Methods for Studying Iron Regulatory Protein 1: An Important Protein in Human Iron Metabolism

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are two cytosolic proteins that maintain cellular iron homeostasis by regulating the expression of genes involved in iron metabolism. IRPs respond to cellular iron deficiency by binding to iron-responsive elements (IREs) found in the mRNAs of iron metabolism transcripts, enhancing iron import, and reducing iron storage, utilization, and export. IRP1, a bifunctional protein, exists in equilibrium between a [Fe₄S₄] cluster containing cytosolic aconitase, and an apoprotein that binds to IREs. At high cellular iron levels, this equilibrium is shifted more toward iron-sulfur cluster containing aconitase, whereas IRP2 undergoes proteasomal degradation by an E3 ubiquitin ligase complex that contains an F-box protein, FBXL5. Irp1^-/- mice develop polycythemia and pulmonary hypertension, whereas Irp2^-/- mice develop microcytic anemia and progressive neurodegeneration, indicating that Irp1 has important functions in the erythropoietic and pulmonary systems, and Irp2 has essential roles in supporting erythropoiesis and nervous system functions. Mice lacking both Irp1 and Irp2 die during embryogenesis, suggesting that functions of Irp1 and Irp2 are redundant. In this review, we will focus on the methods for studying IRP1 activities and function in cells and animals.