Iron regulatory protein-2 (IRP-2) is a RNA-binding protein that regulates intracellular iron homeostasis at the translational level in mammalian cells. Previous work has found that a 73 amino acid motif (near the end of the N-terminal domain of IRP-2) can regulate IRP-2 levels by conferring a property of iron-dependent degradation to the protein, presumably via a redox mechanism resulting from the interaction of Fe(3+) with multiple sulfur-containing amino acids in the motif. The current investigation intends to find out: (1) whether the 73 amino acid motif can confer a similar property (of iron-dependent degradation) to proteins unrelated to IRP-2; (2) whether ROS can be generated via interacting Fe(3+) with the 73 amino acid motif or the whole N-terminal domain of IRP-2; and (3) how the over-expression of the 73 amino acid motif or the whole N-terminal domain may affect redox-regulated signaling pathways in human RD4 cells. Currently, more than 10 expression and/or cloning vectors have been constructed that would express the 73 amino acid motif, the whole N-terminal domain, and several fusion proteins of green fluorescent protein (GFP) and luciferase containing one of the two IRP-2 segments, respectively. Several constructs have been sequence-confirmed and stably-expressed in RD4 cells, the expression being detected by both GFP fluorescence and Western blot.
