Erythroid transporter function in hemoglobin synthesis

描述（由申请人提供）：由于血红素生物合成异常导致的许多疾病影响红细胞生成。在正常的红细胞成熟过程中，血红素前体在线粒体中产生，在细胞质中被修饰，然后返回线粒体与铁进行最终组装。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。然而，血红素产品，副产品和中间体的出口或进口或从线粒体基质的机制还知之甚少。这项建议将集中在这样的机制，利用我们发现的ABC-me，一种新的线粒体红细胞转运蛋白。ABC-me表达受红系转录因子加塔-1控制，并受血红素下调。在分化的红白血病细胞中，ABC-me是血红素生物合成的速率限制。因此，ABC-me是唯一涉及血红素生物合成的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缺陷细胞培养模型和蛋白脂质体中重构转运蛋白的功能测定。我们已经确定，ABC-me活性降低的分化红系细胞产生较少的血红素，并表现出线粒体应激的迹象。我们假设，转运蛋白参与血红素途径作为反应底物，中间体和副产物的看门人，从而夫妇生产的必需的最终产品，以保护有毒中间体。我们提出了一个生物化学和生物物理相结合的方法来研究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胞质和线粒体中的积累吗？3)与ABC-me缺乏相关的线粒体应激的来源是什么？形态和功能后果是什么？4)ABC-me是否运输血红素生物合成中间体、产物或辅因子，以及向哪个方向运输？5)ABC-me是否通过对抗线粒体应激间接促进血红素生物合成？

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.