whereas TiO2 nanoparticles potentiated DC maturation that led towards T(H)1-biased responses, treatment with antioxidant CeO2 nanoparticles induced APCs to secrete the anti-inflammatory cytokine, IL-10, and induce a T(H)2-dominated T cell profile. In subsequent studies, we demonstrate these results are likely explained by the disparate capacities of the nanoparticles to modulate ROS, since TiO2, but not CeO2 NPs, induced inflammatory responses through an ROS/inflammasome/IL-1 beta pathway. This novel capacity of metallic NPs to regulate innate and adaptive immunity in profoundly different directions via their ability to modulate dendritic cell function has strong implications for human health since unintentional exposure to these materials is common in modern societies

Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp PCC 6803

Background: Molecular hydrogen is an environmentally- clean fuel and the reversible ( bidirectional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H(+) + 2e-. H-2 ( g). Results: Hydrogen yields were enhanced up to 41- fold by cloning the bidirectional hydrogenase ( encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well- studied Enterobacter aerogenes HU- 101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone ( small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild- type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm- related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2- fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. Conclusion: Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2

From wrongdoing to imprisonment: Test of a causal-moral model

The authors tested a causal–moral model of punishment in which (a) causal attribution and moral responsibility are distinct precursors of punishment, and (b) dispositional attribution leads to blame which, in turn, determines imprisonment. Specifically, whereas severity of outcome impacts punishment directly, circumstances of the crime and the culture of the observers impact punishment through causal attribution and blame, respectively. In an experiment, Singaporeans and Americans read about a crime that (a) was committed intentionally or under an extenuating circumstance and (b) had low or severe outcome for the victim. They made dispositional attribution to, assigned blame to, and recommended imprisonment for the offender. Results supported the hypotheses and the causal–moral path model that specified a direct effect of severity of outcome, an indirect effect of country via blame, and the indirect effects of circumstance via dispositional attribution to blame on imprisonment

A Selenium-Dependent Xanthine Dehydrogenase Triggers Biofilm Proliferation in Enterococcus faecalis through Oxidant Production

Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with Se-75 is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production

Peptide-Pulsed Dendritic Cells Induce the Hepatitis C Viral Epitope-Specific Responses of Naïve Human T Cells

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8+) and class II-restricted (CD4+) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells