Regulation of mitochondrial heme metabolism by Tmem14c

描述（由申请人提供）：该项目的长期目标是鉴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。本项目对确定红系卟啉症和贫血的基因修饰因子具有重要意义，因此具有公共卫生意义。我们的实验室已经确定Tmem14c是一种新的线粒体蛋白，是脊椎动物红细胞末段分化和血红蛋白化所必需的。血红素合成酶存在于缺乏tmem14c的小鼠红白血病（MEL）细胞中，我们实验室的初步数据表明，缺乏tmem14c的MEL细胞中的铁转运与野生型MEL细胞相当。然而，缺乏tmem14c的MEL细胞合成血红素和原卟啉IX水平降低。这些数据表明，Tmem14c可能促进线粒体卟啉转运，从而在血红素/卟啉代谢中发挥关键作用。在Aim 1中，我们将通过鉴定Tmem14c缺陷细胞中卟啉代谢的阻滞来验证这一假设，比较野生型和Tmem14c缺陷MEL细胞的线粒体和细胞质部分中尿卟啉原III（细胞质）、同比例卟啉原III（线粒体/细胞质）和原卟啉IX（线粒体）的水平。通过这种方式，我们将确定tmem14c缺陷细胞中血红素合成的阻滞是否是卟啉运输缺陷的结果。由于Tmem14c与其他蛋白质形成高阶复合物，我们假设Tmem14c与其伙伴的相互作用在调节其功能中起着至关重要的作用。在Aim 2中，我们将确定Tmem14c伴侣并表征它们在血红素合成途径中调节Tmem14c的作用；我们将鉴定终末红细胞分化、血红素合成和卟啉运输所需的相互作用蛋白，并重点鉴定调节Tmem14c定位和蛋白稳定性的蛋白。总的来说，我们的研究将有助于我们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。这一提议的具体目标具有特别重要的意义，因为对Tmem14c功能的研究将揭示对血红素代谢和红细胞生成调节至关重要的卟啉运输途径。这些实验目标是我在造血方面研究生工作的合理延续，但提供了一个框架，使我能够获得使用斑马鱼作为动物模型的大量培训，并为进一步研究红系生物学提供了宝贵的技术。

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.