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一些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，在细胞质中修饰，然后返回线粒体与铁进行最后的组装。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的关键因素。然而，输往或输往线粒体基质的血红素产物、副产物和中间体的机制尚不清楚。本研究将利用我们发现的一种新的线粒体红系转运蛋白ABC-me来研究这种机制。ABC-me的表达受红系转录因子GATA-1控制，并受血红素下调。在分化红白血病细胞时，ABC-me是血红素生物合成的限速因子。因此，ABC-me是唯一参与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，我们建立了ABC-me缺陷细胞培养模型和重组转运蛋白在蛋白脂质体中的功能测定。我们已经确定，与ABC-me活性降低分化红细胞产生较少的血红素和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作为反应性底物、中间体和副产物的守门人，从而将基本终产物的生产偶联起来，以保护免受有毒中间体的侵害。我们提出了一种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功能失调的后果。我们将解决以下问题：1)血红素生物合成途径中的哪些步骤是由ABC-me促进的？2) ABC-me缺乏是否导致细胞质和线粒体中血红素前体的积累？3)与ABC-me缺乏相关的线粒体应激的来源是什么？形态学和功能上的后果是什么？4) ABC-me是否转运血红素生物合成中间体、产物或辅助因子？转运方向是什么？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.