RESUMO
Although the incidence and severity of atopic dermatitis (AD) is steadily increasing at an alarming rate, its pathogenic mechanisms remain poorly understood yet. Recently, we found that the expression of Grb7 protein was markedly decreased in AD patients using proteomic analysis. In the present study, human Grb7 gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-Grb7 fusion protein. The expressed and purified PEP-1-Grb7 fusion proteins transduced efficiently into skin cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-Grb7 protein was stable for 48 h. In addition, transduced PEP-1-Grb7 fusion protein markedly increased cell viability in macrophage RAW 264.7 cells treated with LPS by inhibition of the COX-2 expression level. These results suggest that the PEP-1-Grb7 fusion protein can be used in protein therapy for inflammatory skin disorders, including AD.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Cisteamina/análogos & derivados , Regulação para Baixo , Proteína Adaptadora GRB7/metabolismo , Lipopolissacarídeos/farmacologia , Peptídeos/metabolismo , Transdução Genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Cisteamina/metabolismo , Fibroblastos/efeitos dos fármacos , Proteína Adaptadora GRB7/genética , Vetores Genéticos , Humanos , Camundongos , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The consequences of ultraviolet (UV) exposure are implicated in skin aging and cell death. The ribosomal protein S3 (rpS3) is one of the major proteins by which cells counteract the deleterious effects of UV and it plays a role in the repair of damaged DNA. In the present study, we investigated the protective effects of PEP-1-rpS3 fusion protein after UV-induced cell injury. A human rpS3 gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-rpS3 fusion protein. The expressed and purified fusion proteins were efficiently transduced into skin cells in a time- and dose-dependent manner. Once inside the cells, transduced PEP-1-rpS3 fusion protein was stable for 48h. We showed that transduced PEP-1-rpS3 fusion protein increased cell viability and dramatically reduced DNA lesions in the UV exposed skin cells. Immunohistochemical analysis revealed that PEP-1-rpS3 fusion protein efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin. These results suggest that PEP-1-rpS3 fusion protein can be used in protein therapy for various disorders related to UV, including skin aging and cancer.
