RESUMO
The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.
Assuntos
Glutationa Peroxidase/genética , Estresse Oxidativo/genética , Fator 1 de Elongação de Peptídeos/ultraestrutura , Phanerochaete/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos/genética , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Glutationa/genética , Dissulfeto de Glutationa/genética , Glutationa Peroxidase/ultraestrutura , Glutationa Transferase/genética , Humanos , Camundongos , Fator 1 de Elongação de Peptídeos/genética , Phanerochaete/ultraestrutura , Príons/ultraestrutura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Fatores de Transcrição/ultraestruturaRESUMO
Construction of catalytic centers on natural protein aggregates is a challenging topic in biomaterial and biomedicine research. Here we report a novel construction of artificial nanoenzyme with glutathione peroxidase (GPx)-like function. By engineering the surface of tobacco mosaic virus (TMV) coat protein, the main catalytic components of GPx were fabricated on TMV protein monomers. Through direct self-assembly of the functionalized viral coat proteins, the multi-GPx centers were installed on these well-defined nanodisks or nanotubes. With the help of muti-selenoenzyme centers, the resulting organized nanoenzyme exhibited remarkable GPx activity, even approaching the level of natural GPx. The antioxidation study on subcell mitochondrial level demonstrated that virus-based nanoenzyme exerted excellent capacity for protecting cell from oxidative damage. This strategy represents a new way to develop artificial nanoenzymes.
Assuntos
Proteínas do Capsídeo/química , Glutationa Peroxidase/química , Mimetismo Molecular , Nanoestruturas/química , Vírus do Mosaico do Tabaco/química , Proteínas do Capsídeo/ultraestrutura , Glutationa Peroxidase/ultraestrutura , Teste de Materiais , Nanoestruturas/ultraestrutura , Tamanho da PartículaRESUMO
The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 microM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.