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- Title
- THE EFFECTS OF TRIVALENT ARSENICALS AND THIOREDOXIN REDUCTASE INHIBITORS ON SELENIUM METABOLISM IN LUNG CELL CULTURE MODELS.
- Creator
-
Talbot, Sarah, Self, William, University of Central Florida
- Abstract / Description
-
Arsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the...
Show moreArsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the selenoprotein thioredoxin reductase (TrxR). Selenium, an essential micronutrient in mammals, is needed in the form of selenocysteine for activity of this enzyme and other selenoproteins. TrxR is part of a key component of the cell's ability to defend against ROS. It has been speculated that TrxR is also involved directly in selenium metabolism, but this has yet to be demonstrated in vivo. The promoter region of the gene encoding the cytosolic TrxR (TrxR1) also contains an antioxidant responsive element (ARE). The ARE is activated by the transcription factor, Nrf2, which is governed by the Nrf2/Keap1 response, and can be triggered by certain oxidants. ATO and arsenite both inhibited incorporation of selenium into selenoproteins. Auranofin, a gold chemotherapeutic inhibitor of TrxR1, also inhibited selenoprotein synthesis. These results seem to support the hypothesis that TrxR1 is needed for selenoprotein synthesis. However, siRNA mediated reduction of TrxR1 did not block incorporation of selenium into selenoproteins. It is likely that ATO and auranofin are forming As-Se and Au-Se complexes, respectively. We also found that exposure of primary lung fibroblasts (WI-38) to MMAIII led to increased synthesis of TrxR1. This increase was dependent on the activation of transcription of the TrxR1 gene, specifically mediated through the ARE element. These results indicate exposure to MMAIII induces the Nrf2 response. The results obtained in these studies aid in both our understanding of the carcinogenic potential of arsenic as well as give new insight into the mechanism of action of emerging cancer drugs.
Show less - Date Issued
- 2007
- Identifier
- CFE0001943, ucf:47467
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001943
- Title
- SELENOTRISULFIDE DERIVATIVE OF ALPHA-LIPOIC ACID: EVALUATION IN A CELL CULTURE MODEL FOR POTENTIAL USE AS A TOPICAL ANTIOXIDANT.
- Creator
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Alonis, Melenie, Self, William, University of Central Florida
- Abstract / Description
-
Selenium is a required micronutrient in mammalian cells. It is incorporated in the form of selenocysteine into selenoenzymes such as glutathione peroxidase and thioredoxin reductase, and is absolutely required for activity. Thioredoxin reductase is necessary for reduction of oxidized thioredoxin and therefore plays a major role in maintaining the redox status of the cell. Glutathione peroxidase is responsible for reducing peroxides into their corresponding alcohols and water. Together, these...
Show moreSelenium is a required micronutrient in mammalian cells. It is incorporated in the form of selenocysteine into selenoenzymes such as glutathione peroxidase and thioredoxin reductase, and is absolutely required for activity. Thioredoxin reductase is necessary for reduction of oxidized thioredoxin and therefore plays a major role in maintaining the redox status of the cell. Glutathione peroxidase is responsible for reducing peroxides into their corresponding alcohols and water. Together, these selenoenzymes constitute a significant part of the cell's arsenal to defend itself against oxidative stress. Exogenous sources of oxidative stress, such as UV radiation, are capable of generating reactive oxygen species (ROS). Elevated levels of ROS can lead to covalent modifications of lipids, nucleic acids, and proteins within a cell. This damage has been implicated in the development of cancer and degenerative diseases. As the skin is the first level of defense for UV radiation, skin cancer is an obvious concern. Previous studies have demonstrated a protective effect against UV-induced cytotoxicity when selenium compounds were administered to skin cells in cell culture models. Topical selenium application to mice has also been shown to reduce UV damage to skin. Although a variety of chemical forms of selenium are available in nutritional supplements, the efficiency by which they are used for selenoprotein synthesis varies greatly. It is debated within the selenium research community which form is best for use as a supplement. In this study, we have focused on a selenotrisulfide derivative of alpha-lipoic acid (LASe). We have examined its utilization for selenoprotein synthesis through radiolabeling studies (75Se) in a human keratinocyte cell line (HaCaT). We have determined that is incorporated into selenoproteins with nearly the same efficiency as selenite and L-selenocysteine. We have also determined that LASe is far more efficient as a supplement in cell culture than selenate or L-selenomethionine, two forms of selenium commonly used as supplements. LASe was also found to protect HaCaT keratinocytes from UV- induced cytotoxicity. Cells pretreated with LASe and exposed to 500J/m2 and 750J/m2 of broadband (UVA/UVB) UV radiation showed greater survival than untreated controls in a dose –dependent manner. Cells pre-treated either with lipoic acid or selenium in the form of selenite alone also observed protection. Nonetheless, these finding are significant given that LASe was previously shown to penetrate the skin better than other forms of selenium. These results indicate that LASe has the potential for use as a topical antioxidant upon further testing in animal studies.
Show less - Date Issued
- 2005
- Identifier
- CFE0000663, ucf:46531
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000663
- Title
- BACTERIAL SELENOPROTEINS: A ROLE IN PATHOGENESIS AND TARGETS FOR ANTIMICROBIAL DEVELOPMENT.
- Creator
-
Rosario, Sarah, Self, William, University of Central Florida
- Abstract / Description
-
Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy...
Show moreSelenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.
Show less - Date Issued
- 2009
- Identifier
- CFE0002790, ucf:48139
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002790
- Title
- Selenium vs. Sulfur: Investigating the Substrate Specificity of a Selenocysteine Lyase.
- Creator
-
Johnstone, Michael, Self, William, Roy, Herve, Moore, Sean, University of Central Florida
- Abstract / Description
-
Selenium is a vital micronutrient in many organisms. While traces are required for survival, excess amounts are toxic; thus, selenium can be regarded as a biological (")double-edged sword("). Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation...
Show moreSelenium is a vital micronutrient in many organisms. While traces are required for survival, excess amounts are toxic; thus, selenium can be regarded as a biological (")double-edged sword("). Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. In this work, we add knowledge to the mechanism for selenium-over-sulfur specificity in hopes of further understanding the controlled regulation of selenium trafficking and the prevention of its toxicity. We have identified SclA, a selenocysteine lyase in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for L-selenocysteine over L-cysteine. Human selenocysteine lyase contains a residue, D146, which plays a significant role in determining its specificity. A D146K mutation eliminated this trait, allowing non-specific L-cysteine degradation. Using computational biology, we identified an orthologous residue in SclA, H100, and generated mutant enzymes with site-directed mutagenesis. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying levels of activity towards L-selenocysteine, hinting at a catalytic role for H100. Additionally, L-cysteine acted as a competitive inhibitor towards all enzymes with higher affinity than L-selenocysteine. Finally, our experiments revealed that SclA possessed extremely poor cysteine desulfurase activity with each mutation exhibiting subtle changes in turnover. Our findings offer key insight into the molecular mechanisms behind selenium-over-sulfur specificity and may further elucidate the role of selenocysteine lyases in vivo.
Show less - Date Issued
- 2019
- Identifier
- CFE0007659, ucf:52481
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007659
