Cloning, overexpression and characterization of iron regulatory proteins from insects

Abstract

Iron is essential for life and iron homeostasis is important for all species. Compared to the understanding of iron metabolisms in vertebrates, we know much less about insect intracellular iron homeostasis. The iron regulatory proteins (IRPs) play central roles in this process by interaction with iron responsive elements (IREs). Here, I report the cloning, sequencing, overexpression, purification and characterization of IRP1s from two insect species, Manduca sexta and Aedes aegypti. Electrophoretic mobility shift assays demonstrated that both IRP1s specifically bind IREs s not only from the same species, but also from human ferritin IRE. Another ferritin subunit also was cloned from Manduca sexta and an IRE was identified in the 5'-untranslated region of the mRNA, and the IRE reacted with Manduca IRP1 specifically. Transcription/translation assays demonstrated that both IRP1s repress ferritin synthesis in vitro, and the repression is IRE dependent. Iron administration to Manduca sexta increased hemolymph ferritin levels and decreased fat body IRP1/IRE binding activities without affecting either the IRP1 mRNA or protein levels. These data indicates that translational control of ferritin synthesis by IRP1/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. The different responses to reducing agent of Manduca sexta and mammalian IRP1s could provide a potential future strategy for designing pesticides in insect control.