RESUMO
Gli3 is a key regulator of development, controlling multiple patterning steps. Here we report the generation of a scFv antibody specific to the repressor domain of human Gli3. We show that this scFv retains the binding capacity of its parent anti-Gli3 monoclonal antibody derived from hybridoma clone 5E1. When expressed in mammalian cells, the anti-Gli3 scFv co-localizes with intracellular Gli3. Immunocytochemical staining of the intrabody in Gli3-positive TM4 cells shows a distinct perinuclear cytoplasmic localization. Such a scFv constitutes a useful tool for studying transcriptional regulation of the hedgehog pathway in mammals and offers a starting point for developing novel Gli-related therapeutic intrabodies.
Assuntos
Proteínas Hedgehog/metabolismo , Hibridomas/metabolismo , Região Variável de Imunoglobulina/química , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Transdução de Sinais , Fatores de Transcrição/química , Proteína Gli3 com Dedos de ZincoRESUMO
Tribbles homolog 3 (TRB3) is a pseudokinase the level of which is increased in response to various stresses. We and other researchers have previously shown that TRB3 interacts with activating transcription factor 4 (ATF4) and may function as a negative feedback regulator of ATF4. In the present study, we investigate the effect of ATF4 and TRB3 on cell growth and viability, using both the enforced expression and silencing of the genes. HEK293 cells overexpressing ATF4 show retarded growth in the complete medium and decreased viability in the glucose-free medium. The enforced expression of ATF4 increases the level of reactive oxygen species (ROS) and the supplementation of the medium with ROS scavenging and reducing compounds supports the growth and survival of cells overexpressing ATF4. The deleterious effects of elevated ATF4 are suppressed by the coexpression of TRB3, which downregulates ATF4 transcriptional activity and results in the decrease of intracellular ROS. Also, the coexpression of TRB3 rescues postmitotic neuronally differentiated PC12 cells from the apoptosis evoked by ATF4 overexpression. The silencing of ATF4 and TRB3 genes by RNA interference reveals that endogenous ATF4 promotes and TRB3 suppresses the death of glucose-deprived SaOS2 cells. Together, the results indicate that TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4.
