Erythroid transporter function in hemoglobin synthesis

红细胞转运蛋白在血红蛋白合成中的功能

• 批准号: 6850115
• 负责人: Orian S Shirihai
• 金额: $ 27.74万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-03 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850115
• 关键词: adenosinetriphosphatase; antioxidants; antisense nucleic acid; biophysics; confocal scanning microscopy; cytoplasm; embryonic stem cell; enzyme activity; erythroid stem cell; erythropoiesis; free radical oxygen; gene expression; hemoglobin; liposomes; membrane potentials; membrane transport proteins; mitochondrial membrane; mutant; oligonucleotides; oxidative stress; porphyrin biosynthesis; protein biosynthesis; protein structure function; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): A number of disorders due to abnormalities in heme biosynthesis influence erythropoiesis. During normal erythroid maturation, heme precursors are generated in the mitochondria, modified in the cytosol and then returned to the mitochondria for final assembly with iron. Remarkably, accumulation of toxic heme intermediates or substrates, and secondary mitochondrial damage, are key elements in the pathophysiology of all heme biosynthesis disorders. However, the mechanisms of export or import of heme products, by-products and intermediates to or from the mitochondrial matrix are as yet poorly understood. This proposal will focus on such mechanisms taking advantage of our discovery of ABC-me, a novel mitochondrial erythroid transporter. ABC-me expression is controlled by the erythroid transcription factor GATA-1 and is down regulated by heme. In differentiating erythroleukemic cells, ABC-me is rate limiting for heme biosynthesis. As such, ABC-me is the only mitochondrial inner membrane transporter implicated in heme biosynthesis. To explore its function in heme biosynthesis, we have developed ABC-me deficient cell culture models and functional assays of reconstituted transporter in proteoliposomes. We have established that differentiating erythroid cells with reduced ABC-me activity produce less heme and exhibit signs of mitochondrial stress. We postulate that transporters involved in the heme pathway serve as gatekeepers for reactive substrates, intermediates and byproducts and thus couple production of essential end products to protection from toxic intermediates. We propose a combined biochemical and biophysical approach to the study of the function of ABC-me in heme biosynthesis and the consequences of its malfunction. We will address the following questions: 1) Which steps in the heme biosynthetic pathway are facilitated by ABC-me? 2) Does ABC-me deficiency result in the accumulation of heme precursors in the cytosol and mitochondria? 3) What is the source of mitochondrial stress associated with ABC-me deficiency and what are the morphological and functional consequences? 4) Does ABC-me transport a heme biosynthesis intermediate, product or co-factor, and in which direction? 5) Does ABC-me facilitate heme biosynthesis indirectly by opposing mitochondrial stress.

描述（由申请人提供）：由于血红素生物合成异常而导致的许多疾病会影响红细胞生成。在正常的红系成熟期间，在线粒体中产生血红素前体，在细胞质中修饰，然后返回线粒体与铁一起进行最终组装。值得注意的是，有毒血红素中间体或底物的积累以及继发性线粒体损伤是所有血红素生物合成障碍的病理生理学中的关键因素。但是，血红素产物的导出或导入机制，副产品和来自线粒体基质的副产品和中间体尚未了解。该建议将集中于利用我们发现ABC-ME的这种机制，ABC-ME是一种新型的线粒体红斑转运蛋白。 ABC-ME表达由红系转录因子GATA-1控制，并由血红素调节。在区分红血病细胞中，ABC-ME是血红素生物合成的速率限制。因此，ABC-ME是与血红素生物合成有关的唯一线粒体内膜转运蛋白。为了探索其在血红素生物合成中的功能，我们开发了ABC-ME缺陷的细胞培养模型和蛋白质脂质体中重构转运蛋白的功能测定。我们已经确定，将红细胞细胞与降低的ABC-ME活性区分开会产生的血红素较少，并且表现出线粒体应激的迹象。我们假设参与血红素途径的转运蛋白是反应性底物，中间体和副产品的守门人，从而将基本最终产物的产生二对造成了毒性中间体的保护。我们提出了一种合并的生化和生物物理方法，以研究ABC-ME在血红素生物合成中的功能及其故障的后果。我们将解决以下问题：1）ABC-ME促进了血红素生物合成途径中的哪些步骤？ 2）ABC-ME缺乏会导致血红素前体在细胞质和线粒体中积累吗？ 3）与ABC-ME缺乏相关的线粒体应力的来源是什么？形态和功能后果是什么？ 4）ABC-ME是否将血红素生物合成中间体，产物或副因素转运？ 5）ABC-ME通过相反的线粒体应激间接促进血红素生物合成。