Regulation Of Intracellular Iron Metabolism

细胞内铁代谢的调节

• 批准号: 6671881
• 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
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6671881
• 关键词: RNA binding protein; active sites; enzyme activity; ferritin; gene targeting; genetic regulation; genetic regulatory element; genetic translation; human genetic material tag; intracellular transport; iron metabolism; iron sulfur protein; microorganism metabolism; molecular cloning; protein sequence; protein structure function; tissue /cell culture; transcription factor; transferrin receptor

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. Animals develop axonal degeneration characterized by iron accumulation in axons. Ferritin over-expression occurs in affected neurons, and ferritin accumulation occurs in axons. Affected cells are those that depend mainly on IRP2 for repression of ferritin translation. Animals that lack both IRP1 and IRP2 are embryonic lethal. Blastocyst harvest may yield cells that completely lack IRPs.

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。铁反应元件（IRES）是在铁蛋白mRNA的5'末端发现的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，铁调节蛋白1（IRP1）和铁调节蛋白2（IRP2。IRP2。当铁水平耗尽时结合IRE的IRES，导致抑制铁蛋白mRNA和其他MRNA的翻译。含有转录蛋白受体mRNA的转录本和半衰期的延长是与线粒体腺苷酶，一种柠檬酸循环酶有关的铁硫蛋白，它作为细胞中的细胞蛋白酶的作用IRP1的RNA结合活性涉及从[4FE-4S]簇结合的IRP1的过渡，与含有蛋白质的[4FE-4S]均失去了铁和刺刺酶活性铁硫簇的降解和诱变表明，铁缺失细胞中RNA结合蛋白的生理相关形式是载脂蛋白的状态。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，这些基因促进了IRP1铁硫群集的组装。 IRP2还结合了铁缺失细胞中的IRE，但是与IRP1不同，IRP2在铁质的细胞中降解。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为特征的进行性神经系统综合征。动物发展为轴突变性，其特征是轴突中铁的积累。铁蛋白过表达发生在受影响的神经元中，铁蛋白的积累发生在轴突中。受影响的细胞是主要依赖于IRP2来抑制铁蛋白翻译的细胞。缺乏IRP1和IRP2的动物是胚胎致死的。胚泡收获可能会产生完全缺乏IRP的细胞。