Regulation Of Intracellular Iron Metabolism

细胞内铁代谢的调节

基本信息

批准号：
10266478
负责人：
TRACEY A. ROUAULT
金额：
$ 175.88万
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/10266478
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Aconitate Hydratase Adult Affect Africa African American Alternative Splicing Anemia Animals Award Axonal Transport Binding Binding Proteins Blood Vessels CRISPR/Cas technology Cancer cell line Cell Death Cell Line Cells Cessation of life Clinical Research Cognitive Collaborations Complex Diet Dietary Supplementation Disease Duodenum Electrophysiology (science)Elements Embryo Enzymes Erythroblasts Erythrocytes Erythroid Cells Erythrophagocytosis Erythropoietin Fatty Acids Ferritin Fibroblasts Fibrosis Functional disorder Gait abnormality Gene Expression Profiling Genes Glycogen Goals Growth H ferritin HIF1A gene Heart Heme Hereditary Leiomyomatosis and Renal Cell Cancer Human Hypoxia Inducible Factor In Vitro Infusion procedures Inherited Iron Iron Overload Iron Regulatory Protein 1 Iron Regulatory Protein 2 Iron-Sulfur Proteins Kidney Knock-in Mouse Knockout Mice Laboratories Lead Leiomyomatosis Life Liver Longevity Lung Maintenance Malaria Malignant Neoplasms Mammals Mediating Messenger RNA Metabolic Metabolism Metformin Mitochondria Molecular Motor Motor Neurons Mucous Membrane Mus Mutation Neoplasm Metastasis Nerve Degeneration Neurodegenerative Disorders Neurologic Neurons Nutrient Oligodendroglia Oxygen Pancreas Parasites Patients Pharmaceutical Preparations Phenotype Physiological Physiology Polycythemia Population Prevalence Production Proteins Pulmonary Hypertension RNA Rare Diseases Regulation Renal carcinoma Repression Resistance Role SLC11A2 gene Spleen Stimulus Sulfur Syndrome System TFRC gene TP53 gene Tissues Transcript Translational Repression Translations United States National Institutes of Health VHL gene Wild Type Mouse Work aerobic glycolysis axonal degeneration axonopathy bench to bedside blastomere structure cancer cell cell type functional loss gain of function mutation heme oxygenase-1 homologous recombination inhibitor/antagonist insight interest interstitial iron deficiency iron metabolism loss of function mutation macrophage malaria infection metal transporting protein 1 microcytic anemia monocyte mouse model multicatalytic endopeptidase complex mutation carrier nutrition overexpression oxidation oxidative damage prevent progressive neurodegeneration protein function sensor small molecule stem tempol tumor metabolism tumor xenograft

项目摘要

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 abnormalities in iron metabolism associated with loss of IRP1 function that include polycythemia, and pulmonary hypertension and heart fibrosis also develop. 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. Two patients with loss of IRP2 develop severe neurological problems that develop in the first year of life and affect cognitive and motor functions. In animals that lack IRP1, IRP 2 partially 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,and 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 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 by suppressing translation of the transcript by binding to the 5' iron-responsive element in the transcript. These insights led us to study Chuvash polycythemia, as Chuvash polycythemia is caused by a mutation in the vHL gene that decreases recognition and degradation of hypoxia inducible factors by the vHL complex. Having discovered that Irp1 is important for translation repression of HIF2 alpha, we treated a Chuvash knockin mouse model with TEMPOL, or with a HIF2 alpha inhibitor and we reversed polycythemia. Crossing with Irp1-/- mice proved that activation of IRE binding of Irp1 by TEMPOL was crucial to reducing erythropoietin production in these mice, and demonstrated that dietary TEMPOL is a good candidate as a treatment for Chuvash polycythemia. We discovered that the iron exporter, ferroportin, is unexpectedly highly expressed in mature red cells, where it exports free iron released by oxidation of heme. We generated mice with a tissue specific loss of ferroportin in erythroid cells. In cells that lacked ferroportin, oxidative damage shortened the life-span of mature red cells and caused a mild microcytic anemia. Conversely, when the exporter has a gain of function mutation, Q248H, clinical studies with our collaborators showed that the Q248 mutation protects from malarial infection by reducing iron available for nutrition of malarial parasites that infect red cells. The mutation is common in malarious regions of Africa (up to 20%) and in African Americans, where its prevalence ranges from 8-12% of African americans. The mutation likely predisposes to iron overload in tissues such as the liver, kidney and pancreas. We are initiating studies on the iron status and potential deleterious effects on carriers of the mutation by evaluating patients and by making a knocking mouse model, supported by an NIH Bench to Bedside award. Continuing our attempts to find a therapy for HMOX1-/- patients, we infused macrophages cultured in vitro into congenial Hmox1-/- mice, and we observed that macrophage infusion prevented disease, and macrophages multiplied in the liver, where they were became more numerous than in wild type mice. These studies indicated that HMOX1-/- patients, a rare disease, might one day be treatable by correcting their mutations in macrophages removed from patients using CRISPR techniques, and infusion of macrophages that express normal HMOX1 could prevent patients from succumbing to the fatal disease that lack of HMOX1 causes. We identified genes that are induces to express upon erythrophagocytosis, and we are analyzing the metabolic remodeling that permits erythrophagocytosis to proceed without resulting in cell death.

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蛋白质所识别的 RNA 特征在前几年已在该实验室得到鉴定和表征。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，铁代谢会出现异常，与 IRP1 功能丧失相关，包括红细胞增多症，还会出现肺动脉高压和心脏纤维化。 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。两名 IRP2 缺失的患者在生命的第一年出现严重的神经系统问题，并影响认知和运动功能。在缺乏 IRP1 的动物中，IRP 2 部分补偿了 IRP1 调节活性的丧失，而 IRP1 和 IRP2 均无效的动物会在早期胚胎时死亡。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。我们发现 Irp1-/- 小鼠会出现促红细胞生成素驱动的红细胞增多症和肺动脉高压，并且 Irp1 通过与 5' 铁反应元件结合来抑制转录物的翻译，对于调节肺内皮和肾间质成纤维细胞中 HIF2 α 的表达非常重要在成绩单中。这些见解促使我们研究楚瓦什红细胞增多症，因为楚瓦什红细胞增多症是由 vHL 基因突变引起的，该突变降低了 vHL 复合物对缺氧诱导因子的识别和降解。发现 Irp1 对于 HIF2 α 的翻译抑制很重要后，我们用 TEMPOL 或 HIF2 α 抑制剂治疗 Chuvash 敲入小鼠模型，并逆转了红细胞增多症。与 Irp1-/- 小鼠杂交证明，TEMPOL 激活 Irp1 的 IRE 结合对于减少这些小鼠中促红细胞生成素的产生至关重要，并证明饮食 TEMPOL 是治疗 Chuvash 红细胞增多症的良好候选药物。我们发现铁转运蛋白在成熟红细胞中出乎意料地高表达，它输出血红素氧化释放的游离铁。我们培育出红系细胞中铁转运蛋白出现组织特异性缺失的小鼠。在缺乏铁转运蛋白的细胞中，氧化损伤会缩短成熟红细胞的寿命并导致轻度小细胞性贫血。相反，当输出端具有功能获得性突变 Q248H 时，我们与合作者的临床研究表明，Q248 突变通过减少可用于感染红细胞的疟疾寄生虫营养的铁来防止疟疾感染。这种突变在非洲疟疾流行地区（高达 20%）和非裔美国人中很常见，其患病率在非裔美国人中为 8-12%。这种突变可能会导致肝脏、肾脏和胰腺等组织铁过载。我们正在通过评估患者和制作敲击小鼠模型来启动关于铁状态和对突变携带者的潜在有害影响的研究，并得到 NIH Bench to Bedside 奖的支持。我们继续尝试寻找治疗 HMOX1-/- 患者的方法，将体外培养的巨噬细胞输注到先天的 Hmox1-/- 小鼠体内，我们观察到巨噬细胞输注可以预防疾病，并且巨噬细胞在肝脏中繁殖，数量变得更多比野生型小鼠。这些研究表明，HMOX1-/- 患者是一种罕见疾病，有一天可能可以通过纠正使用 CRISPR 技术从患者体内取出的巨噬细胞中的突变来治疗，并且输注表达正常 HMOX1 的巨噬细胞可以防止患者死于这种致命疾病。缺乏 HMOX1 的原因。我们鉴定了在吞噬红细胞作用时诱导表达的基因，并且我们正在分析允许吞噬红细胞作用进行而不导致细胞死亡的代谢重塑。