Phillips J D, Guo B, Yu Y, Brown F M, Leibold E A
Department of Medicine, University of Utah, Salt Lake City 84112, USA.
Biochemistry. 1996 Dec 10;35(49):15704-14. doi: 10.1021/bi960653l.
Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms. IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activity when cellular iron is abundant or RNA-binding activity when cellular iron is scarce. In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein. To study and compare the biochemical characteristics of IRP1 and IRP2, we expressed wild-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevisiae. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affinity, resulting in the inhibition of translation of an IRE-reporter mRNA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a mutant IRP1 containing an insertion of this domain bound RNA, but lacked detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity. Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms.
铁调节蛋白（IRP）1和2是胞质RNA结合蛋白，它们与特定的茎环结构结合，这些结构被称为铁反应元件（IRE），位于特定mRNA的非翻译区，这些mRNA编码参与铁代谢的蛋白质。IRP与IRE的结合调节mRNA的翻译或稳定性。尽管IRP1和IRP2是相似的蛋白质，因为它们在全身广泛表达且受铁的负调控，但它们受铁调控的机制不同。IRP1是细胞中特征明确的IRP，是一种双功能蛋白，当细胞铁充足时表现出乌头酸酶活性，当细胞铁缺乏时表现出RNA结合活性。相比之下，IRP2缺乏可检测到的乌头酸酶活性，仅作为RNA结合蛋白发挥作用。为了研究和比较IRP1和IRP2的生化特性，我们在酿酒酵母中表达了野生型和突变型大鼠IRP1和IRP2。在酵母中表达的IRP1和IRP2以高亲和力结合IRE RNA，导致IRE报告mRNA的翻译受到抑制。缺少IRP2特有的73个氨基酸结构域的突变型IRP2和插入了该结构域的突变型IRP1能结合RNA，但缺乏可检测到的乌头酸酶活性，这表明该结构域的存在会阻止乌头酸酶活性。与IRP1一样，IRP2的RNA结合活性对N-乙基马来酰亚胺（NEM）或5,5'-二硫代双（2-硝基苯甲酸）（DTNB）的失活敏感，表明IRP2含有一个对RNA结合至关重要的半胱氨酸。然而，与IRP1不同，IRP1中4Fe-4S簇的重建导致RNA结合活性丧失，而用相同的铁处理时，IRP2的RNA结合活性不受影响。这些数据表明IRP2不包含与IRP1中的簇类似的4Fe-4S簇，这表明它们通过不同的机制感知铁。
