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而引起的一些疾病会影响红细胞生成。在正常的红系成熟过程中，血红素前体在线粒体中产生，在胞浆中被修饰，然后返回到线粒体，与铁进行最终组装。值得注意的是，有毒的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过程中的关键因素。然而，血红素产品、副产品和中间体进出口到线粒体基质或从线粒体基质进口的机制尚不清楚。这项提案将利用我们发现的ABC-Me，一种新型的线粒体红系转运体，专注于这种机制。ABC-Me的表达受红系转录因子GATA-1控制，并受血红素下调。在红白血病细胞的分化过程中，ABC-Me是血红素生物合成的限速因子。因此，ABC-Me是唯一与血红素生物合成有关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作用，我们建立了ABC-Me缺乏的细胞培养模型和蛋白脂质体中重组转运蛋白的功能分析。我们已经证实，分化的红系细胞ABC-Me活性降低，产生的血红素较少，并显示出线粒体应激的迹象。我们推测，参与血红素途径的转运体充当反应底物、中间体和副产品的守门人，从而将必要终端产品的生产与保护免受有毒中间体的影响结合在一起。我们提出了一种生化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促进了血红素生物合成途径中的哪些步骤？2)ABC-Me缺乏是否导致血红素前体在细胞质和线粒体中积累？3)与ABC-Me缺乏相关的线粒体应激的来源是什么？其形态和功能后果是什么？4)ABC-Me是运输血红素生物合成的中间体、产物还是辅助因子，并朝着哪个方向？5)ABC-Me是否通过对抗线粒体应激间接促进血红素的生物合成。

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.