The role of endogenous heme synthesis and degradation domain cysteines in cellular iron-dependent degradation of IRP2
The role of endogenous heme synthesis and degradation domain cysteines in cellular iron-dependent degradation of IRP2
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DOI：
10.1016/s1079-9796(03)00161-x
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发表时间：
2003-09-01
影响因子：
2.3
通讯作者：
Rouault, TA
中科院分区：
文献类型：
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作者：
Bourdon, E;Kang, DK;Rouault, TA
铁调节蛋白2（Iron Regulatory Protein 2，IRP2）是哺乳动物细胞质中的一种铁敏感蛋白，其调节铁代谢蛋白的表达，包括铁蛋白和转铁蛋白受体1。IRP2在铁充足的细胞中被蛋白酶体泛素化和降解，但在铁缺乏的细胞中相对稳定。最近的工作表明，IRP2含有独特的73个氨基酸的结构域，其在体外结合铁并经历铁依赖性氧化和裂解（J.Biol.Chem.278（2003），14857）。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的细胞铁依赖性降解中的作用，我们在全长蛋白质的背景下以各种组合诱变这些半胱氨酸，并产生其中重组IRP2表达是可诱导的细胞系。在细胞中推定降解结构域的任何或所有半胱氨酸处诱变的IRP2的铁依赖性降解与野生型（WT）相当。WT和半胱氨酸突变蛋白都在3%氧气中稳定。硝普钠（SNP），NO+供体的治疗，引起细胞IRP2浓度的下降，但SNP的影响被废除，同时加入铁螯合剂去铁，并没有受到半胱氨酸突变。用琥珀酰丙酮抑制内源性血红素合成显著抑制了IRP 2的铁依赖性降解。添加氯化钴可抑制WT和诱变IRP2的降解。因此，我们不能辨别最近定义的体外半胱氨酸依赖性铁结合位点的IRP2在细胞生理学中的作用。铁依赖性降解IRP2的早期分子事件仍有待阐明。
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.