Activation of iron regulatory protein-1 by oxidative stress in vitro.
Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-gammaS and GTP-gammaS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.
Pantopoulos
K
K
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Hentze
M W
MW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D051379:Mice; D018384:Oxidative Stress; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015398:Signal Transduction
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D000469:Alkaline Phosphatase; D035941:Iron Regulatory Protein 1