The Biological Role of Heme in Nutrition

描述（由申请人提供）：本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稳态。血红素是一种含铁的有机环，是一种重要的辅助因子，负责多种生物功能 除了作为人体营养中生物可利用铁的主要来源之外。血红素是在线粒体中合成的疏水性和细胞毒性辅因子，尽管受体血红素蛋白存在于不同的细胞隔室中。血红素是如何通过细胞膜和细胞器运输的？在上一个资助周期中，我们率先使用秀丽隐杆线虫，一种不合成血红素但利用环境血红素制造血红素蛋白的遗传动物模型，鉴定了七种新分子，并建立了动物血红素稳态和贩运模型。在下一个资助周期中，我们将采用上一个资助周期中开发的创新工具，以弥合在描绘组织和细胞如何传达其血红素状态以调节活体动物中有机体血红素稳态方面的重大知识差距。虽然C.尽管在线虫中发现了血红素营养缺陷型的基因，但在人类、小鼠和斑马鱼中的几条证据表明，这种途径也必须存在于脊椎动物中。在此，我们建议阐明组织间通讯的机制，并确定血红素信号的分子身份使用C。优雅本提案中的研究将检验以下假设：HRG-7和HRG-8通过启动反馈反应来调节全身血红素稳态，从而与肠外组织沟通和整合肠血红素状态。为了阐明HRG-7在分子和生物体水平上的功能机制，我们将确定HRG-7的催化活性、肠分泌和亚细胞定位是否对系统血红素稳态至关重要。为了阐明HRG-8在分子和生物体水平上的功能机制，我们将使用HRG-8的异位表达和膜锚定版本通过HRG-8突变体的功能互补来定义其遗传要求，并评估HRG-8和HRG-7对血红素信号传导功能的相互依赖性。为了鉴定HRG-7和HRG-8的分子相互作用物或底物，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物，并具体表征在人类中具有同源物的相互作用物。我们在C. elegans将克服由哺乳动物模型系统造成的几个障碍，即通过外部手段控制细胞内血红素水平、在亚细胞分辨率下对动物的实验操作、以及用于在发育期间体内监测组织之间的血红素信号的光学透明度。我们提出的项目是变革性的，因为在真核生物中血红素的运输和分布以调节系统血红素稳态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为解决其他微量营养素的类似问题提供启发式范例。

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.