Oliveira L, Bouton C, Drapier J C
Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
J Biol Chem. 1999 Jan 1;274(1):516-21. doi: 10.1074/jbc.274.1.516.
Iron regulatory proteins (IRP1 and IRP2) are redox-sensitive RNA-binding proteins that modulate the expression of several genes encoding key proteins of iron metabolism. IRP1 can also exist as an aconitase containing a [4Fe-4S] cluster bound to three cysteines at the active site. We previously showed that biosynthesis of nitric oxide (NO) induces the transition of IRP1 from aconitase to apoprotein able to bind RNA. This switch is also observed when cytosolic extracts are exposed to NO donors. However, the activation of IRP1 under these conditions is far from maximal. In this study we examined the capacity of physiological reducing systems to cooperate with NO in the activation of IRP1. Cytosolic extracts from the macrophage cell line RAW 264.7 or purified IRP1 were incubated with NO donors and subsequently exposed to glutathione or to thioredoxin (Trx), a strong protein disulfide reductase. Trx was the most effective, inducing a 2-6-fold enhancement of the RNA binding activity of NO-treated IRP1. Furthermore, the effect of NO on IRP1 from cytosolic extracts was abolished in the presence of anti-Trx antibodies. We also studied the combined effect of NO and Trx on IRP2, which exhibits constitutive RNA binding activity. We observed an inhibition of IRP2 activity following exposure to NO donors which was restored by Trx. Collectively, these results point to a crucial role of Trx as a modulator of IRP activity in situations of NO production.
铁调节蛋白（IRP1和IRP2）是对氧化还原敏感的RNA结合蛋白，可调节多个编码铁代谢关键蛋白的基因的表达。IRP1也可以作为一种乌头酸酶存在，其活性位点含有一个与三个半胱氨酸结合的[4Fe-4S]簇。我们之前表明，一氧化氮（NO）的生物合成会诱导IRP1从乌头酸酶转变为能够结合RNA的脱辅基蛋白。当细胞溶质提取物暴露于NO供体时也会观察到这种转变。然而，在这些条件下IRP1的激活远未达到最大值。在本研究中，我们研究了生理还原系统与NO协同激活IRP1的能力。将巨噬细胞系RAW 264.7的细胞溶质提取物或纯化的IRP1与NO供体一起孵育，随后暴露于谷胱甘肽或硫氧还蛋白（Trx，一种强大的蛋白质二硫键还原酶）。Trx最为有效，可使经NO处理的IRP1的RNA结合活性提高2至6倍。此外，在存在抗Trx抗体的情况下，NO对细胞溶质提取物中IRP1的作用被消除。我们还研究了NO和Trx对IRP2的联合作用，IRP2表现出组成型RNA结合活性。我们观察到暴露于NO供体后IRP2活性受到抑制，而Trx可使其恢复。总的来说，这些结果表明Trx在NO产生的情况下作为IRP活性调节剂起着关键作用。
