Hanson E S, Leibold E A
Eccles Program in Human Molecular Biology and Genetics and the Department of Medicine, University of Utah, Salt Lake City, Utah 84112, USA.
J Biol Chem. 1998 Mar 27;273(13):7588-93. doi: 10.1074/jbc.273.13.7588.
Given the important relationship between O2 and iron (Fenton chemistry) a study was undertaken to characterize the effects of hypoxia, as well as subsequent reoxygenation, on the iron-regulatory proteins 1 and 2 (IRP1 and IRP2) in a rat hepatoma cell line. IRP1 and IRP2 are cytosolic RNA-binding proteins that bind RNA stem-loops located in the 5'- or 3'-untranslated regions of specific mRNAs encoding proteins that are involved in iron homeostasis. In cells exposed to hypoxia, IRP1 RNA binding was decreased approximately 2. 8-fold after a 6-h exposure to 3% O2. Hypoxic inactivation of IRP1 was abolished when cells were pretreated with the iron chelator desferrioxamine, indicating a role for iron in inactivation. IRP1 inactivation was reversible since re-exposure of hypoxically-treated cells to 21% O2 increased RNA binding activity approximately 7-fold after 21 h with an increase in activity seen as early as 1-h post-reoxygenation. IRP1 protein levels were unaffected during hypoxia as well as during reoxygenation. Whereas the protein synthesis inhibitor cycloheximide did not block IRP1 inactivation during hypoxia, it completely blocked IRP1 reactivation during subsequent reoxygenation. Reactivation of IRP1 during reoxygenation was also partially blocked by the phosphatase inhibitor okadaic acid. Finally, reactivated IRP1 was found to be resistant to inactivation by exogenous iron known to down-regulate its activity during normoxia. These data demonstrate that IRP1 RNA binding activity is post-translationally regulated during hypoxia and hypoxia/reoxygenation. Regulation of IRP1 by changing oxygen tension may provide a novel mechanism for post-transcriptionally regulating gene expression under these stresses.
鉴于氧气（O₂）与铁之间的重要关系（芬顿化学），我们开展了一项研究，以表征缺氧以及随后的复氧对大鼠肝癌细胞系中铁调节蛋白1和2（IRP1和IRP2）的影响。IRP1和IRP2是胞质RNA结合蛋白，它们与位于特定mRNA的5'-或3'-非翻译区的RNA茎环结合，这些mRNA编码参与铁稳态的蛋白质。在暴露于缺氧环境的细胞中，在3% O₂ 中暴露6小时后，IRP1的RNA结合能力下降了约2.8倍。当用铁螯合剂去铁胺预处理细胞时，IRP1的缺氧失活被消除，这表明铁在失活过程中起作用。IRP1的失活是可逆的，因为将缺氧处理的细胞重新暴露于21% O₂ 中21小时后，RNA结合活性增加了约7倍，早在复氧后1小时就可见活性增加。在缺氧和复氧过程中，IRP1蛋白水平均未受到影响。虽然蛋白质合成抑制剂放线菌酮在缺氧期间并未阻止IRP1的失活，但它完全阻止了随后复氧期间IRP1的再激活。复氧期间IRP1的再激活也被磷酸酶抑制剂冈田酸部分阻断。最后，发现重新激活的IRP1对已知在常氧期间下调其活性的外源性铁的失活具有抗性。这些数据表明，IRP1的RNA结合活性在缺氧和缺氧/复氧过程中受到翻译后调控。通过改变氧张力对IRP1的调节可能为在这些应激条件下转录后调节基因表达提供一种新机制。
