RESUMO
Thioredoxin reductase (TrxR) has been considered a potential target for cancer chemotherapy. It acts by controlling the redox homeostasis of human cells and, therefore, interfering in its function may trigger apoptosis, which is a crucial tumor suppression mechanism. Despite the great effort in the search for TrxR inhibitors, none was approved for human therapy. In the present study a virtual screening for natural organic compounds is discussed for a set of 72 compounds with known IC-50 for TrxR inhibition. The results suggest the Pleurotin, a naphthoquinone obtained from Hohenbuehelia grisea fungus, as a potential TrxR inhibitor, which acts by binding to the active site of the enzyme, between the N- and C-terminal domains. The presence of the ligand blocks the approximation of the C-terminal arm to the N-terminal, which is an essential step of the enzyme function. Besides, the two equivalent binding sites of TrxR were explored, by docking two ligands simultaneously. The results indicate that both sites have an allosteric correlation and, the presence of the ligand in one site may interfere, or even prevent, the binding of the second ligand at the other site. All these findings are quantitatively discussed based on the analysis of long molecular dynamics trajectories, which provides a full description of the ligand-receptor binding modes, average binding energies and conformational changes.Communicated by Ramaswamy H. Sarma.
Assuntos
Simulação de Dinâmica Molecular , Tiorredoxina Dissulfeto Redutase , Humanos , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Ligantes , Inibidores Enzimáticos/química , Simulação de Acoplamento MolecularRESUMO
Excessive production of reactive species in living cells usually has pathological effects. Consequently, the synthesis of compounds which can mimic the activity of antioxidant enzymes has inspired great interest. In this study, a variety of diselenoamino acid derivatives from phenylalanine and valine were tested to determine whether they could be functional mimics of glutathione peroxidase (GPx) and substrates for liver thioredoxin reductase (TrxR). Diselenides C and D showed the best GPx mimicking properties when compared with A and B. We suppose that the catalytic activity of diselenide GPx mimics depends on the steric effects, which can be influenced by the number of carbon atoms between the selenium atom and the amino acid residue and/or by the amino acid lateral residue. Compounds C and D stimulated NADPH oxidation in the presence of partially purified hepatic mammalian TrxR, indicating that they are substrates for TrxR. Our study indicates a possible dissociation between the two pathways for peroxide degradation (i.e., via a substrate for TrxR or via mimicry of GPx) for compounds tested in this study, except for PhSeSePh, and the antioxidant activity of diselenoamino acids can also be attributed to their capacity to mimic GPx and to be a substrate for mammalian TrxR.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Simulação por Computador , Glutationa Peroxidase/metabolismo , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Animais , Domínio Catalítico , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredução , Ratos , Tiorredoxina Dissulfeto Redutase/químicaRESUMO
This paper addresses the identification, cloning, expression, purification and functional characterization of thioredoxin reductase from Babesia bovis, the etiological agent of babesiosis. The work deals with in vitro steady state kinetic studies and other complementary analyses of the thioredoxin reductase found in the pathogenic protist. Thioredoxin reductase from B. bovis was characterized as a homodimeric flavoprotein that catalyzes the NADPH-dependent reduction of Trx with a high catalytic efficiency. Moreover, the enzyme exhibited a disulfide reductase activity using DTNB as substrate, being this activity highly sensitive to inhibition by Eosin B. The thioredoxin reductase/thioredoxin system can reduce oxidized glutathione and S-nitrosoglutathione. Our in vitro data suggest that antioxidant defense in B. bovis could be supported by this enzyme. We have performed an enzymatic characterization, searching for targets for rational design of inhibitors. This work contributes to the better understanding of the redox biochemistry occurring in the parasite.
Assuntos
Babesia bovis/enzimologia , Proteínas de Protozoários/química , Tiorredoxina Dissulfeto Redutase/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antiprotozoários/química , Clonagem Molecular , Sequência Conservada , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fluoresceínas/química , Dissulfeto de Glutationa/química , Canamicina/farmacologia , Cinética , Dados de Sequência Molecular , NADP/química , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/biossíntese , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/biossínteseRESUMO
The antioxidant properties of organoselenium compounds have been extensively investigated because oxidative stress is a hallmark of a variety of chronic human diseases. Here, we reported the influence of substituent groups in the antioxidant activity of ß-selenoamines. We have investigated whether they exhibited glutathione peroxidase-like (GPx-like) activity and whether they could be substrate of thioredoxin reductase (TrxR). In the DPPH assay, the ß-selenium amines did not exhibit antioxidant activity. However, the ß-selenium amines with p-methoxy and tosyl groups prevented the lipid peroxidation. The ß-selenium amine compound with p-methoxy substituent group exhibited thiol-peroxidase-like activity (GPx-like activity) and was reduced by the hepatic TrxR. These results contribute to understand the influence of structural alteration of non-conventional selenium compounds as synthetic mimetic of antioxidant enzymes of mammalian organisms.
Assuntos
Sequestradores de Radicais Livres/química , Compostos Organosselênicos/química , Peroxidases/química , Animais , Compostos de Bifenilo/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Catálise , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/química , Peroxidação de Lipídeos , Fígado/enzimologia , Masculino , NADP/química , Compostos Organosselênicos/farmacologia , Oxirredução , Peroxidases/farmacologia , Picratos/química , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/isolamento & purificaçãoRESUMO
The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M ). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.
Assuntos
Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Dissulfetos/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/química , Flavinas/metabolismo , Cinética , Modelos Moleculares , Mutagênese , NADP/metabolismo , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Tolueno/análogos & derivados , Tolueno/químicaRESUMO
Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen species during tissue invasion. In this work, we report the molecular cloning, from E. histolytica genomic DNA, of the genes ehtrxr and ehtrx41, respectively coding for thioredoxin reductase (EhTRXR) and thioredoxin (EhTRX41). The genes were expressed in Escherichia coli cells, and the corresponding recombinant proteins were purified and characterized. EhTRXR catalyzed the NADPH (Km=4.5 microM)-dependent reduction of 5,5'-dithiobis-(2-nitrobenzoic) acid (Km=1.7 mM), EhTRX41 (Km=3.6 microM), and E. coli TRX (Km=4.6 microM). EhTRXR and EhTRX41 could be assayed as a functional redox pair that, together with peroxiredoxin, mediate the NADPH-dependent reduction of hydrogen peroxide and tert-butyl hydroperoxide. It is proposed that this detoxifying system could be operative in vivo. Results add value to the genome project information and advise reconsideration of key metabolic pathways operating in E. histolytica.
Assuntos
Entamoeba histolytica/metabolismo , Proteínas de Protozoários/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Entamoeba histolytica/genética , Escherichia coli/genética , Genes de Protozoários , Genoma de Protozoário , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , NADP/metabolismo , Oxirredução , Proteínas de Protozoários/genética , Alinhamento de Sequência , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
Thioredoxin reductase 1 (Trr1) from Saccharomyces cerevisiae is a member of the family of pyridine nucleotide-disulfide oxidoreductases capable of reducing the redox-active disulfide bond of the cytosolic thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2). NADPH, Trr1 and Trx1 (or Trx2) comprise the thioredoxin system, which is involved in several biological processes, including the reduction of disulfide bonds and response to oxidative stress. Recombinant Trr1 was expressed in Escherichia coli as a His6-tagged fusion protein and purified by nickel-affinity chromatography. The protein was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3000 as precipitant after treatment with hydrogen peroxide. X-ray diffraction data were collected to a maximum resolution of 2.4 A using a synchrotron-radiation source. The crystal belongs to the centred monoclinic space group C2, with unit-cell parameters a = 127.97, b = 135.41, c = 75.81 A, beta = 89.95 degrees. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.