STRUCTURE/FUNCTION OF HEME REGULATED ELF-2ALPHA KINASE

血红素调节的 ELF-2α 激酶的结构/功能

• 批准号: 2446303
• 负责人: JANE-JANE CHEN
• 金额: $ 20.66万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-15 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2446303
• 关键词: X ray crystallography; alkylation; cell differentiation; cell growth regulation; crosslink; enzyme activity; enzyme inhibitors; enzyme structure; erythroid stem cell; erythropoiesis; heme; hemoglobin; hemoprotein biosynthesis; intermolecular interaction; mass spectrometry; molecular site; phosphorylation; protein binding; protein kinase; protein sequence; protein structure function; proteolysis; reticulocytes; site directed mutagenesis; stress proteins

DESCRIPTION: The long-term objectives are the elucidation of the regulation of hemoglobin synthesis and erythropoiesis in normal and abnormal hematologic conditions. The focus of this research is to study the mechanisms of regulation of heme-regulated EIF-2a kinase (HRI) at the molecular structural level. HRI is a potent inhibitor of protein synthesis in immature erythroid cells. Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. In heme-deficiency, protein synthesis is inhibited at the level of initiation due to the activation of HRI. HRI is a heme-regulated protein kinase that phosphorylates the a-subunit of EIF-2 impairs its recycling in translational initiation and results in the cessation of protein synthesis. HRI is mainly expressed in erythroid cells and the physiological role of HRI is to maintain the balanced synthesis of heme and globin chains. The specific aims of this proposed study are (1) Regulation of HRI by phosphorylation; (2) Molecular mechanisms in the regulation of HRI by heme, the prosthetic group of hemoglobin which is the principal proteins in reticulocytes; ( 3) Determination of HRI structure by X-ray crystallography. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, synthesis of oligodeoxynucleotides, high pressure liquid chromatography, amino acid microsequencing, in vitro transcription and translation, protein kinase assays, gel electrophoresis, deoxynucleotide sequencing, expression of HRI and other proteins using recombinant baculovirus, immunoaffinity column chromatography, immunoprecipitation, Western blot analysis, and X-ray crystallography. HRI is a key regulator of protein synthesis, a process which is essential for the regulation of cell growth, differentiation, antiviral response and hormonal responses. Structure-function analysis of HRI will advance our knowledge not only on the regulation of protein kinases which are vital in the regulation of various cellular processes, but also on the regulation of heme proteins.

描述：长期目标是阐明法规 血红蛋白合成和红细胞生成在正常和异常 血液状况。 本研究的重点是研究 血红素调节的EIF-2a激酶（HRI）的调节机制 分子结构水平。 HRI是蛋白质合成的有效抑制剂 在未成熟的红细胞中。 完整网织红细胞中的蛋白质合成， 它们的裂解物依赖于血红素的可用性。 在血红素缺乏症中， 蛋白质合成在起始水平受到抑制， 激活HRI。 HRI是血红素调节的蛋白激酶， 磷酸化EIF-2的α-亚基损害其在翻译中的再循环， 引发并导致蛋白质合成的停止。 HRI主要是 在红系细胞中表达，HRI的生理作用是 维持血红素和球蛋白链的平衡合成。 具体 本研究的目的是：（1）磷酸化对HRI的调控; (2)血红素调节HRI的分子机制 一组血红蛋白，是网织红细胞中的主要蛋白质;（3） 通过X射线晶体学确定HRI结构。 所采用的方法是重组DNA技术、聚合酶链反应、 反应，寡脱氧核苷酸合成，高压液相 层析、氨基酸微测序、体外转录和 翻译，蛋白激酶测定，凝胶电泳，脱氧核苷酸 测序、使用重组蛋白表达HRI和其它蛋白 杆状病毒，免疫亲和柱层析，免疫沉淀， 蛋白质印迹分析和X射线晶体学。 HRI是一个关键的监管机构， 蛋白质合成，这是一个过程，这是必不可少的调节细胞 生长、分化、抗病毒反应和激素反应。 HRI的结构-功能分析不仅可以提高我们对HRI的认识， 蛋白激酶的调节，这是至关重要的调节 各种细胞过程，而且对血红素蛋白的调节。