Philpott C C, Haile D, Rouault T A, Klausner R D
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Aug 25;268(24):17655-8.
The iron-responsive element-binding protein (IRE-BP) binds to specific RNA stem-loop structures called iron-responsive elements (IREs), which mediate the post-transcriptional regulation of a variety of genes involved in iron metabolism. The IRE-BP is cytosolic aconitase, and a [4Fe-4S] cubane cluster is required for aconitase activity but is associated with loss of IRE binding affinity. Chemical modification of the IRE-BP can abrogate RNA binding and the 3 cysteines predicted to coordinate the Fe-S cluster in the IRE-BP could be targets for modification. We report the expression of recombinant IRE-BP in which the three putative cluster cysteines (Cys-437, Cys-503, and Cys-506) have been mutated to serine residues. Replacement of any or all of these cysteine residues results in a complete loss of aconitase activity. While all of the mutants bind RNA, substitution of Cys-437 specifically renders the IRE-BP resistant to inactivation by low concentrations of N-ethylmaleimide or diamide. These results identify Cys-437 as the target of in vitro regulation of RNA binding in the IRE-BP and suggest that, in the RNA-binding form of the protein, Cys-437 is free and therefore available for modifications that inhibit RNA binding.
铁反应元件结合蛋白（IRE-BP）与称为铁反应元件（IREs）的特定RNA茎环结构结合，这些结构介导参与铁代谢的多种基因的转录后调控。IRE-BP是胞质乌头酸酶，乌头酸酶活性需要一个[4Fe-4S]立方烷簇，但该簇与IRE结合亲和力的丧失有关。IRE-BP的化学修饰可消除RNA结合，预计在IRE-BP中协调Fe-S簇的3个半胱氨酸可能是修饰的靶点。我们报道了重组IRE-BP的表达，其中三个假定的簇半胱氨酸（Cys-437、Cys-503和Cys-506）已突变为丝氨酸残基。这些半胱氨酸残基中的任何一个或全部被取代都会导致乌头酸酶活性完全丧失。虽然所有突变体都能结合RNA，但Cys-437的取代使IRE-BP对低浓度的N-乙基马来酰亚胺或二酰胺失活具有抗性。这些结果确定Cys-437是IRE-BP中RNA结合体外调控的靶点，并表明，在蛋白质的RNA结合形式中，Cys-437是游离的，因此可用于抑制RNA结合的修饰。
