The Biological Role of Heme in Nutrition

描述(由申请人提供)：这项提案的长期目标是描述人类如何在生物体水平上调节和整合血红素动态平衡。血红素是一种含铁的有机环，是一种重要的辅因子，具有多种生物学功能。 除了作为人体营养中生物可利用铁的主要来源之外。血红素是一种在线粒体中合成的疏水性和细胞毒性的辅因子，尽管受体血红素蛋白存在于不同的细胞室。血红素是如何通过细胞膜和细胞器运输的？在上一个赠款周期中，我们率先使用了秀丽线虫，这是一种遗传动物模型，它不合成血红素，但利用环境中的血红素制造血红素蛋白，以识别七个新的分子，并建立一个血红素动态平衡和动物贩运的模型。在下一个资助周期中，我们将使用在上一个资助周期中开发的创新工具，以弥补在描绘组织和细胞如何传递其血红素状态以调节活体动物中的有机血红素动态平衡方面的重大知识空白。尽管在线虫(一种血红素营养缺陷型动物)中可能存在系统性的血红素交流和运输系统，但在人类、小鼠和斑马鱼中的几条证据表明，脊椎动物也一定存在这样的途径。在这里，我们建议阐明组织间通讯的机制，并利用线虫鉴定血红素信号的分子同一性。这项建议中的研究将检验这一假设，即HRG-7和HRG-8通过启动反馈反应来调节全身血红素稳态，并将肠道血红素状态与肠外组织联系和整合。为了在分子和组织水平上阐明HRG-7的作用机制，我们将确定HRG-7的催化活性、肠道分泌和亚细胞定位是否对全身血红素的动态平衡是必不可少的。为了在分子和组织水平上阐明HRG-8的功能机制，我们将通过使用异位表达和膜锚定版本的HRG-8突变体的功能互补来确定其遗传需求，并评估HRG-8和HRG-7对血红素信号功能的相互依赖。为了鉴定HRG-7和HRG-8的分子相互作用或底物，我们将使用多维蛋白质鉴定技术，通过RNAi敲除验证候选相互作用，并具体表征在人类中具有同源物的相互作用。我们对线虫的研究将克服哺乳动物模型系统带来的几个障碍，即通过外部手段控制细胞内血红素水平，以亚细胞分辨率对动物进行实验操作，以及在体内监测发育过程中组织之间的血红素信号的光学透明度。我们提出的项目具有变革性，因为在真核生物中，调节系统内血红素动态平衡的血红素运输和分配的分子和机制在很大程度上是未知的。我们的研究结果可能会为解决其他微量营养素的类似问题提供一个启发式范例。 与公共卫生相关：铁缺乏是世界上头号营养失调，而血红素是人类消费中生物可利用的铁的形式。确定饮食中的血红素是如何运输的，并与全身的血红素水平相协调，这将使专门针对缺铁个人的合成血红素“营养食品”的设计成为可能。缺铁会因血红素营养缺乏性寄生虫(如肠道钩虫)的失血而加剧，这些寄生虫依赖宿主血红素生存。一种针对蠕虫中的血红素运输和调控途径的药物将是一种强大的驱虫剂。因此，确定跨物种的血红素运输途径将对我们对基本、临床和应用过程的理解产生深远而持久的影响。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to delineate how humans regulate and integrate heme homeostasis at the organismal level. Heme, an iron containing organic ring, functions as a vital cofactor responsible for diverse biological functions in addition to serving as the major source of bioavailable iron in human nutrition. Heme is a hydrophobic and cytotoxic cofactor synthesized in the mitochondria although acceptor hemoproteins reside in different cellular compartments. How is heme transported through cellular membranes and organelles? In the previous grant cycle, we pioneered the use of Caenorhabditis elegans, a genetic animal model which does not synthesize heme but utilizes environmental heme to manufacture hemoproteins, to identify seven new molecules and establish a model for heme homeostasis and trafficking in animals. In the next funding cycle, we will employ innovative tools developed in the previous grant cycle to bridge significant knowledge gaps in delineating how tissues and cells communicate their heme status to regulate organismal heme homeostasis in live animals. Although a systemic heme communication and transport system may be expected in C. elegans, a heme auxotroph, several lines of evidence in humans, mice and zebrafish indicate that such pathways must also exist in vertebrates. Herein, we propose to elucidate the mechanisms of inter-tissue communication and identify the molecular identity of the heme signal using C. elegans. The studies in this proposal will test the hypothesis that HRG-7 and HRG-8 communicate and integrate intestinal heme status with extraintestinal tissues by initiating a feedback response to regulate systemic heme homeostasis. To elucidate the mechanisms of HRG-7 function at the molecular and organismal level, we will determine if catalytic activity, intestinal secretion, and subcellular location of HRG-7 is essentil for systemic heme homeostasis. To elucidate the mechanisms of HRG-8 function at the molecular and organismal level, we will define its genetic requirement by functional complementation of hrg-8 mutants using ectopically expressed and membrane anchored versions of HRG-8, and assess the inter-dependence of HRG-8 and HRG-7 for heme signaling function. To identify the molecular interactors or substrates of HRG-7 and HRG-8, we will use multidimensional protein identification technology, validate candidate interactors with RNAi knockdown, and specifically characterize interactors which have homologs in humans. Our studies in C. elegans will overcome several obstacles posed by mammalian model systems namely the control of intracellular heme levels by external means, experimental manipulation of animals at subcellular resolution, and optical transparency for in vivo monitoring of heme signals between tissues during development. Our proposed project is transformative because the molecules and mechanisms for trafficking and distribution of heme to regulate systemic heme homeostasis in eukaryotes are largely unknown. Results from our studies may provide a heuristic paradigm to tackle similar questions for other micronutrients. PUBLIC HEALTH RELEVANCE: Iron deficiency is the world's number one nutritional disorder, and heme is the most bioavailable form of iron for human consumption. Identification of how dietary heme is transported and coordinated with systemic heme levels will permit the design of synthetic heme-based "nutraceuticals" specifically targeted to iron-deficient individuals. Iron deficiency is exacerbated by blood-loss due to heme auxotrophic parasites such as intestinal hookworms which depend on host heme to survive. A drug that targets the heme transport and regulatory pathway in helminths would be a powerful anthelminthic. Thus, identification of heme trafficking pathways across species will have a profound and long-lasting impact on our understanding of fundamental, clinical and applied processes.