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REGULATION OF INTRACELLULAR IRON METABOLISM

细胞内铁代谢的调节

基本信息

批准号：
6432572
负责人：
TRACEY A. ROUAULT
金额：
--
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2. IRPs bind IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other IRE containing transcripts and prolongation of the half-life of the transferrin receptor mRNA. IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. Regulation of RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound, to a form that loses both iron and aconitase activity. The [4Fe-4S] containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to determine whether IRP1 will bind RNA. Recently, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron-sulfur cluster of IRP1. IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are iron-replete. Experimental evidence indicates that IRP2 binds iron and undergoes iron-catalyzed oxidation. The oxidized protein is then selectively ubiquitinated and degraded by the proteasome. Indirect evidence suggests that numerous other proteins will be degraded by a pathway in which oxidative modification is followed by ubiquitination and proteasomal degradation of the ubiquitinated substrate. To approach questions about the physiology of iron metabolism, loss of function mutations of IRP1 and IRP2 have been generated in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, there are no abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/- mice develop a progressive neurologic syndrome characterized by gait abnormalities and tremor. Loss of function of both IRPs appears to be lethal. Characterization of the roles of the genes for hereditary hemochromatosis and murine microcytic anemia are ongoing in normal and knockout mice.

该项目旨在了解细胞内铁代谢的分子基础。在过去的几年中，本实验室已经确定并表征了介导铁蛋白和转铁蛋白受体丰度的铁依赖性改变的顺式和反式元素。铁反应元件（Iron-responsive elements，IREs）是存在于铁蛋白mRNA的5'端和转铁蛋白受体mRNA的3'端的RNA茎环。我们已经克隆、表达并鉴定了两种必需的铁敏感蛋白，铁调节蛋白1（IRP 1）和铁调节蛋白2（IRP 2）。当铁水平耗尽时，IRP结合IRE，导致抑制铁蛋白mRNA和其它含IRE的转录物的翻译，并延长转铁蛋白受体mRNA的半衰期。IRP 1是一种与线粒体顺乌头酸酶（一种柠檬酸循环酶）相关的铁硫蛋白，它在铁充足的细胞中发挥细胞质顺乌头酸酶的作用。IRP 1的RNA结合活性的调节涉及从结合[4Fe-4S]簇的IRP 1形式到失去铁和顺乌头酸酶活性的形式的转变。含[4Fe-4S]的蛋白质不结合IRE。铁-硫簇的受控降解和诱变揭示了铁耗竭细胞中RNA结合蛋白的生理相关形式是脱辅基蛋白。簇的状态似乎决定了IRP 1是否会结合RNA。最近，我们已经确定了哺乳动物的铁-硫簇组装的酶是同源的NifS和Nif U基因参与细菌的铁-硫簇组装，我们已经表明，这些基因产物促进装配的铁-硫簇IRP 1。IRP 2也结合铁耗尽细胞中的IRE，但与IRP 1不同，IRP 2在铁充足的细胞中降解。实验证据表明，IRP 2结合铁并经历铁催化氧化。氧化的蛋白质然后被蛋白酶体选择性地泛素化和降解。间接证据表明，许多其他蛋白质将被降解的途径，其中氧化修饰，其次是泛素化和蛋白酶体降解的泛素化底物。为了探讨铁代谢的生理学问题，通过胚胎细胞系中的同源重组，在小鼠中产生了IRP 1和IRP 2的功能丧失突变。在没有刺激的情况下，没有与IRP 1功能丧失相关的铁代谢异常。IRP 2-/-小鼠发生以步态异常和震颤为特征的进行性神经综合征。两个IRP的功能丧失似乎是致命的。遗传性血色素沉着症和小鼠小细胞性贫血的基因的作用的特点正在正常和敲除小鼠。