Regulation of Protein Synthesis and Erythropoiesis

描述（由申请人提供）：我们的长期目标是促进对正常和异常血液学条件下血红蛋白合成和红细胞分化的调节的理解，特别强调血红素和蛋白质合成在这些过程中的作用。完整网织红细胞及其裂解物中的蛋白质合成依赖于血红素的可用性。血红素作为血红蛋白的辅基，血红蛋白是红细胞及其晚期前体细胞中的主要蛋白质。生物化学研究表明，在血红素缺乏或铁缺乏的条件下，由于血红素调节的翻译抑制剂（HRI）的激活，蛋白质合成在起始水平被抑制。HRI是一种血红素调节的蛋白激酶，其磷酸化真核起始因子2（eIF 2a）的α-亚基。eIF 2的α亚基的磷酸化损害其在翻译起始中的再循环，并导致蛋白质合成的停止。本研究设计为（1）阐明HRI调节红细胞生成的分子机制，（2）评估HRI在红细胞疾病中的保护作用，（3）阐明HRI应激激活的分子机制。所采用的方法是遗传修饰小鼠的产生、血液学分析、组织学和电子显微镜检查、红系祖细胞的集落培养测定、Western印迹分析和蛋白激酶测定、细胞培养、小鼠胚胎的表型检查、重组DNA技术。我们最近的研究表明，HRI不仅是球蛋白合成所必需的，也是血红素生物合成所必需的。HRI对地中海贫血和红细胞生成性原卟啉症小鼠的存活也很重要。我们提出的研究可能导致HRI在治疗人类红细胞疾病患者中的潜在应用。

DESCRIPTION (provided by applicant): Our long-term objective is to contribute to the understanding of the regulation of hemoglobin synthesis and erythroid differentiation in normal and abnormal hematological conditions, with particular emphasis on the roles of heme and protein synthesis in these processes. Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. Heme serves as the prosthetic group of the hemoglobin, the predominant protein in red blood cell (RBC) and its late precursor cells. Biochemical studies have shown that, under conditions of heme-deficiency or iron-deficiency, protein synthesis is inhibited at the level of initiation due to the activation of the heme-regulated translational inhibitor (HRI). HRI is a heme-regulated protein kinase that phosphorylates the a-subunit of eukaryotic initiation factor 2 (eIF2a). Phosphorylation of the a-subunit of eIF2 impairs its recycling in translational initiation and results in the cessation of protein synthesis. The research designs are (1) To Elucidate the Molecular Mechanism by which HRI Regulates Red Blood Cell Production, (2) To Assess the Protective Role of HRI in Red Blood Cell Disorders, and (3) To Delineate the Molecular Mechanism of Stress-Activation of HRI. The methods to be employed are the generation of genetically modified mice, hematological analysis, histological and electron microscopic examinations, colony culture assays for erythroid progenitors, Western-blot analysis and protein kinase assays, cell culture, phenotypic examination of mouse embryos, recombinant DNA techniques. Our recent study indicates that HRI is not only necessary for globin synthesis, but also for heme biosynthesis. HRI is also important for the survival of mice with thalassemia and erythropoietic protoporphria. Our proposed study may lead to potential application of HRI in treating human patients with red cell diseases.