HRI-eIF2a Phosphorylation signaling in oxidative stress and erythropoiesis

描述(由申请人提供)：我们对这项拟议研究的长期目标是帮助更全面地了解应激条件下和红细胞紊乱情况下的血红蛋白合成和红细胞生成的调节。在这项建议中，我们重点关注在氧化应激和应激性红细胞生成中由血红素调节的eIF2？激酶(HRI)进行的翻译控制。我们的实验室已经证明，在缺铁期间，HRI对于减少无效的红细胞生成和维持红系前体细胞适当的基因表达是必要的。HRI对于降低地中海贫血的表型严重程度也是必不可少的。HRI对eIF2的磷酸化不仅在整体上抑制翻译，而且选择性地增加原始红系前体中ATF4mRNA的翻译。这种HRI激活的ATF4应激反应通路对于减轻氧化应激和促进红系分化是必要的。最近，HRI-eIF2？P通路被证明在人CD34细胞红系分化过程中诱导胎儿血红蛋白(HBF)的产生。虽然红细胞生成过程中的转录调控已经得到了广泛的研究，但对翻译调控在这一过程中的作用却知之甚少。我们假设eIF2？P介导的翻译在应激性红细胞生成过程中对于减轻氧化应激和促进红系分化是必要的。我们将使用最近开发的全基因组方法，核糖体图谱，来研究血红素缺乏症和地中海贫血患者在正常和应激性红细胞生成过程中的翻译调节。将产生一种新的红系特异性eIF2？Ser51Ala敲门小鼠(红系-A/A)，这些小鼠在红系谱系中存在eIF2？P信号缺陷。在铁充足和缺铁条件下，用流式细胞仪分选野生型(Wt)、HRI-/-和红系-A/A小鼠胎肝中的红细胞，研究血红素、HRI和eIF2？P在体内全基因组翻译调控中的作用。此外，还将在地中海贫血小鼠的脾嗜碱性红细胞中进行核糖体分析。这些研究的结果将阐明血红素和eIF2？P介导的翻译在应激条件下的红细胞生成中的重要作用。这项拟议的研究还将揭示翻译调控的新分子机制和红系分化过程中产生的新蛋白质。 从这些研究中获得的新信息将极大地促进红细胞生成领域的发展，并将对血红蛋白疾病的新疗法的开发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.