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1.  A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major 
The FEBS journal  2007;274(21):5643-5658.
Summary
The genome of Leishmania major, the causative agent of cutaneous Leishmaniasis, contains three nearly identical genes encoding putative glutathione peroxidases, which only differ at their N- and C-termini. Since the gene homologues are essential in trypanosomes, they may also represent potential drug targets in leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as electron donor indicating that it is not a bone fide glutathione peroxidase. In contrast, high peroxidase activity was obtained with tryparedoxin indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in formation of an intramolecular disulphide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than to t-butyl hydroperoxide whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 M−1s−1 whereas TryP1 exhibits higher rate constants ~106 M−1s−1. All three TDPX proteins together constitute about 0.05 % of the Leishmania major promastigote protein content whereas the TryPs are ~40 times more abundant. Possible specific functions of TDPXs are discussed.
doi:10.1111/j.1742-4658.2007.06087.x
PMCID: PMC3430366  PMID: 17922848
Glutathione peroxidase; tryparedoxin peroxidase; peroxiredoxin; trypanothione; Leishmania
2.  The Crystal Structures of the Tryparedoxin-Tryparedoxin Peroxidase Couple Unveil the Structural Determinants of Leishmania Detoxification Pathway 
Leishmaniasis is a neglected disease caused by Leishmania, an intracellular protozoan parasite which possesses a unique thiol metabolism based on trypanothione. Trypanothione is used as a source of electrons by the tryparedoxin/tryparedoxin peroxidase system (TXN/TXNPx) to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is not only unique to the parasite but is also essential for its survival; therefore, it constitutes a most attractive drug target. Several forms of TXNPx, with very high sequence identity to one another, have been found in Leishmania strains, one of which has been used as a component of a potential anti-leishmanial polyprotein vaccine. The structures of cytosolic TXN and TXNPx from L. major (LmTXN and LmTXNPx) offer a unique opportunity to study peroxide reduction in Leishmania parasites at a molecular level, and may provide new tools for multienzyme inhibition-based drug discovery. Structural analyses bring out key structural features to elucidate LmTXN and LmTXNPx function. LmTXN displays an unusual N-terminal α-helix which allows the formation of a stable domain-swapped dimer. In LmTXNPx, crystallized in reducing condition, both the locally unfolded (LU) and fully folded (FF) conformations, typical of the oxidized and reduced protein respectively, are populated. The structural analysis presented here points to a high flexibility of the loop that includes the peroxidatic cysteine which facilitates Cys52 to form an inter-chain disulfide bond with the resolving cysteine (Cys173), thereby preventing over-oxidation which would inactivate the enzyme. Analysis of the electrostatic surface potentials of both LmTXN and LmTXNPx unveils the structural elements at the basis of functionally relevant interaction between the two proteins. Finally, the structural analysis of TXNPx allows us to identify the position of the epitopes that make the protein antigenic and therefore potentially suitable to be used in an anti-leishmanial polyprotein vaccine.
Author Summary
Leishmania spp. are protozoa responsible for Leishmaniases, neglected diseases killing up to 60,000 people every year. Current therapies rely mainly on antimonial drugs that are inadequate due to poor drug efficacy and safety, combined with increasing drug resistance. To overcome these problems, there is an urgent need to find new and more affordable drugs. Leishmania reduces the hydrogen peroxide produced by macrophages during the infection by means of the tryparedoxin/tryparedoxin peroxidase couple. The two enzymes are potentially suitable drug targets since they are both necessary for parasite survival and absent in the human host. To understand the molecular basis of peroxide reduction in the Leishmania parasites, we have solved the X-ray crystal structures of both enzymes. Structural analyses highlight oligomerization of the two proteins and allow the regions responsible for their interaction to be identified. Moreover, based on the X-ray structures and on electronic microscopy data present in literature for the homologous proteins from Trypanosoma brucei, we have generated a model of interaction between tryparedoxin and tryparedoxin peroxidase from L. major. From the X-ray structure and from this model, we have identified the epitopes of tryparedoxin peroxidase, which is part of a potential threecomponent vaccine that is presently being studied in animal models and in human.
doi:10.1371/journal.pntd.0001781
PMCID: PMC3424247  PMID: 22928053
3.  Glutathione Peroxidase-1 in Health and Disease: From Molecular Mechanisms to Therapeutic Opportunities 
Antioxidants & Redox Signaling  2011;15(7):1957-1997.
Abstract
Reactive oxygen species, such as superoxide and hydrogen peroxide, are generated in all cells by mitochondrial and enzymatic sources. Left unchecked, these reactive species can cause oxidative damage to DNA, proteins, and membrane lipids. Glutathione peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Certain reactive oxygen species, such as hydrogen peroxide, are also essential for growth factor-mediated signal transduction, mitochondrial function, and maintenance of normal thiol redox-balance. Thus, by limiting hydrogen peroxide accumulation, GPx-1 also modulates these processes. This review explores the molecular mechanisms involved in regulating the expression and function of GPx-1, with an emphasis on the role of GPx-1 in modulating cellular oxidant stress and redox-mediated responses. As a selenocysteine-containing enzyme, GPx-1 expression is subject to unique forms of regulation involving the trace mineral selenium and selenocysteine incorporation during translation. In addition, GPx-1 has been implicated in the development and prevention of many common and complex diseases, including cancer and cardiovascular disease. This review discusses the role of GPx-1 in these diseases and speculates on potential future therapies to harness the beneficial effects of this ubiquitous antioxidant enzyme. Antioxid. Redox Signal. 15, 1957–1997.
I. Introduction
II. GPx‐1 Activity
A. Enzymatic mechanisms of GPx
B. Structure and function: analysis of the active site
C. Inhibitors of GPx
D. Comparison among mammalian GPxs 1–4
III. Regulation of GPx‐1 Expression and Activity
A. Transcriptional regulation
B. Post‐transcriptional and translational regulation
1. Basic mechanisms of Sec incorporation
2. Selenium, nonsense‐mediated decay of GPx‐1 mRNA, and translational repression
3. Post‐transcriptional upregulation of GPx‐1
4. Inhibition of GPx‐1 translation
C. Post‐translational regulation
1. Sec oxidation
2. Stimulation by signal transduction and/or protein–protein interactions
IV. GPx‐1 and Oxidant‐Dependent Cellular Processes
A. Oxidative damage and cell death, apoptosis, and injury
1. Role of oxidants in cell death and apoptosis
2. Role of GPx‐1 in cell death and apoptosis
3. GPx‐1 and response to in vivo ROS
B. Redox‐dependent cell signaling, growth, and survival
V. GPx‐1 and Cancer
A. GPx‐1 and the mechanisms of cancer susceptibility
B. GPx‐1 and genetic polymorphisms
C. GPx‐1: genetic polymorphisms and cancer risk
1. Breast cancer
2. Lung cancer
3. Prostate cancer
4. Bladder cancer
5. Other cancers
VI. GPx‐1, Diabetes, and Cardiovascular Disease
A. GPx‐1 and the mechanisms of susceptibility to diabetes and cardiovascular disease
1. Diabetes mellitus
2. Cardiac dysfunction and toxicity
3. Ischemia/reperfusion injury, angiogenesis, and EPC function
4. Endothelial dysfunction and vascular tone
5. Inflammation and atherogenesis
B. Epidemiologic and genetic studies of GPx‐1 and cardiovascular disease
VII. GPx‐1 and Future Directions for Therapeutic Applications
doi:10.1089/ars.2010.3586
PMCID: PMC3159114  PMID: 21087145
4.  Cytosolic Peroxidases Protect the Lysosome of Bloodstream African Trypanosomes from Iron-Mediated Membrane Damage 
PLoS Pathogens  2014;10(4):e1004075.
African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I–II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I–II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I–II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.
Author Summary
In many cell types, mitochondria are the main source of intracellular reactive oxygen species but iron-induced oxidative lysosomal damage has been described as well. African trypanosomes are the causative agents of human sleeping sickness and the cattle disease Nagana. The parasites are obligate extracellular pathogens that multiply in the bloodstream and body fluids of their mammalian hosts and as procyclic forms in their insect vector, the tsetse fly. Bloodstream Trypanosoma brucei in which the genes for cytosolic lipid hydroperoxide-detoxifying peroxidases have been knocked out undergo an extremely rapid membrane peroxidation and lyse within less than two hours when they are cultured without an exogenous antioxidant. Here we show that the primary site of intracellular damage is the single terminal lysosome of the parasites. Disintegration of the lysosome clearly precedes damage of the mitochondrion and parasite death. Iron, acquired by the endocytosis of iron-loaded host transferrin, induces cell lysis. Contrary to the cytosolic enzymes, the respective mitochondrial peroxidase is dispensable for both in vitro proliferation and mouse infectivity. This is the first report demonstrating that cytosolic thiol peroxidases are responsible for protecting the lysosome of a cell.
doi:10.1371/journal.ppat.1004075
PMCID: PMC3983053  PMID: 24722489
5.  Antitumor Quinol PMX464 Is a Cytocidal Anti-trypanosomal Inhibitor Targeting Trypanothione Metabolism* 
The Journal of Biological Chemistry  2011;286(10):8523-8533.
Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class (“quinols”) and identified several with potent trypanocidal activity (EC50 < 100 nm). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)2), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)2, TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and l-cysteine bound in a 2:1 ratio with PMX464 with Kd values of 6 and 27 μm, respectively, whereas T(SH)2 bound more tightly in a 1:1 ratio with a Kd value of 430 nm. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)2 is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.
doi:10.1074/jbc.M110.214833
PMCID: PMC3048735  PMID: 21212280
Drug Action; Metabolism; Peroxidase; Thiol; Trypanosome; Quinol; Trypanothione; Tryparedoxin Peroxidase
6.  Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite 
The Biochemical journal  2008;410(2):359-368.
There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k = 7.2 × 105 M-1 · s-1) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein-DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.
doi:10.1042/BJ20071138
PMCID: PMC2441817  PMID: 17973627
cytosolic peroxiredoxin; macrophages; mitochondrial peroxiredoxin; peroxynitrite; Trypanosoma cruzi; virulence
7.  Conformational and oligomeric effects on the cysteine pKa of typaredoxin peroxidase 
Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (Cp) supported by a lowered pKa in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that Cp (C52) pKa values are highly affected by oligomeric state; an unshifted Cp pKa (~ 8.3, comparable to the pKa of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the Cps evolve dynamically asymmetric pKas during the simulations; one subunit’s Cp pKa is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same Cp, showing no preference for a lowered pKa in either subunit. Two conserved pathways that communicate the asymmetric pKas between Cps of different subunits can be identified. In the decamer, all the Cp pKas are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent Cp pKa shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the Cp pKa. In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the Cp in dimers relative to the monomers. These side chain and backbone interactions which contribute to pKa modulation indicate the importance of oligomerization to the function of the typical 2-Cys Prxs.
PMCID: PMC2874197  PMID: 20476795
peroxiredoxins; MD simulations; MEAD
8.  Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium 
Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival, and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active-sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24–48 hrs significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.
doi:10.1016/j.aquatox.2012.02.025
PMCID: PMC3660139  PMID: 22446825
GPx4; cadmium; olfactory injury; Coho salmon
9.  Susceptibility of the antioxidant selenoenyzmes thioredoxin reductase and glutathione peroxidase to alkylation-mediated inhibition by anticancer acylfulvenes 
Chemical research in toxicology  2011;24(5):726-736.
Selenium, in the form of selenocysteine, is a critical component of some major redox-regulating enzymes, including thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). TrxR has emerged as an anticancer target for drug development due to its elevated expression level in many aggressive human tumors. Acylfulvenes (AFs) are semisynthetic derivatives of the natural product illudin S, and display improved cytotoxic selectivity profiles. AF and illudin S alkylate cellular macromolecules. Compared to AFs, illudin S more readily reacts with thiol-containing small molecules such as cysteine, glutathione and cysteine-containing peptides. However, a previous study indicates the reactivity of AFs and illudin S with glutathione reductase, a thiol-containing enzyme, are inversely correlated with reactivity toward small molecule thiols. In this study, we investigate mechanistic aspects underlying the enzymatic and cellular effects of the AFs and illudin S on thioredoxin reductase. Both AF and HMAF were found to inhibit mammalian TrxR in the low- to sub-micromolar range, but illudin S was significantly less potent. TrxR inhibition by AFs was shown to be irreversible, concentration- and time-dependent, and mediated by alkylation of C-terminus active site Sec/Cys residues. In contrast, neither AFs nor illudin S inhibit Gpx, demonstrating that enzyme structure-specific small molecule interactions have a significant influence over the inherent reactivity of the Sec residue. In human cancer cells, TrxR activity can be inhibited by low micromolar concentrations of all three drugs. Finally, it was demonstrated that preconditioning cells by addition of selenite to the cell culture media results in an enhancement in cell sensitivity towards AFs. These data suggest potential strategies for increasing drug activity by combination treatments that promote selenium enzyme activity.
doi:10.1021/tx2000152
PMCID: PMC3210965  PMID: 21443269
thioredoxin reductase; glutathione peroxidase; illudin S; acylfulvene; redox enzyme inhibition
10.  Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals 
Background
Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown.
Results
We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH)-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx)-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms.
Conclusion
Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic expansion/deletion and exon-intron remodeling. The differentiated enzymatic properties might be acquired by the evolutionary relaxation of selection pressure and/or biochemical adaptation to the acting environments. Our present study would be beneficial to get detailed insights into the complex GPx evolution, and to understand the molecular basis of the specialized physiological implications of this antioxidant system in their respective donor organisms.
doi:10.1186/1471-2148-9-72
PMCID: PMC2679728  PMID: 19344533
11.  Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage 
Biochemical Journal  2011;441(Pt 1):305-316.
Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl−, Br− and SCN− by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×103–5.8×106 M−1·s−1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M−1·s−1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.
doi:10.1042/BJ20101762
PMCID: PMC3242511  PMID: 21892922
eosinophil peroxidase; glutathione peroxidase; hypothiocyanous acid (HOSCN); myeloperoxidase (MPO); selenium; thiocyanate; thioredoxin reductase; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; Fmoc, fluoren-9-ylmethoxycarbonyl; GPx, glutathione peroxidase; GR, glutathione reductase; LPO, lactoperoxidase; MetSeO, methionine selenoxide; MPO, myeloperoxidase; RBC, red blood cell; Sec, selenocysteine; SeMet, selenomethionine; t-BOOH, t-butyl hydroperoxide; TNB, 5-thio-2-nitrobenzoic acid; TrxR, thioredoxin reductase; UPLC, ultra-performance liquid chromatography
12.  Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein¶ 
Free radical biology & medicine  2011;51(1):197-204.
Se-dependent glutathione peroxidase-1 (GPX1) and Cu,Zn-superoxide dismutase (SOD1) are two major intracellular antioxidant enzymes. This study was to elucidate biochemical mechanisms for the 40% loss of hepatic GPX1 activity in SOD1−/− mice. Compared with the wild-type (WT), the SOD1−/− mice showed no change in the total amount of GPX1 protein. However, their total enzyme protein exhibited a 31 and 38% decrease (P < 0.05) in the apparent kcat for hydrogen peroxide and tert-butyl peroxide (at 2 mM GSH), respectively. Most striking, mass spectrometry revealed two chemical forms of the 47th residue of GPX1: the projected native selenocysteine (Sec) and the Se-lost dehydroalanine (DHA). The hepatic GPX1 protein of the SOD1−/− mice contained 38% less Sec and 77% more DHA than that of WT, respectively, and showed aggravated dissociation of the tetramer structure. In conclusion, knockout of SOD1 elevated the conversion of Sec to DHA in the active site of hepatic GPX1, leading to proportional decreases in the apparent kcat and activity of the enzyme protein as a whole. Our data reveal a structural and kinetic mechanism for the in vivo functional dependence of GPX1 on SOD1 in mammals, and provide a novel mass spectrometric method for the assay of oxidative modification of the GPX1 protein.
doi:10.1016/j.freeradbiomed.2011.03.018
PMCID: PMC3109192  PMID: 21420488
Dehydroalanine; glutathione peroxidase; liver; mass spectrometry; mouse; selenocysteine; superoxide dismutase
13.  Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity 
PLoS ONE  2010;5(9):e12607.
Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfur-containing peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently.
doi:10.1371/journal.pone.0012607
PMCID: PMC2935891  PMID: 20838623
14.  Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA 
Molecular and Cellular Biology  1998;18(5):2932-2939.
The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an ∼50-fold reduction in Se-GPx1 activity and an ∼20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.
PMCID: PMC110672  PMID: 9566912
15.  Unglycosylated recombinant human glutathione peroxidase 3 mutant from Escherichia coli is active as a monomer 
Scientific Reports  2014;4:6698.
Glutathione peroxidase 3 (GPx3) is a glycosylated member of GPx family and can catalyze the reaction of different types of peroxides with GSH to form their corresponding alcohols in vitro. The active center of GPx3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. In this study, we prepared a recombinant human GPx3 (rhGPx3) mutant with all Cys changed to Ser from a Cys auxotrophic strain of E. coli, BL21(DE3)cys. Although lacking post-translational modification, rhGPx3 mutant still retained the ability to reduce H2O2 and PLPC-OOH. Study on the quaternary structure suggested that rhGPx3 mutant existed as a monomer in solution, which is different from native tetrameric GPx3. Loss of the catalytic activity was considered to be attributed to both the absence of glycosylation and the failure of the tetramer. Further analysis was performed to compare the structures of rhGPx3 and GPx4 mutant, which were quite similar except for oligomerization loop. The differences of amino acid composition and electrostatic potentials on the oligomerization loop may affect the binding of large substrates to rhGPx3 mutant. This research provides an important foundation for biosynthesis of functionally selenium-containing GPx3 mutant in E.coli.
doi:10.1038/srep06698
PMCID: PMC4204031  PMID: 25331785
16.  Pulmonary effects of short term selenium deficiency. 
Thorax  1996;51(5):479-483.
BACKGROUND: Selenium dependent glutathione peroxidase (GPx) reduces hydrogen peroxide (H2O2) and organic hydrogen peroxides in both normal and pathological states. Chronic dietary deficiency of selenium results in a gradual decrease in GPx and altered response to environmental stress. However, glutathione-S-transferase (GST) isozymes may increase and compensate for chronic GPx deficiency. The pattern of antioxidant enzyme activity and immunolocalisation of various enzymes in rat lung has not been described in short term (< 3 weeks) acute selenium deficiency. METHODS: The time course of GPx depletion from rat lung (measured every five days in subgroups of rats) during acute dietary selenium deficiency was evaluated. After 20 days of depletion, enzyme activity of lung GPx, catalase, superoxide dismutase (SOD), glutathione reductase (GR), glucose-6-phosphodiesterase (G-6-PD), and GST were determined. Immunohistochemical localisation of GPx and SOD was also performed. The response to lethal hyperoxia (> 95%) in control and selenium deficient rats was then established. RESULTS: At 20 days, lung GPx activity in the rats fed a selenium deficient diet was one third less than in control animals who received a normal diet, while changes in blood enzymes between control and deficient animals were similar. Other lung enzyme activities remained normal with the exception of cyanide inhibited SOD activity measured in selenium deficient rat lungs which declined to approximately 50% of normal. Immunohistochemical localisation of GPx showed a generalised loss of the enzyme throughout the lung parenchyma with some possible sparing of activity in epithelial cells of the bronchioles. When exposed to lethal hyperoxia, selenium deficient animals were more susceptible than control rats. CONCLUSIONS: This is the earliest time at which dietary selenium deficiency has been shown to produce moderate loss of GPx activity. This change in activity was associated with increased susceptibility to pulmonary oxidant stress. However, the role of decreased SOD activity (presumed to represent copper, zinc SOD), although unexpected, may have been a major contributor to increased damage from hyperoxia. These results emphasise the complex potential interaction of elemental deficiency with the natural antioxidant response to lethal hyperoxia.
Images
PMCID: PMC473591  PMID: 8711674
17.  Glutathione Peroxidase-3 Deficiency Promotes Platelet-dependent Thrombosis in vivo 
Circulation  2011;123(18):1963-1973.
Background
Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk in young individuals.
Methods and Results
We recently developed a genetic mouse model to assess platelet function in hemostasis and thrombosis in the setting of GPx-3 deficiency. GPx-3(−/−) mice showed an attenuated bleeding time compared with wild-type mice. Platelet aggregation studies revealed an enhanced aggregation response to the agonist ADP in GPx-3(−/−) compared to wild-type mice. We also found an increase in the plasma levels of soluble P-selectin and a decrease in plasma cyclic GMP in GPx-3(−/−) mice compared with wild-type mice. ADP was infused into the right ventricle of mice to induce platelet aggregation in the pulmonary vasculature, and produced a more robust platelet activation response in the GPx-3(−/−) mice than in wild-type mice; histological sections from the pulmonary vasculature of GPx-3(−/−) compared with wild-type mice show increased platelet-rich thrombi and a higher percentage of occluded vessels. Endothelial function studies using a cremaster muscle preparation revealed dysfunction in the GPx-3(−/−) compared to wild-type mice. Using a no-flow ischemia-reperfusion stroke model, GPx-3(−/−) mice had significantly larger cerebral infarctions compared with wild-type mice. To investigate the effect of platelet inhibition on stroke size in GPx-3 deficiency, we found that clopidogrel treatment reduced stroke size significantly in GPx-3(−/−) mice compared with vehicle-treated controls. To assess the neuroprotective role of antioxidants in this model, we found that MnTBAP treatment reduced stroke size in GPx-3(−/−) mice compared with vehicle-treated controls.
Conclusions
These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.
doi:10.1161/CIRCULATIONAHA.110.000034
PMCID: PMC3107543  PMID: 21518981
Glutathione peroxidase-3; GPx-3; antioxidant; reactive oxygen species; platelet-dependent thrombosis; stroke
18.  Selenium supplementation improves antioxidant capacity in vitro and in vivo in patients with coronary artery disease: The SElenium Therapy in Coronary Artery disease Patients (SETCAP) Study 
American heart journal  2008;156(6):1201.e1-1201.11.
Background
Selenium is a central determinant of antioxidative glutathione peroxidase 1 (GPx-1) expression and activity. The relevance of selenium supplementation on GPx-1 in coronary artery disease (CAD) needs to be established. We assessed the effect of selenium supplementation on GPx-1 in cell culture and on endothelial function in a prospective clinical trial.
Methods
Human coronary artery endothelial cells were incubated with 5.78 to 578 nmol/L sodium selenite, Se-methyl-selenocysteine hydrochloride, or seleno-L-methionine. Glutathione peroxidase 1 mRNA and protein expression and activity were measured. Coronary artery disease patients (n = 465) with impaired endothelial function (flow-mediated dilation [FMD] <8%) were randomly assigned to receive 200 or 500 μg sodium selenite daily or matching placebo during a 12-week period. We tested the effect on red blood cell GPx-1 activity and brachial artery FMD. Furthermore, differences in biomarkers of oxidative stress and inflammation were measured.
Results
Sodium selenite and Se-methyl-selenocysteine hydrochloride increased GPx-1 protein and activity in a dose-dependent manner (P< .0001). The intention-to-treat groups comprised 433 CAD patients. Glutathione peroxidase 1 activity increased from 37.0 U/gHb (31.3–41.7) to 41.1 U/gHb (35.2–48.4) (P < .0001) in the 200 μg and from 38.1 U/gHb (33.2–43.8) to 42.6 U/gHb (35.0–49.1) (P< .0001) in the 500 μg sodium selenite group treated for 12-weeks. No relevant changes were observed for FMD or biomarkers of oxidative stress and inflammation.
Conclusions
Sodium selenite supplementation increases GPx-1 activity in endothelial cells and in CAD patients. Future studies have to demonstrate whether long-term CAD outcome can be improved.
doi:10.1016/j.ahj.2008.09.004
PMCID: PMC3624729  PMID: 19033020
19.  The Roles of Glutathione Peroxidases during Embryo Development 
Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4.
doi:10.3389/fnmol.2011.00012
PMCID: PMC3148772  PMID: 21847368
reactive oxygen species; teratogenesis; anti-oxidative defense; selenium
20.  A mechanistic mathematical model for the catalytic action of glutathione peroxidase 
Free radical research  2014;48(4):487-502.
Glutathione peroxidase (GPx) is a well-known seleno-enzyme that protects cells from oxidative stress (e.g., lipid peroxidation and oxidation of other cellular proteins and macromolecules), by catalyzing the reduction of harmful peroxides (e.g., hydrogen peroxide: H2O2) with reduced glutathione (GSH). However, the catalytic mechanism of GPx kinetics is not well characterized in terms of a mathematical model. We developed here a mechanistic mathematical model of GPx kinetics by considering a unified catalytic scheme and estimated the unknown model parameters based on different experimental data from the literature on the kinetics of the enzyme. The model predictions are consistent with the consensus that GPx operates via a ping-pong mechanism. The unified catalytic scheme proposed here for GPx kinetics clarifies various anomalies, such as what are the individual steps in the catalytic scheme by estimating their associated rate constant values and a plausible rationale for the contradicting experimental results. The developed model presents a unique opportunity to understand the effects of pH and product GSSG on the GPx activity under both physiological and pathophysiological conditions. Although model parameters related to the product GSSG were not identifiable due to lack of product-inhibition data, the preliminary model simulations with the assumed range of parameters show that the inhibition by the product GSSG is negligible, consistent with what is known in the literature. In addition, the model is able to simulate the bi-modal behavior of the GPx activity with respect to pH with the pH-range for maximal GPx activity decreasing significantly as the GSH levels decrease and H2O2 provides a key component for an integrated model of H2O2 levels increase (characteristics of oxidative stress). The model balance under normal and oxidative stress conditions.
doi:10.3109/10715762.2014.886775
PMCID: PMC4068149  PMID: 24456207
enzyme kinetics; glutathione peroxidase; hydrogen peroxide; redox biology; mathematical modeling
21.  Effects of Different Selenium Levels on Gene Expression of a Subset of Selenoproteins and Antioxidative Capacity in Mice 
Biological Trace Element Research  2013;154(2):255-261.
This study aimed to evaluate how excess selenium induces oxidative stress by determining antioxidant enzyme activity and changes in expression of selected selenoproteins in mice. BALB/c mice (n = 20 per group) were fed a diet containing 0.045 (Se-marginal), 0.1 (Se-adequate), 0.4 (Se-supernutrition), or 0.8 (Se-excess) mg Se/kg. Gene expression was quantified in RNA samples extracted from the liver, kidney, and testis by real-time quantitative reverse transcription-polymerase chain reaction. We found that glutathione peroxidase (GPx) and catalase activities decreased in livers of mice fed the marginal or excess dose of Se as compared to those in the Se-adequate group. Additionally, superoxide dismutase and glutathione reductase activities were significantly reduced only in mice fed the excess Se diet, compared to animals on the adequate Se diet. Se-supernutrition had no effect on hepatic mRNA levels of GPx isoforms 1 and 4 (GPx1 and GPx4), down-regulated GPx isoform 3 (GPx3), and upregulated selenoprotein W (SelW) mRNA expression. The excess Se diet led to decreased hepatic mRNA levels of GPx1, GPx3 and GPx4 but no change in testicular mRNA levels of GPx1, GPx3 or SelW. Dietary Se had no effect on testicular mRNA levels of GPx4. Thus, our results suggest that Se exposure can reduce hepatic antioxidant capacity and cause liver dysfunction. Dietary Se was found to differentially regulate mRNA levels of the GPx family or SelW, depending on exposure. Therefore, these genes may play a role in the toxicity associated with Se.
doi:10.1007/s12011-013-9710-z
PMCID: PMC3703305  PMID: 23760574
Selenium; Antioxidant; Overexposure; mRNA
22.  Irreversible Inactivation of Glutathione Peroxidase 1 and Reversible Inactivation of Peroxiredoxin II by H2O2 in Red Blood Cells 
Antioxidants & Redox Signaling  2010;12(11):1235-1246.
Abstract
Catalase, glutathione peroxidase1 (GPx1), and peroxiredoxin (Prx) II are the principal enzymes responsible for peroxide elimination in RBC. We have now evaluated the relative roles of these enzymes by studying inactivation of GPx1 and Prx II in human RBCs. Mass spectrometry revealed that treatment of GPx1 with H2O2 converts the selenocysteine residue at its active site to dehydroalanine (DHA). We developed a blot method for detection of DHA-containing proteins, with which we observed that the amount of DHA-containing GPx1 increases with increasing RBC density, which is correlated with increasing RBC age. Given that the conversion of selenocysteine to DHA is irreversible, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime. Prx II is inactivated by occasional hyperoxidation of its catalytic cysteine to cysteine sulfinic acid during catalysis. We believe that the activity of sulfiredoxin in RBCs is sufficient to counteract the hyperoxidation of Prx II that occurs in the presence of the basal level of H2O2 flux resulting from hemoglobin autoxidation. If the H2O2 flux is increased above the basal level, however, the sulfinic Prx II begins to accumulate. In the presence of an increased H2O2 flux, inhibition of catalase accelerated the accumulation of sulfinic Prx II, indicative of the protective role of catalase. Antioxid. Redox Signal. 12, 1235–1246.
doi:10.1089/ars.2009.2701
PMCID: PMC2875961  PMID: 20070187
23.  High-Resolution Imaging of Selenium in Kidneys: A Localized Selenium Pool Associated with Glutathione Peroxidase 3 
Antioxidants & Redox Signaling  2012;16(3):185-192.
Abstract
Aim: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. Results: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA[Ser]Sec and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. Innovation: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. Conclusion: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution. Antioxid. Redox Signal. 16, 185–192.
doi:10.1089/ars.2011.3997
PMCID: PMC3234661  PMID: 21854231
24.  Functional and physical interaction between the selenium-binding protein 1 (SBP1) and the glutathione peroxidase 1 selenoprotein 
Carcinogenesis  2010;31(8):1360-1366.
Selenium-binding protein (SBP) 1 is present in reduced levels in several cancer types as compared with normal tissues, and lower levels are associated with poor clinical prognosis. Another selenium-containing protein, glutathione peroxidase 1 (GPX1), has been associated with cancer risk and development. The interaction between these representatives of different classes of selenoproteins was investigated. Increasing SBP1 levels in either human colorectal or breast cancer cells by transfection of an expression construct resulted in the reduction of GPX1 enzyme activity. Increased expression of GPX1 in the same cell types resulted in the transcriptional and translational repression of SBP1, as evidenced by the reduction of SBP1 messenger RNA and protein and the inhibition of transcription measured using an SBP1 reporter construct. The opposing effects of SBP1 and GPX1 on each other were also observed when GPX1 was increased by supplementing the media of these tissue culture cells with selenium, and the effect of selenium on SBP1 was shown to be GPX1 dependent. Decreasing or increasing GPX1 levels in colonic epithelial cells of mice fed a selenium-deficient, -adequate or -supplemented diet resulted in the opposing effect on SBP1 levels. These data are explained in part by the demonstration that SBP1 and GPX1 form a physical association, as determined by coimmunoprecipitation and fluorescence resonance energy transfer assay. The results presented establish an interaction between two distinct selenium-containing proteins that may enhance the understanding of the mechanisms by which selenium and selenoproteins affect carcinogenesis in humans.
doi:10.1093/carcin/bgq114
PMCID: PMC2915633  PMID: 20530237
25.  Polymorphonuclear Leukocyte Bactericidal Activity and Oxidative Metabolism During Glutathione Peroxidase Deficiency 
Infection and Immunity  1977;18(1):78-84.
Glutathione peroxidase (GPx) deficiency has been proposed as a cause of some instances of chronic granulomatous disease (CGD). GPx activity varies greatly among species, and specific deficiency of this selenium-dependent enzyme can be produced by dietary selenium deficiency in rats. Bactericidal activity of polymorphonuclear (PMN) leukocytes from normal rats, humans, and guinea pigs (GPx high, intermediate, and nearly absent, respectively), selenium-deficient rats (GPx absent), and a patient with CGD were compared. There was no correlation between natural levels of GPx and bactericidal activity; only CGD was associated with inability to kill a Proteus mirabilis strain in vitro (killing known to be dependent on oxidative mechanisms). Postphagocytic metabolism was examined in normal and GPx-deficient rats. Both demonstrated normal iodination and superoxide production during phagocytosis and gave similar histochemical reduction of nitroblue tetrazolium dye under either resting or endotoxin-stimulation conditions. Postphagocytic hexose monophosphate shunt activity was somewhat lower in PMN from GPx-deficient animals as compared with normal but was substantially (10-fold) higher than that observed in resting cells. Thus, postphagocytic oxidative responses and subsequent bactericidal activity of PMN leukocytes were not compromised by complete absence of GPx, even in the species with the highest natural level of this enzyme. These results are not compatible with the hypothesis that CGD can be caused by a deficiency of GPx.
PMCID: PMC421196  PMID: 198376

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