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.

该项目旨在理解细胞内铁代谢的分子基础。在该实验室的前几年中，已经确定并表征了介导铁依赖铁和转移蛋白受体的依赖铁依赖性改变的顺式和跨元件。铁响应元件（IRES）是在铁蛋白mRNA的5端发现的RNA干循环和转铁蛋白受体mRNA的3'末端。我们已经克隆，表达和表征了两种必需的铁感应蛋白，即铁调节蛋白1（IRP1）和铁调节蛋白2（IRP2。IRP2。IRPS结合IRE时，IRPER的含量耗尽，导致抑制了铁蛋白mRNA和其他含有IRE的转移量的转移和延长的转移液的转移液的转移液的转移，这会抑制IRNA的转移和延长。蛋白质与线粒体刺激性酶，一种柠檬酸循环酶，它在铁中的细胞中作为胞质刺激性酶的作用。 IRES。最近，我们已经确定了与细菌铁硫簇组装有关的NIFS和NIF U基因同源的铁硫簇组装的哺乳动物酶，我们已经表明，这些基因产物有助于IRP1的铁硫簇组装。 IRP2还结合了铁缺失的细胞中的IRE，但是与IRP1不同，IRP2在铁含量的细胞中脱落。实验证据表明，IRP2结合铁并经历铁催化的氧化。然后，氧化蛋白被选择性地泛素化并被蛋白酶体降解。间接证据表明，许多其他蛋白质将被一条途径降解，其中氧化修饰之后是泛素化和蛋白酶体降解的泛素化底物。为了解决有关铁代谢生理的问题，通过胚胎细胞系中的同源重组，已经在小鼠中产生了IRP1和IRP2功能突变的丧失。在没有挑衅性刺激的情况下，与IRP1功能丧失有关的铁代谢没有异常。 IRP2 - / - 小鼠发展出一种以步态异常和震颤为特征的进行性神经系统综合征。两种IRP的功能丧失似乎都是致命的。在正常和敲除小鼠中，基因在遗传性血色素沉着症和鼠微细胞贫血中的作用的表征正在持续。