Regulation Of Intracellular Iron Metabolism

细胞内铁代谢的调节

基本信息

批准号：
8553882
负责人：
TRACEY A. ROUAULT
金额：
$ 100.08万
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

来源：
https://reporter.nih.gov/project-details/8553882
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Aconitate Hydratase Adult Affect Anemia Animal Model Animals Axonal Transport Beryllium Binding Blood Vessels Bone Marrow Transplantation Cancer cell line Cell Line Cells Cessation of life Disease Elements Embryo Enzymes Erythroblasts Fatty Acids Ferritin Functional disorder Gait abnormality Gene Expression Glycogen Goals Growth H ferritin HIF1A gene Heme Human Indium Inherited Iron Iron Regulatory Protein 1 Iron Regulatory Protein 2 Iron-Sulfur Proteins Knockout Mice Laboratories Lead Leiomyomatosis Liver 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 Binding Protein p53 Proteins RNA RNA Splicing Regulation Renal carcinoma Role SLC11A2 gene Spleen Staging Stimulus Sulfur Syndrome System Testing Tissues Transcript Transferrin Receptor Translations Tumor Suppressor Proteins Work aerobic glycolysis axonal degeneration axonopathy base blastomere structure cancer cell heme oxygenase-1 homologous recombination human disease human subject insight interest iron deficiency iron metabolism loss of function mutation macrophage metal transporting protein 1 mouse model multicatalytic endopeptidase complex oxidative damage prevent progressive neurodegeneration protein function research study sensor small molecule stem tempol tumor xenograft

项目摘要

This project aims to understand the molecular basis for regulation 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 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. We are culturing neurons from Irp null mice to better characterize their metabolic abnormalities. 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.

该项目旨在了解调节细胞内铁代谢的分子基础。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件已在本实验室前几年被鉴定和表征。铁反应元件 (IRE) 是在铁蛋白 mRNA 的 5' 端和转铁蛋白受体 mRNA 的 3' 端发现的 RNA 茎环。我们之前克隆、表达并表征了两种重要的铁感应蛋白：铁调节蛋白 1 (IRP1) 和铁调节蛋白 2 (IRP2)。当铁水平耗尽时，IRP 与 IRE 结合，从而抑制铁蛋白 mRNA 和其他在 5' 非翻译区含有 IRE 的转录物的翻译，或者稳定转铁蛋白受体 mRNA 和可能在 3' 非翻译区含有 IRE 的其他转录物。 'UTR。 IRP1 的 IRE 结合活性取决于该蛋白是否含有铁硫簇（参见项目 HD008814-01）。 IRP2 也在缺铁细胞中结合 IRE，但与 IRP1 不同的是，IRP2 在铁充足的细胞中会被降解。有九种主要的 IRE mRNA，其中许多具有非常重要的功能，例如铁输出蛋白、铁转运蛋白和氧传感器、HIF2 α。我们发现铁输出蛋白铁转运蛋白的一种选择性剪接转录物缺乏 IRE，并且十二指肠粘膜和成红细胞中非 IRE 形式的表达解释了生理学的几个重要方面。在铁充足的细胞中，IRP2 被 FBXL5 选择性泛素化并被蛋白酶体降解。为了解决有关铁代谢生理学的问题，通过胚胎细胞系中的同源重组在小鼠体内产生了 IRP1 和 IRP2 的功能丧失突变。在没有刺激的情况下，铁代谢会出现与 IRP1 功能丧失相关的微妙异常。 IRP2-/- 小鼠会出现一种进行性神经系统综合征，其特征是步态异常和轴突变性。铁蛋白过度表达发生在受影响的神经元中，以及少突胶质细胞突出到轴突变性产生的空间中。我们正在培养 Irp 缺失小鼠的神经元，以更好地表征它们的代谢异常。在缺乏 IRP1 的动物中，IRP 2 补偿了 IRP1 调节活性的损失，而 IRP1 和 IRP2 均无效的动物则在早期胚胎时死亡。当 IRP1 的一个拷贝也被删除时，成年 IRP2-/- 小鼠的成年期神经变性会加剧。 IRP2-/- 小鼠提供了成人自发性缓慢进行性神经变性的独特例子，并且正在对疾病各个阶段的基因表达和铁状态进行分析。我们发现下运动神经元受到非常不利的影响，导致轴突病变和死亡。此外，使用稳定的硝基氧 Tempol 进行小分子治疗可预防 IRP2-/- 动物的神经变性。我们对 HLRCC 细胞系的代谢进行了表征，发现 AMPK 下降，导致 p53 和铁输入蛋白 DMT1 减少。由此产生的缺铁促进了有氧糖酵解的转变。只有 HIF1 α 显着升高，而 HIF2 α 表达受到 IRP 激活的抑制。这些代谢变化导致糖原和脂肪酸的大量储存，这使得这些癌细胞能够储存大量能量，这些能量可以在它们转移和暂时失去营养时为它们提供燃料。我们发现，用二甲双胍与干扰血管生长的实验药物联合治疗细胞可以消除 HLRCC 细胞系形成的小鼠异种移植肿瘤的生长。我们还致力于在动物模型和人类受试者中描述楚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。