RESUMO
Manduca sexta IRP1 was cloned and sequenced. The deduced amino acid sequence of Manduca IRP1 shows high similarity to other IRP1 proteins. The Cys residues required as ligands for the iron sulfur cluster, as well as all residues necessary for aconitase activity are conserved in the insect protein. Purified recombinant Manduca IRP1 binds specifically to transcripts of the iron responsive element (IRE) of Manduca or human ferritin subunit mRNA. Binding activity of the recombinant protein was not influenced by the presence of beta-mercaptoethanol. However, IRP/IRE binding activity of cytoplasmic extracts from fat body was decreased by reducing agents in a dose-responsive manner. Fat body IRP1/IRE binding activity was reduced for Manduca sexta larvae injected with low doses of iron, while IRP1 mRNA and protein levels remained stable. At higher iron doses, binding activity increased and stabilized. Hemolymph ferritin levels showed an inverse relationship to IRP1/IRE binding activity. These data suggest that the Manduca IRP1 is likely involved in translational control of ferritin synthesis in a manner similar to that found in vertebrates. However, factors other than iron can influence IRP/IRE interaction and hemolymph ferritin levels in insects.
Assuntos
Proteínas de Transporte/metabolismo , Ferritinas/genética , Proteínas de Insetos , Proteína 1 Reguladora do Ferro , Ferro/metabolismo , Manduca/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , DNA Complementar , Humanos , Manduca/genética , Dados de Sequência Molecular , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de RespostaAssuntos
Proteínas Ferro-Enxofre/química , Proteínas de Ligação a RNA/química , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Análise de Fourier , Humanos , Ferro/química , Proteínas Reguladoras de Ferro , Proteínas Ferro-Enxofre/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria por Raios X/métodos , Enxofre/químicaRESUMO
Mammalian ferritin subunit synthesis is controlled at the translational level by the iron regulatory protein 1 (IRP1)/iron responsive element (IRE) interaction. Insect haemolymph ferritin subunit messages have an IRE in the 5'-untranslated region (UTR). We have shown that recombinant M. sexta IRP1 represses the in vitro translation of both the heavy and light chain ferritin subunits from this species without altering transcription. Deletion of either the 5'-UTR or the IRE from the mRNA abolishes IRP1 repression. Our studies indicated that the translational control of ferritin synthesis by IRP/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge, this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. Furthermore, this is the first indication that the synthesis of a secreted ferritin subunit can also be controlled in this manner.
