Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions , In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aco nitase, IRE binding activity is restored by cluster loss in response to iro n starvation, NO, or extracellular H2O2. Here, we study the effects of intr acellular quinone-induced oxidative stress on IRP-1. Treatment of murine Be fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, c auses a modest activation of IRP-1 to bind to IREs within 15-30 min. Howeve r, IRE binding drops to basal levels within 60 min. Surprisingly, a remarka ble loss of both IRE binding and aconitase activities of IRP-1 follows trea tment with MSB for 1-2 h, These effects do not result from alterations in I RP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated ce lls to increase transferrin receptor mRNA levels is inhibited, and MSB incr eases the translation of a human growth hormone indicator mRNA bearing an I RE in its 5'-untranslated region, Nonetheless, MSB inhibits ferritin synthe sis. Thus, menadione-induced oxidative stress leads to post-translational i nactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple e ffects on cellular iron metabolism.