HRI-eIF2a Phosphorylation signaling in oxidative stress and erythropoiesis

描述（由申请人提供）：我们这项拟议研究的长期目标是促进更全面地了解应激条件下和红细胞疾病中血红蛋白合成和红细胞生成的调节。在这个建议中，我们专注于翻译控制血红素调节eIF2 <$激酶（HRI）在氧化应激和应激红细胞生成。我们的实验室已经证明，HRI是必要的，以减少无效的红细胞生成和维持适当的基因表达在红细胞前体在缺铁。HRI也是降低地中海贫血表型严重程度的关键。eIF 2的磷酸化¶ HRI不仅全面抑制了ATF 4 mRNA的翻译，而且选择性地增加了原代红系前体细胞中ATF 4 mRNA的翻译。这种HR1激活的ATF4应激反应途径对于减轻氧化应激和促进红细胞分化是必需的。最近，HRI-eIF2？P途径已显示在经历红系分化的人CD 34+细胞中诱导胎儿血红蛋白（HbF）产生。虽然红细胞生成过程中的转录调控已被广泛研究，但对翻译调控在这一过程中的作用知之甚少。我们假设eIF2？P-介导的翻译是必要的，以减轻氧化应激和促进红系分化在应激红细胞生成。我们将采用最近开发的全基因组方法，核糖体分析，研究血红素缺乏和地中海贫血的正常和应激红细胞生成过程中的翻译调控。将产生红系特异性eIF2 <$Ser51Ala敲入（红系-<$A/A）小鼠的新品系，其在红系谱系中特异性地在eIF2 <$P信号传导中有缺陷。在铁充足和缺乏的条件下，通过FACS分选从野生型（Wt）、Hri-/-和红系-A/A小鼠的胎肝分离的成红细胞将用于研究血红素、HRI和eIF2 μ P在调节体内全基因组翻译中的作用。此外，还将在地中海贫血小鼠的脾嗜碱性成红细胞中进行核糖体分析。这些拟议研究的结果将阐明血红素和eIF 2 µ P介导的翻译在应激条件下红细胞生成中的重要作用。这项拟议的研究还将揭示新的分子机制，在翻译调节和新的蛋白质产生的红系分化过程中。 从这些研究中获得的新信息将大大推进红细胞生成领域，并将对血红蛋白病新疗法的发展产生非常重要的影响。

DESCRIPTION (provided by applicant): Our long-term objective for this proposed research is to contribute to the more comprehensive understanding of the regulation of hemoglobin synthesis and erythropoiesis under stress conditions and in red cell disorders. In this proposal, we focus on translational control by the heme-regulated eIF2¿ kinase (HRI) in oxidative stress and stress erythropoiesis. Our laboratory has demonstrated that HRI is necessary to reduce ineffective erythropoiesis and to maintain proper gene expression in erythroid precursors during iron deficiency. HRI is also essential for reducing the phenotypic severities of ¿-thalassemia. Phosphorylation of eIF2¿ by HRI not only inhibits translation globally, but also selectively increases the translation of ATF4 mRNA in primary erythroid precursors. This HRI-activated ATF4 stress response pathway is necessary to mitigate oxidative stress and to promote erythroid differentiation. Most recently, HRI-eIF2¿P pathway has been shown to induce fetal hemoglobin (HbF) production in human CD34+ cells undergoing erythroid differentiation. While transcriptional regulation during erythropoiesis has been studied extensively, much less is known about the role of translational control in this process. We hypothesize that eIF2 ¿P-mediated translation is necessary to mitigate oxidative stress and to promote erythroid differentiation during stress erythropoiesis. We will employ a recently developed genome-wide approach, ribosome profiling, to study translational regulation during normal and stress erythropoiesis in heme deficiency and in ¿-thalassemia. A novel line of erythroid-specific eIF2¿Ser51Ala knockin (erythroid- ¿A/A) mice, which are defective in eIF2¿P signaling specifically in the erythroid lineage, will be generated. Erythroblasts isolated by FACS sorting from fetal livers of wild type (Wt), Hri-/- and erythroid-A/A mice under iron sufficient and deficint conditions, will be used to study the role of heme, HRI and eIF2¿P in regulating in vivo translation genome-wide. In addition, ribosome profiling will also be performed in splenic basophilic erythroblasts of ¿-thalassemic mice. The outcomes of these proposed studies will elucidate the essential role of heme and eIF2¿P-mediated translation in erythropoiesis under stress conditions. This proposed research will also uncover novel molecular mechanisms in translational regulation and new proteins produced in the erythroid lineage during differentiation. The novel information obtained from these studies will advance the field of erythropoiesis greatly and will have a very significant impact on the development of new therapies for hemoglobinopathies.