Regulation Of Intracellular Iron Metabolism

细胞内铁代谢的调节

• 批准号: 9550320
• 负责人: TRACEY A. ROUAULT
• 金额: $ 104.13万
• 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/9550320
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Aconitate Hydratase; Adult; Affect; Alternative Splicing; Anemia; Animal Model; Animals; Axonal Transport; Binding; Binding Proteins; Blood Vessels; Bone Marrow Transplantation; Cell Line; Cells; Cessation of life; Disease; Duodenum; Electrophysiology (science); Elements; Embryo; Enzymes; Erythroblasts; Erythropoietin; Fatty Acids; Ferritin; Fibroblasts; Functional disorder; Gait abnormality; Gene Expression Profiling; Glycogen; Goals; Growth; H ferritin; HIF1A gene; Heme; Hereditary Leiomyomatosis and Renal Cell Cancer; Human; Infusion procedures; Inherited; Iron; Iron Regulatory Protein 1; Iron Regulatory Protein 2; Iron-Sulfur Proteins; Kidney; Knockout Mice; Laboratories; Lead; Leiomyomatosis; Liver; Lung; Maintenance; Malignant Neoplasms; Mammals; Mediating; Messenger RNA; Metabolic; Metabolism; Metformin; Mitochondria; Molecular; Motor Neurons; Mucous Membrane; Mus; Neoplasm Metastasis; Nerve Degeneration; Neurologic; Neurons; Nutrient; Oligodendroglia; Oxygen; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Polycythemia; Production; Protein p53; Proteins; Pulmonary Hypertension; RNA; Regulation; Renal carcinoma; Role; SLC11A2 gene; Spleen; Stimulus; Sulfur; Syndrome; System; TFRC gene; TP53 gene; Tissues; Transcript; Translational Repression; Translations; Tumor Suppressor Proteins; Work; aerobic glycolysis; axonal degeneration; axonopathy; blastomere structure; cancer cell; cell type; heme oxygenase-1; homologous recombination; human subject; insight; interest; interstitial; iron deficiency; iron metabolism; loss of function mutation; macrophage; metal transporting protein 1; monocyte; mouse model; multicatalytic endopeptidase complex; overexpression; oxidative damage; prevent; progressive neurodegeneration; protein function; sensor; small molecule; stem; tempol; tumor metabolism; tumor xenograft

This project aims to understand the molecular basis for regulation of intracellular iron metabolism. The RNA features recognized by proteins that mediate the iron-dependent alterations in abundance of ferritin and the transferrin receptor were 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 previously cloned, expressed, and characterized two essential iron- sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2). IRPs bind IREs when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and other transcripts that contain an IRE in the 5' untranslated regions, or in stabilization of the transferrin receptor mRNA and possibly other transcripts that contain IREs in the 3'UTR. The IRE-binding activity of IRP1 depends on whether the protein contains an iron-sulfur cluster (see project HD008814-01). IRP2 also binds IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are iron- replete. There are nine major IRE-containing mRNAs, and many have very important functions, such as the iron exporter, ferroportin, and the oxygen sensor, HIF2 alpha. We discovered that one alternatively spliced transcript of the iron exporter, ferroportin, lacks an IRE, and expression of the non-IRE form in duodenal mucosa and erythroblasts explains several important aspects of physiology. In iron-replete cells, IRP2 is selectively ubiquitinated by FBXL5 and degraded by the proteasome. 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 subtle abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/- mice develop a progressive neurologic syndrome characterized by gait abnormalities and axonal degeneration. Ferritin over-expression occurs in affected neurons, and in protrusions of oligodendrocytes into the space created by axonal degeneration. In animals that lack IRP1, IRP 2 compensates for loss of IRP1 regulatory activity, whereas animals that null for both IRP1 and IRP2 die as early embryos. The adult-onset neurodegeneration of adult IRP2-/- mice is exacerbated when one copy of IRP1 is also deleted. IRP2-/- mice offer a unique example of spontaneous adult-onset slowly progressive neurodegeneration, and analyses of gene expression and iron status at various stages of disease are ongoing. We have found that lower motor neurons are very adversely affected, developing axonopathy and death. In addition, small molecule treatment with the stable nitroxide, Tempol, prevents neurodegeneration in IRP2-/- animals. We characterized metabolism in an HLRCC cell line and discovered that AMPK is down, which leads to reduced p53 and DMT1, an iron importer. The iron deficiency that arises as a consequence promotes the switch to aerobic glycolysis. Only HIF1 alpha is significantly elevated, whereas HIF2 alpha expression is repressed by IRP activation. These metabolic changes lead to high storage of glycogen and fatty acids, which enables these cancer cells to store large amounts of energy that may fuel them during when they metastasize and temporarily lose access to nutrients. We discovered that treatment of cells with metformin in combination with an experimental drug that interferes with vascular growth eliminates growth of mouse xenograft tumors formed from the HLRCC cell line. We are also working to characterize Chuvash polycythemia in animal models and human subjects with particular emphasis on the underlying molecular pathophysiology. We discovered that Irp1-/- mice develop erythropoietin-driven polycythemia and pulmonary hypertension, and Irp1 is important for modulating expression of HIF2 alpha in pulmonary endothelia and renal interstitial fibroblasts.

本项目旨在了解细胞内铁代谢调节的分子基础。在过去的几年里，本实验室鉴定和表征了介导铁蛋白和转铁蛋白受体丰度变化的蛋白质所识别的RNA特征。铁反应元件(IRES)是位于铁蛋白mRNA5‘端和转铁蛋白受体mRNA3’端的RNA茎环。我们已经克隆、表达并鉴定了两种重要的铁敏感蛋白：铁调节蛋白1(Irp1)和铁调节蛋白2(IRP2)。当铁水平耗尽时，IRPS与IRES结合，导致铁蛋白mRNA和5‘非翻译区中包含IRE的其他转录本的翻译受到抑制，或者转铁蛋白受体mRNA和可能在3’UTR中包含IRES的其他转录本稳定。IRP1的IRE结合活性取决于蛋白质是否含有铁-硫簇(见项目HD008814-01)。IRP2也结合缺铁细胞中的IRES，但与IRP1不同的是，IRP2在铁充足的细胞中被降解。有九个主要的含有IRE的mRNAs，其中许多具有非常重要的功能，如铁出口蛋白，铁转运蛋白，和氧气传感器，HIF2α。我们发现铁出口蛋白的一个选择性剪接转录本缺乏IRE，而非IRE形式在十二指肠粘膜和红细胞中的表达解释了几个重要的生理学方面。在铁含量丰富的细胞中，IRP2被FBXL5选择性泛素化，并被蛋白酶体降解。为了探讨铁代谢的生理学问题，已经在小鼠胚胎细胞系中通过同源重组产生了IRP1和IRP2的功能缺失突变。在没有刺激性刺激的情况下，与Irp1功能丧失有关的铁代谢会出现微妙的异常。IRP2-/-小鼠出现以步态异常和轴突变性为特征的进行性神经综合征。铁蛋白过度表达发生在受影响的神经元中，以及由轴突变性形成的突起中的少突胶质细胞。在缺乏irp1的动物中，irp2弥补了irp1调节活性的丧失，而同时缺失irp1和irp2的动物作为早期胚胎死亡。当IRP1的一个拷贝也被缺失时，成年IRP2-/-小鼠的成年性神经变性加剧。IRP2-/-小鼠提供了一个独特的自发性成人起病缓慢进行性神经变性的例子，对疾病不同阶段的基因表达和铁状态的分析正在进行中。我们发现，下运动神经元受到非常不利的影响，发展为轴索病变和死亡。此外，用稳定的氮氧化物Tempoll进行小分子治疗，可以防止IRP2-/-动物的神经退化。 我们研究了HLRCC细胞系的代谢，发现AMPK下调，导致P53和铁导入蛋白DMT1减少。由此产生的铁缺乏会促进有氧糖酵解的转变。只有HIF1α显著升高，而HIF2α的表达被IRP激活抑制。这些代谢变化导致糖原和脂肪酸的高度储存，这使得这些癌细胞能够储存大量的能量，这些能量可能会在它们转移时为它们提供燃料，并暂时失去获得营养的途径。我们发现，二甲双胍联合一种干扰血管生长的实验药物治疗细胞，可以消除由HLRCC细胞系形成的小鼠异种移植瘤的生长。我们还致力于在动物模型和人类受试者中描述丘瓦什红细胞增多症的特征，特别强调潜在的分子病理生理学。我们发现，Irp1-/-小鼠发生了促红细胞生成素引起的红细胞增多症和肺动脉高压，Irp1对肺内皮细胞和肾间质成纤维细胞中HIF2α的表达具有重要的调节作用。