32] Activation of iron regulatory protein-1 by oxidative stress

Abstract

Publisher Summary Iron regulatory protein 1 (IRP1) posttranscriptionally controls the expression of proteins implicated in iron and energy metabolism. IRE/IRP1 interactions modulate mRNA translation or stability and result in homeostatic adaptations to changes in iron availability. IRP1 belongs to the family of iron–sulfur isomerases. Its genetic activity is regulated by the iron dependent assembly–disassembly of a cubane, aconitase-type [4Fe–4S] cluster. Direct administration of hydrogen peroxide to cells leads to a rapid activation of IRP1 to its IRE-binding form—IRPI activation. This chapter describes the basic methods that have been developed and applied to study the activation of IRPI by hydrogen peroxide. These include the electrophoretic mobility shift assay to detect IRE-binding activity, the chemiluminescence luminol/hypochlorite assay to detect extracellular hydrogen peroxide, the method for enzymatic generation of hydrogen peroxide at steady-state levels, and the fluorometric assay to monitor relative intracellular hydrogen peroxide levels. In addition, the chapter describes key experiments that have provided insights regarding the mechanism and the physiological implications of IRP1 activation by hydrogen peroxide in cultured B6 fibroblasts, in permeabilized B6 fibroblasts, and in the intact rat liver.