Cell Cycle Regulation of IRP2 Phosphorylation During Hematopoiesis

造血过程中 IRP2 磷酸化的细胞周期调控

• 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
• 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' 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

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.

摘要 铁是细胞生长所必需的，因为它是参与DNA合成的蛋白质中的辅因子， 线粒体呼吸和血红蛋白的产生。脊椎动物的铁代谢是在 铁调节蛋白2（Irp 2）。Irp 2与mRNA中的铁响应元件（IREs）结合 参与铁摄取（转铁蛋白受体1）、螯合（铁蛋白）和输出（膜铁转运蛋白）的蛋白质，以及 调节这些mRNA的翻译或稳定性。在缺铁时，Irp 2结合IRE调节mRNA 在铁充足期间，Irp 2被FBXL 5-SCF泛素连接酶降解。 在这里，我们发现Irp 2在G2/M期受到Ser 157的铁非依赖性磷酸化的调节， 它在有丝分裂期间与铁蛋白IRE mRNA相互作用并抑制铁蛋白翻译。Irp 2-S157 A的表达 在Irp 2KO-MEFs中，导致G2/M延迟并减缓增殖。S157磷酸化的意义在于： 在小鼠中研究，其中Ser 157突变为Ala 157（Irp 2A/A））。Irp 2A/A小鼠表现出大红细胞性贫血， 红系终末分化缺陷、脾肿大和全身铁代谢失调。我们 提出铁蛋白和其他IRE-mRNA的细胞周期调节可能提供了一种调节细胞周期的机制。 细胞周期中的细胞不稳定铁库。我们的总体目标是确定S157磷酸化的丧失 扰乱红细胞生成并引起贫血。我们的目标是：1）确定Irp 2的细胞自主作用， S157磷酸化在骨髓移植红细胞生成中的作用及流式细胞术分析 细胞周期状态、增殖、铁和其他参数，2）确定失调的潜在机制 使用WT和Irp 2A/A BM的小鼠成红细胞体外分化系统在Irp 2A/A小鼠中进行红细胞生成 细胞和成红细胞群体的蛋白质组学分析，以鉴定WT和Irp 2A/A之间的蛋白质变化 细胞，以及3）产生红系特异性Irp 2A/A敲入小鼠模型以研究红系末端 分化我们预计，我们的研究将提供新的见解正常和失调 红细胞生成