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有助于更全面地了解应激条件和红细胞疾病下血红蛋白合成和红细胞生成的调节。在这一建议中，我们关注血红素调控的eIF2¿激酶（HRI）在氧化应激和应激性红细胞生成中的翻译控制。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细胞前体中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¿的磷酸化不仅抑制了ATF4 mRNA的翻译，而且选择性地增加了ATF4 mRNA在原发性红系前体中的翻译。这种由hri激活的ATF4应激反应途径是减轻氧化应激和促进红细胞分化所必需的。最近，HRI-eIF2¿P通路已被证明可诱导红系分化的人CD34+细胞产生胎儿血红蛋白（HbF）。虽然红细胞生成过程中的转录调控已被广泛研究，但对翻译控制在这一过程中的作用知之甚少。我们假设eIF2¿p介导的翻译对于缓解应激性红细胞生成过程中的氧化应激和促进红细胞分化是必要的。我们将采用最近开发的全基因组方法，核糖体分析，研究血红素缺乏症和地中海贫血患者正常和应激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敲入蛋白（红系- A/A）小鼠，它在红系谱系中特异性地存在eIF2¿P信号缺陷。通过FACS分选从野生型（Wt）、Hri-/-和erythroid-A/A小鼠的胎儿肝脏中分离出红母细胞，用于研究血红素、Hri和eIF2¿P在体内全基因组翻译调控中的作用。此外，核糖体分析也将在地中海贫血小鼠的脾嗜碱性红细胞中进行。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。本研究还将揭示红系分化过程中翻译调控的新分子机制和新蛋白的产生。

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.