DIFFERENCES IN THE REGULATION OF IRON REGULATORY PROTEIN-1 (IRP-1) BYEXTRACELLULAR AND INTRACELLULAR OXIDATIVE STRESS

We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROI s). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron met abolism following its activation by iron deficiency. nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respirator y chain (Hentze, M. W., and Kuhn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 mu m H2O2 suffice for complet e IRP-1 activation within 60 min when H2O2 is generated extracellularl y at steady-state. By contrast, rapid cellular H2O2 degradation necess itates a 5-10-fold higher bollus dose. To study IRP-1 responses to int racellular oxidative stress, mitochondrial respiration was inhibited w ith antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H2O2 degradation); in parallel, 2',7'-dichloradihydrofluoresc ein diacetate was used as a redox-sensitive probe to monitor intracell ular H2O2 levels by fluorescence-activated cell sorting. Catalase inhi bition elevates intracellular H2O2, but surprisingly does not cause co ncomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase i n detectable intracellular H2O2. IRP-1 is thus activated both by extra - and intracellular generation of ROIs. While extracellular H2O2 rapid ly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activat ion. IRP-1 thus represents a novel example of are H2O2-regulated prote in that responds differentially to alterations of extra- and intracell ular H2O2 levels. Our data also suggest that a direct attack on the 4F e-4S cluster of IRP-1 by H2O2 (or an H2O2-derived reactive species) re presents an unlikely explanation for IRP-1 activation by oxidative str ess.