Bourdon Emmanuel, Kang Dae-Kyung, Ghosh Manik C, Drake Steven K, Wey Jane, Levine Rodney L, Rouault Tracey A
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
Blood Cells Mol Dis. 2003 Sep-Oct;31(2):247-55. doi: 10.1016/s1079-9796(03)00161-x.
Iron regulatory protein 2 (IRP2) is a mammalian cytosolic iron-sensing protein that regulates expression of iron metabolism proteins, including ferritin and transferrin receptor 1. IRP2 is ubiquitinated and degraded by the proteasome in iron-replete cells but is relatively stable in iron-depleted cells. Recent work has shown that IRP2 contains a unique 73-amino-acid domain that binds iron in vitro and undergoes iron-dependent oxidation and cleavage (J. Biol. Chem. 278 (2003), 14857). Several cysteines in the 73-amino-acid domain function as an in vitro iron-binding site. To assess the role of these cysteines in cellular iron- dependent degradation of IRP2, we mutagenized these cysteines in various combinations in the context of full-length protein and generated cell lines in which recombinant IRP2 expression was inducible. Iron-dependent degradation of IRP2 mutagenized at any or all of the cysteines of the putative degradation domain in cells was comparable to wild-type (WT). Both WT and cysteine mutant protein were stabilized in 3% oxygen. Treatment with sodium nitroprusside (SNP), an NO+ donor, caused a decrease in cellular IRP2 concentrations, but the SNP effect was abrogated by simultaneous addition of the iron chelator desferal and was not affected by cysteine mutations. Inhibition of endogenous heme synthesis with succinylacetone significantly inhibited iron- dependent degradation of IRP2. Addition of cobalt chloride inhibited degradation of both WT and mutagenized IRP2. Thus, we could not discern a role for the recently defined in vitro cysteine-dependent iron-binding site of IRP2 in cellular physiology. The early molecular events in iron-dependent degradation of IRP2 remain to be elucidated.
铁调节蛋白2（IRP2）是一种哺乳动物胞质铁感应蛋白，可调节铁代谢蛋白的表达，包括铁蛋白和转铁蛋白受体1。在铁充足的细胞中，IRP2会被蛋白酶体泛素化并降解，但在缺铁细胞中相对稳定。最近的研究表明，IRP2包含一个独特的73个氨基酸的结构域，该结构域在体外与铁结合，并经历铁依赖性氧化和裂解（《生物化学杂志》278卷（2003年），第14857页）。73个氨基酸结构域中的几个半胱氨酸作为体外铁结合位点发挥作用。为了评估这些半胱氨酸在IRP2细胞铁依赖性降解中的作用，我们在全长蛋白的背景下以各种组合对这些半胱氨酸进行诱变，并生成了重组IRP2表达可诱导的细胞系。在细胞中，对假定降解结构域的任何一个或所有半胱氨酸进行诱变的IRP2的铁依赖性降解与野生型（WT）相当。野生型和半胱氨酸突变蛋白在3%氧气中均得到稳定。用一氧化氮供体硝普钠（SNP）处理导致细胞IRP2浓度降低，但同时添加铁螯合剂去铁胺可消除SNP的作用，且不受半胱氨酸突变的影响。用琥珀酰丙酮抑制内源性血红素合成可显著抑制IRP2的铁依赖性降解。添加氯化钴可抑制野生型和诱变型IRP2的降解。因此，我们无法确定IRP2最近定义的体外半胱氨酸依赖性铁结合位点在细胞生理学中的作用。IRP2铁依赖性降解的早期分子事件仍有待阐明。
