Modulation of iron regulatory protein functions. Further insights into the role of nitrogen- and oxygen-derived reactive species.
Iron regulatory protein (IRP) is a cytosolic bifunctional [Fe-S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA. The modulators of these activities are the intracellular concentration of iron and, as recently described, NO synthase activity. In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO-, the product of the reaction between NO and O2-.), as well as oxygen-derived radicals (O2-. and H2O2) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophagelike RAW 264.7 cells. Neither the addition of a bolus of ONOO- or H2O2 nor O2-. generation significantly affected IRE binding even though they inhibited its aconitase activity. Moreover, we show that 3-morpholinosydnonimine (SIN-1), a chemical which releases both NO and O2-. enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus gluthathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O2-. nor ONOO- can convert [4Fe-4S] IRP into IRE-binding protein but rather suggest that an active redox form of NO converts IRP into its IRE binding form by targeting the [Fe-S] cluster.
Bouton
C
C
U 365 INSERM, Section de Recherche, Institut Curie, Paris, France.
Raveau
M
M
Drapier
J C
JC
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors; D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D005293:Ferritins / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D035925:Iron-Regulatory Proteins; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000737:chemistry; D009566:Nitrates / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D004791:Enzyme Inhibitors; D006454:Hemoglobins; D035925:Iron-Regulatory Proteins; D009566:Nitrates; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D013481:Superoxides; C039642:peroxynitric acid; C002385:linsidomine; D005293:Ferritins; D006861:Hydrogen Peroxide; D008981:Molsidomine; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase