Iron regulatory protein-2 (IRP-2) is an RNA-binding protein that regulates intracellular iron homeostasis at the translational level in mammalian cells. Previous work established that a 73-amino acid motif regulates IRP-2 levels through an iron-dependent protein degradation. This oxygen-dependent process probably depends on the interaction of iron with multiple sulfur-containing amino acids in the motif. Recombinant peptide, including the motif, has been successfully expressed in E. coli and shown to be sensitive to oxidative modification by an iron-dependent mechanism, thus providing a model of this functional region of the protein. In addition, the chemical nature of the modified residue in the peptide was determined. Based on the above results, 10 IRP-2 mutants have been constructed, their sequences confirmed, and transfected in human 293 Tet-on cell lines to elucidate the site and chemical nature of the modified residue in the full-length IRP-2.
