RESUMO
Glutathione peroxidase (GPx) is an antioxidant protein containing selenium. Owing to the limitations of native GPx, considerable efforts have been made to develop GPx mimics. Here, a short 5-mer peptides (5P) was synthesized and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Enzyme coupled assays were used to evaluate GPx activity. The cell viability and apoptosis of H22 cells were tested, and mice bearing H22 cell-derived tumors were used to determine the effects of 5P on tumor inhibition. In comparison with other enzyme models, 5P provided a suitable substrate with proper catalytic site positions, resulting in enhanced catalytic activity. In our mouse model, 5P showed excellent inhibition of tumor growth and improved immunity. In summary, our findings demonstrated the design and synthesis of the small 5P molecule, which inhibited tumor growth and improved immunity. Notably, 5P could inhibit tumor growth without affecting normal growth. Based on these advantages, the novel mimic may have several clinical applications.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Imunidade Inata/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Oligopeptídeos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antioxidantes/síntese química , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Biocatálise , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Mimetismo Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Especificidade de Órgãos , Fagocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Resultado do Tratamento , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/imunologiaRESUMO
By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp(29)-Sec(52)-Gln(72)). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser(52)/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe(29)/Trp-Val(72)/Gln mutations enhance the reaction rate via functional cooperation with Sec(52). Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr(101) occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr(54), Asn(55), and Gly(56) to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK(a) shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole-dipole interactions among Trp(29)-Sec(52)-Gln(72) lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.
Assuntos
Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Mutação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Anticorpos Catalíticos/química , Anticorpos Catalíticos/genética , Anticorpos Catalíticos/metabolismo , Domínio Catalítico , Glutationa Peroxidase/genética , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Anticorpos de Cadeia Única/genética , TermodinâmicaRESUMO
BACKGROUND: The generation of harmful reactive oxygen species (ROS) induced by UVB irradiation could induce cell apoptosis and change the cell cycle. 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-ß-cyclodextrin (6-CySeCD) is a novel glutathione peroxidase (GPx; EC 1.11.1.9) mimic. The aim of this study was to investigate the anti-oxidative effects of 6-CySeCD in cultured immortalised human keratinocyte cells (HaCaT). METHODS: HaCaT cells were treated with 30 mJ/cm(2) UVB to establish a damage model. The cultured HaCaT cells were randomly assigned to the control, UVB and treatment groups. The treatment group was incubated with 20 µmol/L of GPx mimics before UVB irradiation. Cell viability was detected by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the level of lipid peroxidation was determined by the formation of malondialdehyde (MDA), DNA fragmentation was observed using agarose gel electrophoresis and the levels of intracellular ROS and cell cycle progression were measured by flow cytometry. RESULTS: The levels of cytotoxicity, intracellular ROS, lipid peroxidation and oxidative DNA damage significantly increased after UVB irradiation in the HaCaT cells. UVB irradiation caused pre-G1 -phase arrest in HaCaT cells and significantly reduced the number of HaCaT cells in the S phase. The GPx mimics 6-CySeCD and 2-phenyl-l,2-benzisoselenazol-3(2H)-one (ebselen) significantly blocked UVB-induced apoptosis and changed the cell cycle of the HaCaT cells. The blocked effect of pretreatment 6-CySeCD in UVB-irradiated HaCaT cells was better than that of pretreatment with ebselen. CONCLUSION: 6-CySeCD can relieve the damage induced by UVB irradiation in HaCaT cells.
Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Compostos Organosselênicos/farmacologia , Protetores contra Radiação/farmacologia , beta-Ciclodextrinas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Azóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Isoindóis , Queratinócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiaçãoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor activity via membrane receptors on cancer cells without deleterious side effects for normal tissue. Unfortunately, breast cancer cells, as many other cancer types, develop resistance to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. 2-Tellurium-bridged ß-cyclodextrin (2-TeCD) is a synthetic organotellurium compound, with both glutathione peroxidase-like catalytic ability and thioredoxin reductase inhibitor activity. In the present study, we reported that 2-TeCD sensitized TRAIL-resistant human breast cancer cells and xenograft tumors to undergo apoptosis. In vitro, 2-TeCD efficiently sensitized MDA-MB-468 and T47D cells, but not untransformed human mammary epithelial cells, to TRAIL-mediated apoptosis, as evidenced by enhanced caspase activity and poly (adenosine diphosphate-ribose) polymerase cleavage. From a mechanistic standpoint, we showed that 2-TeCD treatment of breast cancer cells significantly upregulated the messenger RNA and protein levels of TRAIL receptor, death receptor (DR) 5, in a transcription factor Sp1-dependent manner. 2-TeCD treatment also suppressed TRAIL-induced nuclear factor-κB (NF-κB) prosurvival pathways by preventing cytosolic IκBα degradation, as well as p65 nuclear translocation. Consequently, the combined administration suppressed anti-apoptotic molecules that are transcriptionally regulated by NF-κB. In vivo, 2-TeCD and TRAIL were well tolerated in mice and their combination significantly inhibited growth of MDA-MB-468 xenografts and promoted apoptosis. Upregulation of DR5 and downregulation of NF-κB by the dual treatment were also observed in tumor tissues. Overall, 2-TeCD sensitizes resistant breast cancer cells to TRAIL-based apoptosis in vitro and in vivo. These findings provide strong evidence for the therapeutic potential of this combination against breast cancers.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Ciclodextrinas/farmacologia , Inibidores Enzimáticos/farmacologia , NF-kappa B/antagonistas & inibidores , Compostos Organometálicos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator de Transcrição Sp1/fisiologiaRESUMO
Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.
Assuntos
Glutationa Peroxidase/química , Peptídeos/química , Superóxido Dismutase/química , Animais , Chlorocebus aethiops , Cobre/química , Glutationa Peroxidase/síntese química , Glutationa Peroxidase/farmacologia , Cinética , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Selênio/química , Superóxido Dismutase/síntese química , Superóxido Dismutase/farmacologia , Células VeroRESUMO
Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.
Assuntos
Glutationa Peroxidase/genética , Glutationa Transferase/química , Proteínas de Helminto/química , Proteínas Recombinantes de Fusão/química , Schistosoma japonicum/enzimologia , Superóxido Dismutase/química , Animais , Glutationa Peroxidase/química , Glutationa Transferase/genética , Proteínas de Helminto/genética , Espécies Reativas de Oxigênio/química , Proteínas Recombinantes de Fusão/genética , Schistosoma japonicum/genética , Superóxido Dismutase/genéticaRESUMO
Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se-scFv-B3 (selenium-containing single-chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv-B3 was carried out. A three-dimensional (3D) structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization (EM) calculations of the antibody-GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni(2+)-immobilized metal affinity chromatography (IMAC), then transformed to selenium-containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se-scFv-B3-A180S was significantly increased compared to the original Se-scFv-B3.
Assuntos
Anticorpos Catalíticos/química , Anticorpos Catalíticos/metabolismo , Glutationa Peroxidase/metabolismo , Região Variável de Imunoglobulina/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Selênio/metabolismo , Sequência de Aminoácidos , Anticorpos Catalíticos/genética , Anticorpos Catalíticos/isolamento & purificação , Sítios de Ligação , Western Blotting , Células Clonais , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Glutationa/química , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Humanos , Ligação de Hidrogênio , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Selenocisteína/metabolismo , Alinhamento de Sequência , TermodinâmicaRESUMO
A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.
Assuntos
Cisteína/análogos & derivados , Glutationa Peroxidase/metabolismo , Glutationa Transferase/química , Catálise , Cisteína/síntese química , Cisteína/química , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/química , OxirreduçãoRESUMO
Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.
Assuntos
Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Selênio/metabolismo , Catálise/efeitos dos fármacos , Humanos , Cinética , Serina/metabolismo , Análise Espectral , Compostos de Tosil/farmacologiaRESUMO
For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.
Assuntos
Glutationa Peroxidase/metabolismo , Mimetismo Molecular , Selênio/metabolismo , Sítios de Ligação , Catálise , Eletroforese em Gel de Poliacrilamida , Especificidade por SubstratoRESUMO
S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.
Assuntos
Metionina Adenosiltransferase/metabolismo , Pichia/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Catálise , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Cinética , Metionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , TemperaturaRESUMO
A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.
Assuntos
Cloro/química , Ciclodextrinas/química , Ciclodextrinas/metabolismo , Glutationa Peroxidase/metabolismo , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Selênio/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , Animais , Catálise , Bovinos , Ciclodextrinas/síntese química , Cinética , Mitocôndrias Cardíacas/metabolismo , Estrutura Molecular , Compostos Organosselênicos/síntese química , Estresse Oxidativo , beta-Ciclodextrinas/síntese químicaRESUMO
Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged beta-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/mumol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H(2)O(2) contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.
Assuntos
Antioxidantes/farmacologia , Células NIH 3T3/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Estresse Oxidativo , beta-Ciclodextrinas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Células NIH 3T3/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Protetores contra Radiação/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.
Assuntos
Anticorpos/metabolismo , Perfilação da Expressão Gênica/métodos , Região Variável de Imunoglobulina/metabolismo , Biblioteca de Peptídeos , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Animais , Anticorpos/química , Biomarcadores/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/imunologia , Colífagos/imunologia , Escherichia coli , Humanos , Camundongos , Ligação Proteica , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.
Assuntos
Glutationa Peroxidase/metabolismo , Glutationa Transferase/química , Glutationa/metabolismo , Engenharia de Proteínas/métodos , Selênio/metabolismo , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Catálise , Glutationa Peroxidase/síntese química , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/química , Cinética , Oxirredução , Selênio/química , Serina/química , Especificidade por SubstratoRESUMO
Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.
Assuntos
Antioxidantes/metabolismo , Dípteros/enzimologia , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Selenocisteína/metabolismo , Animais , Sítios de Ligação , Catálise , Cinética , Selênio/metabolismoRESUMO
The antioxidant activity of a novel artificial glutathione peroxidase-like enzyme, selenium-containing glutathione 5-transferase from Lucilia cuprina (seleno-LuGST1-1), was studied by using a ferrous sulfate/ascorbate-induced mitochondrial damage model system. Swelling of mitochondria, lipid peroxidation, and cytochrome-c oxidase activity were selected to evaluate the preservation of mitochondrial integrity in this system. Seleno-LuGST1-1 could effectively protect the mitochondria against oxidative damage in a dose-dependent manner and exhibited both higher catalytic activity and greater antioxidant ability than the classic mimic, 2-phenyl-1,2-benziososelenazol-3(2H)-one (Ebselen). This novel artificial biocatalyst therefore may have great potential for pharmacologic application in the treatment of reactive oxygen species-related diseases.
Assuntos
Antioxidantes/farmacologia , Glutationa Transferase/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Selenoproteínas/farmacologia , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacosRESUMO
Glutathione peroxidase (GPX) is a well-known selenoenzyme that functions as an antioxidant and catalyzes the reduction of harmful peroxide by glutathione and protects cells against oxidative damage. Because many diseases are related to oxidative stress, GPX is an ancient foe of many diseases. Antioxidants are very useful for biological bodies, and considerable effort has been spent to find compounds that could imitate the properties of GPX. This paper reviews GPX mimics developed so far and describes a new, more effective strategy for fabricating them. Although many GPX mimics have been made, they possess serious disadvantages: low activity, low solubility in water, and, in some cases, toxicity. In order to overcome these drawbacks, we have proposed a new strategy of imitating GPX. First, a receptor with a substrate binding site is generated. Next, a catalytic group is incorporated into the receptor near the substrate binding site, allowing the catalytic group access to the functional group of the substrate. Finally, a highly efficient enzyme mimic is obtained. Using this strategy, we successfully fabricated GPX mimics that use antibodies, cyclodextrins, some enzymes and proteins as receptors and chemical modification to incorporate the catalytic group, selenocysteine (Sec). The general principle of combining a functional group involved in catalysis with a specific binding site for the substrate is an approach that could be applied to the generation of other efficient semisynthetic biocatalysts. We describe the antioxidant activities of these GPX mimics and the reasons of their being promising candidates for medicinal applications.
Assuntos
Glutationa Peroxidase/química , Mimetismo Molecular , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/química , Animais , Sítios de Ligação , Catálise , Células Cultivadas , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/farmacologia , Glutationa Peroxidase/fisiologia , Humanos , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade por SubstratoRESUMO
Cell proliferation and cell death of keratinocytes are tightly regulated to ensure epidermal homeostasis. UV-B induces keratinocyte apoptosis. UV-B also induces lipid peroxidation of keratinocytes to increase their amount of malondialdehyde (MDA). These phenomena can be explained by the production of reactive oxygen species (ROS) induced by UV-B radiation. We synthesized 2-selenium-bridged beta-cyclodextrin (2-SeCD) to imitate glutathione peroxidase (GPX), an important antioxidant and established a damage system, in which keratinocytes can be damaged by Ultraviolet B (UV-B) radiation. Using this damage system we studied 2-SeCD protection of keratinocytes against injury induced by UV-B. Experimental results showed that 2-SeCD could protect keratinocytes from apoptosis. Moreover, 2-SeCD inhibits lipid peroxidation of keratinocytes and scavenges ROS. 2-SeCD inhibits the UV-B induced apoptotic signal transduction. This antiapoptotic mechanism may be partly related to the elimination of hydrogen peroxide.