Cell Cycle Regulation of IRP2 Phosphorylation During Hematopoiesis

造血过程中 IRP2 磷酸化的细胞周期调控

基本信息

批准号：
10639952
负责人：
Elizabeth Ann Leibold
金额：
$ 55.5万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10639952
关键词：
3&apos Untranslated Regions Affect Alanine Alleles Anemia Binding Binding Proteins Blood Cell Count Bone Marrow Bone Marrow Cells Bone Marrow Transplantation CRISPR/Cas technology Cell Cycle Cell Cycle Arrest Cell Cycle Regulation Cell Death Cell Differentiation process Cell Lineage Cell Survival Cells Characteristics Codon Nucleotides DNA biosynthesis Development Duodenum Dysmyelopoietic Syndromes Elements Embryo Engineering Enterobacteria phage P1 Cre recombinase Enterocytes Erythroblasts Erythrocytes Erythroid Erythroid Cells Erythropoiesis Exhibits Ferritin Fibroblasts Flow Cytometry Functional disorder G2 Phase Gene Expression Generations Goals Hela Cells Hematological Disease Hematology Hematopoiesis Hematopoietic stem cells Heme Hemoglobin In Vitro Inherited Iron Iron Regulatory Protein 2 Knock-in Mouse LoxP-flanked allele Macrocytic Anemia Mediating Messenger RNA Mitochondria Mitosis Molecular Mouse Strains Mus Mutate Oxidative Stress Oxygen Pathway interactions Persons Phenotype Phosphorylation Physiological Population Process Production Proliferating Proteins Proteomics RNA RNA Binding Reporting Respiration Role S phase SKP Cullin F-Box Protein Ligases Serine Serum Spleen Splenomegaly Supporting Cell System TFRC gene Testing Tissues Translations Transplantation cell growth cell growth regulation cofactor erythroid differentiation in vivo insight iron deficiency iron metabolism mRNA Stability mRNA Translation metal transporting protein 1 mouse model mutant novel posttranscriptional single-cell RNA sequencing stem stem cell proliferation transplant model ubiquitin ligase uptake

项目摘要

ABSTRACT Iron is essential for cellular growth due to its role as a cofactor in proteins involved in DNA synthesis, mitochondrial respiration and hemoglobin production. Vertebrate iron metabolism is controlled post- transcriptionally by iron-regulatory protein 2 (Irp2). Irp2 binds to iron-responsive elements (IREs) in the mRNAs of proteins involved in iron uptake (transferrin receptor 1), sequestration (ferritin) and export (ferroportin), and regulates the translation or stability of these mRNAs. During iron deficiency, Irp2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency, Irp2 is degraded by the FBXL5-SCF ubiquitin ligase. Here we show that Irp2 is regulated by iron-independent phosphorylation of Ser157 during G2/M that disrupts its interaction with ferritin IRE mRNA during mitosis and depresses ferritin translation. Expression of Irp2-S157A in Irp2KO-MEFs causes a G2/M delay and slows proliferation. The significance of S157 phosphorylation was investigated in mice where Ser157 was mutated to Ala157 (Irp2A/A)). Irp2A/A mice exhibit macrocytic anemia, defective erythroid terminal differentiation, splenomegaly, and dysregulated systemic iron metabolism. We propose that cell-cycle regulation of ferritin and other IRE-mRNAs may provide a mechanism to modulate the cellular labile iron pool during the cell cycle. Our overall goal is to determine how loss of S157 phosphorylation perturbs erythropoiesis and causes anemia. Our aims are to 1) determine the cell-autonomous role for Irp2- S157 phosphorylation in erythropoiesis by bone marrow (BM) transplantation and flow cytometric analysis of cell-cycle status, proliferation, iron and other parameters, 2) identify mechanisms underlying dysregulated erythropoiesis in Irp2A/A mice using a mouse erythroblast in vitro differentiation system with WT and Irp2A/A BM cells, and proteomic analysis of erythroblast populations to identify protein changes between WT and Irp2A/A cells, and 3) generate an erythroid-specific Irp2A/A knockin mouse model to study erythroid terminal differentiation. We anticipate that our studies will provide new insights of normal and dysregulated erythropoiesis.

抽象的铁对于细胞生长至关重要，因为它是参与DNA合成的蛋白质中的辅因子的作用，线粒体呼吸和血红蛋白产生。脊椎动物铁代谢受到控制。通过铁调节蛋白2（IRP2）在转录上。 IRP2与mRNA中的铁响应元件（IRE）结合涉及铁摄取（转铁蛋白受体1），固相（铁蛋白）和出口（铁蛋白）的蛋白质的蛋白质，以及调节这些mRNA的翻译或稳定性。在铁缺乏期间，IRP2结合IRES以调节mRNA 翻译或稳定性，而在铁充足期间，IRP2被FBXL5-SCF泛素连接酶降解。在这里，我们表明IRP2受G2/m期间Ser157的铁无依赖性磷酸化调节它与铁蛋白IRE mRNA的相互作用在有丝分裂过程中并降低铁蛋白翻译。 IRP2-S157A的表达在IRP2KO-MEF中会导致G2/m延迟并减慢增殖。 S157磷酸化的重要性是在将Ser157突变为ALA157（IRP2A/A）的小鼠中进行了研究。 IRP2A/A小鼠表现出大细胞性贫血，红细胞终末分化，脾肿大和全身铁代谢失调的缺陷。我们提出铁蛋白和其他IRE-MRNA的细胞周期调节可能会提供一种调节的机制细胞周期中的细胞不稳定铁池。我们的总体目标是确定S157磷酸化的损失葡萄球菌性红细胞生成并引起贫血。我们的目的是1）确定IRP2-的细胞自主作用 S157通过骨髓（BM）移植和流式细胞仪分析的红细胞生成磷酸化细胞周期状态，增殖，铁和其他参数，2）识别失调的机制 IRP2A/A小鼠中使用小鼠红细胞在体外分化系统中使用WT和IRP2A/A BM的促红细胞生成细胞，以及红细胞种群的蛋白质组学分析，以鉴定WT和IRP2A/A之间的蛋白质变化细胞和3）生成红细胞特异性IRP2A/敲蛋白小鼠模型来研究红系终端分化。我们预计我们的研究将提供正常和失调的新见解红细胞生成。