Modulation of iron regulatory protein functions - Further insights into the role of nitrogen- and oxygen-derived reactive species
Modulation of iron regulatory protein functions - Further insights into the role of nitrogen- and oxygen-derived reactive species
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DOI：
10.1074/jbc.271.4.2300
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发表时间：
1996-01-26
影响因子：
4.8
通讯作者：
Drapier, JC
中科院分区：
文献类型：
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作者：
Bouton, C;Raveau, M;Drapier, JC
铁调节蛋白 (IRP) 是一种胞质双功能 [Fe-S] 蛋白，具有顺乌头酸酶活性或在特定 mRNA 的非翻译区域结合铁反应元件 (IRE)。这些活性的调节剂是细胞内铁浓度，以及最近描述的 NO 合酶活性。在本研究中，我们尝试在体外实验中使用巨噬细胞样 RAW 264.7 细胞的胞质提取物确定过氧亚硝酸盐（ONOO-，NO 和 O-2（自由基阴离子）之间反应的产物）以及氧衍生自由基（O-2（自由基阴离子）和 H2O2）和各种 NO 供体是否允许 IRP 结合 IRE。添加 ONOO- 或 H2O2 或 O-2（自由基阴离子）产生的推注均不会显着影响 IRE 结合，即使它们抑制了乌头酸酶活性。此外，我们还发现，3-吗啉代亚胺 (SIN-1)（一种同时释放 NO 和 O-2（自由基阴离子）的化学物质）仅在超氧化物歧化酶 (SOD) 存在的情况下才增强 IRP 的 IRE 结合活性。 S-亚硝基硫醇和 NONOate sper/NO 加谷胱甘肽 (GSH) 通过 IRP 激活 IRE 结合，而氧合血红蛋白则阻止 SIN-1/SOD 和 sper/NO 加 GSH 增强这种结合。 cis-Aconitate（一种乌头酸酶底物）也消除了 SIN-1/SOD 对 IRP 与 IRE 结合的影响。这些结果意味着 O-2（自由基阴离子）和 ONOO- 都不能将 [4Fe-4S] IRP 转化为 IRE 结合蛋白，而是表明 NO 的活性氧化还原形式通过靶向 [Fe-S] 簇将 IRP 转化为其 IRE 结合形式。
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 O-2(radical anion)), as well as oxygen-derived radicals (O-2(radical anion) and H2O2) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophage-like RAW 264.7 cells. Neither the addition of a bolus of ONOO- or H2O2 nor O-2(radical anion) 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 O-2(radical anion), enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus glutathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, an aconitase substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O-2(radical anion) 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.