细胞间血红素分子伴侣HRG-3的功能机理研究

Heme is a critical cofactor for proteins such as hemoglobin, myoglobin, cytochromes and peroxidases. It plays essential roles in various biological processes including oxygen transport, electron transport, gas sensing, xenobiotic detoxification, signal transduction, microRNA processing, and circadian clock control. Since free heme is cytotoxic due to its peroxidase activity, cells have developed specific trafficking pathways to import, transport, sequester, and utilize heme. In most eukaryotes, heme is synthesized via a conserved eight-step biosynthetic pathway, which is regulated by many effectors. It is difficult to study heme trafficking pathways due to the complexity of intrinsic heme production. The free-living roundworm C. elegans is unable to synthesize heme, despite requiring this iron-containing compound nutritionally. Thus, this animal provides an ideal genetic system for dissecting heme trafficking pathways in metazoa. Research on C. elegans has led to the discoveries of the heme importer HRG-1 and the first intercellular heme chaperone HRG-3. Our preliminary work has shown that HRG-3 is required for the delivery of maternal heme to oocytes and developing embryos in the worm. However, the mechanisms responsible for HRG-3-mediated heme transport are largely unknown. For example, what is the receptor of HRG-3 in oocytes or embryos? What molecules escort HRG-3 to move from the intestine to the uterus? What is the fate of HRG-3 after it delivers heme? The main objective of this proposal is to identify and characterize novel proteins involved in intercellular heme trafficking using C. elegans as the model organism. We propose to purify the HRG-3-associated complexes from worms and identify the peptides by mass spectrometry. The functions of HRG-3 partners in heme metabolism will be interrogated using the following approaches. First, we will synthesize transcriptional and translational reporter constructs to determine the temporal and spatial expression pattern of candidate genes in the worm. Second, the candidate genes will be expressed in mammalian cells for the analysis of subcellular localization. Third, we will utilize the loss-of-function studies in C. elegans to evaluate the specific roles of candidate genes in: a) heme-dependent growth and development, b) organismal heme homeostasis, and c) intercellular heme trafficking. Collectively, characterization of intercellular heme trafficking pathways will improve the understanding of heme metabolism. Results from this proposal will also provide a molecular blueprint to model parasitic heme acquisition systems, which could serve as potential drug targets for helminthic control. In addition, the findings in this study will facilitate the identification of the homologous heme trafficking pathways in mammals, and provide new genetic basis for treating human blood disorders such as anemias and porphyrias.

血红素是血红蛋白、肌红蛋白、细胞色素等蛋白的功能辅基，在氧运输、电子传递、昼夜节律调控等细胞活动中起重要作用。游离态的血红素具有过氧化物酶活性和细胞毒性，因此，动物细胞必须利用特定的分子机制吸收、转运和贮存血红素。秀丽线虫自身不能合成血红素，但其细胞代谢和生长繁殖又需要血红素，因此是研究血红素运输的理想动物模型。通过在秀丽线虫上的研究，我们发现了首个细胞间血红素伴侣-HRG-3，它负责将肠道吸收的血红素通过循环系统运输至卵细胞和虫胚，因此对胚胎的营养发育十分重要。为了阐明HRG-3的作用机理，本项目拟通过蛋白质组学、细胞生物学、遗传学等研究手段，筛选与HRG-3相互作用的蛋白分子，并鉴定它们在血红素运输过程中的功能。我们将进一步寻找这些分子在寄生虫和哺乳动物上的同源蛋白。预期发现的HRG-3受体及相关蛋白将完善对细胞间血红素运输通道的认识，并为防治寄生虫病、贫血、卟啉症等疾病提供理论依据。

血红素是自然界所有生命体都必需的一种卟啉化合物，它在细胞中的稳态代谢受特定分子通路调控。我们通过前期研究，在模式动物秀丽线虫上发现了细胞间血红素分子伴侣HRG-3，但其作用机制还不清楚。本项目提出以HRG-3为切入点，鉴定新的血红素代谢相关分子，深入探究动物细胞中的血红素运输通路。我们通过遗传筛选，在秀丽线虫上发现了两个同源的血红素感应基因tngo-a和tngo-b，它们的表达同时受血红素水平和HRG-3影响，研究结果表明TNGO很可能是一个细胞内血红素分子伴侣。本项目取得的主要研究结果包括：（1）秀丽线虫tngo-a和tngo-b主要表达于肠细胞，线虫TNGO蛋白定位于顶端细胞膜和细胞核，而人的同源蛋白TANGO定位于线粒体和细胞质。（2）tngo-a和tngo-b调控线虫肠细胞的血红素稳态。我们构建了tngo基因敲除线虫，发现敲除tngo影响了秀丽线虫对血红素的感应，对血红素类似物GaPP毒性的耐受，以及对荧光血红素类似物ZnMP的吸收。（3）哺乳动物TANGO调控细胞及线粒体内的血红素稳态。我们在人的体外细胞系中敲除了TANGO，发现TANGO缺失后，线粒体中血红素累积，并且细胞能够耐受较高浓度血红素的毒性。（4）我们通过体外蛋白纯化和生化实验发现TANGO是一个血红素结合蛋白。综合遗传学、细胞生物学、生物化学等实验的结果，我们推测TNGO很可能是一个血红素分子伴侣，负责调控细胞内血红素稳态。TNGO及其同源蛋白的发现有望填补细胞内血红素稳态领域的空白，为血红素代谢学科提供新的认识和发展方向，并为认识及防治相关遗传性贫血及血红素代谢疾病提供潜在的理论依据。