Regulation of mitochondrial heme metabolism by Tmem14c

描述(申请人提供)：该项目的长期目标是确定和表征调节线粒体血红素代谢的新蛋白质，特别是在线粒体血红素/卟啉运输中发挥作用的蛋白质。该项目对确定红系卟啉症和贫血的遗传修饰因素具有重要意义，因此具有公共卫生意义。我们的实验室已经发现Tem14c是一种新的线粒体蛋白，它是脊椎动物红系终末分化和血红素化所必需的。缺陷型小鼠红白血病细胞中存在血红素合成酶，我们实验室的初步数据表明，缺陷型小鼠红白血病细胞中铁的转运与野生型相当。然而，缺乏Tem14c的MEL细胞合成的血红素和原卟啉IX水平降低。这些数据表明，Tem14c可能促进线粒体的卟啉转运，从而在血红素/卟啉代谢中发挥关键作用。在目标1中，我们将通过比较野生型和Tem14c缺陷型MEL细胞线粒体和细胞质部分中的尿卟啉原III(细胞质)、共比例卟啉原III(线粒体/细胞质)和原卟啉IX(线粒体)的水平来验证这一假设。通过这种方式，我们将确定Tem14c缺陷细胞中的血红素合成受阻是否是有缺陷的卟啉转运的结果。由于Tem14c与其他蛋白质形成高阶复合体，我们假设Tem14c与其合作伙伴的相互作用在调节其功能方面起着至关重要的作用。在目标2中，我们将确定Tem14c的合作伙伴，并研究它们在血红素合成途径中调节Tem14c的作用；我们将确定与红系终末分化、血红素合成和卟啉运输所需的相互作用蛋白，并重点研究调节Tem14c的定位和蛋白质稳定性的蛋白质。总而言之，我们的研究将有助于我们的长期目标，即了解控制血红素代谢的调节机制，以及血红素代谢与红细胞生成的相互作用。这项建议中的具体目标具有特别重要的意义，因为对Tem14c功能的研究将揭示对血红素代谢和红细胞生成的调节至关重要的、鲜为人知的卟啉转运途径。这些实验目标是我在造血学研究生工作中合乎逻辑的延续，但也提供了一个框架，可以在使用斑马鱼作为动物模型和其他技术方面获得实质性培训，这些技术将被证明对红系生物学的进一步研究非常有价值。

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and characterize novel proteins that regulate mitochondrial heme metabolism, particularly proteins that play a role in mitochondrial heme/porphyrin transport. This project has implications for identifying genetic modifiers for erythroid porphyria and anemia, and thus has public health significance. Our lab has identified Tmem14c as a novel mitochondrial protein that is required for terminal erythroid differentiation and hemoglobinization in vertebrates. Heme synthesis enzymes are present in Tmem14c-deficient murine erythroleukemia (MEL) cells, and preliminary data from our lab indicate that iron transport in Tmem14c-deficient MEL cells is comparable to wild-type MEL cells. However, Tmem14c-deficient MEL cells synthesize decreased levels of heme and protoporphyrin IX. These data suggest that Tmem14c may facilitate mitochondrial porphyrin transport, thereby playing a key role in heme/porphyrin metabolism. In Aim 1, we will test this hypothesis by identifying the block in porphyrin metabolism in Tmem14c deficient cells, comparing the levels of uroporphyrinogen III (cytoplasmic), coproporphyrinogen III (mitochondrial/cytoplasmic) and protoporphyrin IX (mitochondrial) in the mitochondrial and cytoplasmic fractions of wild-type and Tmem14c-deficient MEL cells. In this manner, we will determine if the block in heme synthesis in Tmem14c-deficient cells is a result of defective porphyrin trafficking. As Tmem14c forms higher-order complexes with other proteins, we hypothesize that Tmem14c's interactions with its partners play a crucial role in regulating its function. In Aim 2, we will identify Tmem14c partner and characterize their role in regulating Tmem14c in the heme synthetic pathway; we will identify interacting proteins that are required for terminal erythroid differentiation, heme synthesis and porphyrin transport, and focus on characterizing proteins that regulate Tmem14c's localization and protein stability. Collectively, our studies will contribute to our lon-term objective of understanding regulatory mechanisms controlling heme metabolism, and the interaction of heme metabolism with erythropoiesis. The specific aims in this proposal are of particular significance because studies of Tmem14c function will shed light on the poorly understood porphyrin trafficking pathways that are central to the regulation of heme metabolism and erythropoiesis. These experimental aims are a logical continuation of my graduate work in hematopoiesis, but provide a framework with which to obtain substantial training in the use of the zebrafish as an animal model and additional techniques that will prove invaluable for further studies in erythroid biology.