Cairo G, Castrusini E, Minotti G, Bernelli-Zazzera A
Centro di Studio sulla Patologia Cellulare CNR, Università di Milano, Italy.
FASEB J. 1996 Sep;10(11):1326-35. doi: 10.1096/fasebj.10.11.8836047.
Cellular iron homeostasis is regulated by the cytoplasmic iron regulatory protein (IRP), which binds to iron-responsive elements (IRE) of mRNAs, modulating iron uptake and sequestration, respectively. When iron is scarce, IRP binds to IRE and coordinately increases the synthesis of transferrin receptor and decreases that of ferritin, thus providing the cell with readily available free iron. When iron is in excess, IRP does not bind and iron sequestration prevails over iron uptake. We have found that incubation of rat liver lysates with xanthine oxidase (XO), which generates superoxide (O2-.) and hydrogen peroxide (H2O2), caused a remarkable but reversible inhibition of IRP activity, as the formation of IRE-IRP decreased by 70-80% but returned to baseline values upon exposure to a reducing agent like 2-mercaptoethanol. IRP inhibition was prevented by separate or simultaneous addition of superoxide dismutase and catalase, showing that both O2-. and H2O2 were involved. By contrast, iron chelators and hydroxyl radical scavengers did not impede the inhibition of IRP, suggesting that O2-. and H2O2 acted independently of free iron sources. Ferritin enhanced IRP inhibition, but this process involved tightly bound iron centers that shunted reducing equivalents from XO and returned them to oxygen, thus increasing the formation of O2-. In agreement with the exclusive role of O2-. and H2O2, XO also inhibited recombinant human IRP in the absence of iron. These results demonstrate that O2-. and H2O2 can directly but reversibly down-regulate the RNA-binding activity of IRP, causing transient decrease of free iron that otherwise would convert them into more potent oxidants such as hydroxyl radicals or equally aggressive iron-peroxo complexes. This establishes a novel protective stratagem against oxidative injury under pathophysiologic conditions characterized by the excessive generation of O2-. and H2O2.
细胞铁稳态由细胞质铁调节蛋白（IRP）调控，该蛋白与mRNA的铁反应元件（IRE）结合，分别调节铁的摄取和螯合。当铁缺乏时，IRP与IRE结合，协同增加转铁蛋白受体的合成并减少铁蛋白的合成，从而为细胞提供易于获取的游离铁。当铁过量时，IRP不结合，铁螯合作用超过铁摄取作用。我们发现，用产生超氧化物（O2-·）和过氧化氢（H2O2）的黄嘌呤氧化酶（XO）孵育大鼠肝脏裂解物，会导致IRP活性显著但可逆的抑制，因为IRE-IRP的形成减少了70-80%，但在暴露于2-巯基乙醇等还原剂后恢复到基线值。单独或同时添加超氧化物歧化酶和过氧化氢酶可防止IRP抑制，表明O2-·和H2O2都参与其中。相比之下，铁螯合剂和羟基自由基清除剂并不妨碍IRP的抑制，这表明O2-·和H2O2的作用独立于游离铁源。铁蛋白增强了IRP抑制，但这一过程涉及紧密结合的铁中心，这些铁中心将还原当量从XO转移并使其回到氧，从而增加O2-·的形成。与O2-·和H2O2的独特作用一致，XO在无铁的情况下也抑制重组人IRP。这些结果表明，O2-·和H2O2可直接但可逆地下调IRP的RNA结合活性，导致游离铁的短暂减少，否则游离铁会将它们转化为更具活性的氧化剂，如羟基自由基或同样具有侵蚀性的铁过氧络合物。这确立了一种在以O2-·和H2O2过度产生为特征的病理生理条件下对抗氧化损伤的新保护策略。
