The Transport of Nutritional Heme in Animal Development

描述(申请人提供)：我们研究的长期目标是确定人类营养中的血红素动态平衡的细胞和分子决定因素。缺铁是最常见的营养失调。膳食中的血红素(铁-原卟啉IX)是生物可利用铁的重要来源，但人类肠道细胞中负责血红素运输和利用的基因和途径仍然难以捉摸。在人类中，超过60%的全身铁以血红素的形式存在于血红蛋白中。血红素中的铁通过吞噬衰老的红细胞和消化巨噬细胞中的血红蛋白来循环利用。与肠细胞类似，巨噬细胞中血红素通过吞噬小体膜运输和利用的途径是未知的。由于血红素是一个疏水的、细胞毒性的大循环，我们断言血红素不会被动地通过膜扩散，而是通过特定的细胞内和细胞间途径进行主动运输，这些途径包括血红素的摄取、运输和封存。我们已经证明线虫是鉴定血红素转运途径的一个很好的动物模型，因为它合成了大量与人类同源的血红素蛋白，而不是从新合成的血红素。蠕虫的生长和繁殖需要食物中的血红素。因此，蠕虫模型提供了一个干净的遗传背景，没有内源性血红素，也没有外部操纵细胞内血红素代谢流量的能力。利用线虫作为血红素营养缺乏症的遗传动物模型，我们确定了第一个真核血红素进口者/转运者HRG-1(自然，2008)。HRG-1是一种在人类中保守的渗透酶，它结合和运输血红素。蠕虫和人类的HRG-1蛋白共同定位于内溶酶体室。在斑马鱼中敲除HRG-1会导致脑积水、卵黄管缺陷和贫血--这些表型完全可以被蠕虫HRG-1拯救。这些研究建立了细胞内血红素运输的保守模型，并验证了线虫是鉴定血红素稳态途径的真实模型。这项建议中的研究旨在阐明HRG-1在分子、细胞和生物水平上转运血红素的确切机制。我们试图测试这一假设，即由HRG-1介导的血红素运输是动物体内血红素稳态调节网络的一个组成部分。我们将利用结构-功能方法来确定HRG-1负责血红素运输的功能元件，用细胞生物学方法来确定HRG-1作为血红素-铁循环的囊泡转运体，以及用系统方法来确定将HRG-1与生物体的血红素运输相结合的复杂调控电路。我们的目标是全面了解在哺乳动物中调节血红素动态平衡的途径，到目前为止，这些途径仍然知之甚少。 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。确定血红素是如何被运输来合成含铁和血红素的蛋白质的，将有助于设计专门针对包括怀孕母亲和婴儿在内的铁缺乏症患者的以血红素为基础的合成“营养食品”。确定血红素-铁循环产生血红蛋白的分子基础将为人工血的合成带来新的策略。

DESCRIPTION (provided by applicant): The long-term objective of our studies is to define the cellular and molecular determinants of heme homeostasis in human nutrition. Iron deficiency is the most common nutritional disorder. Dietary heme (iron-protoporphyrin IX) is a significant source of bioavailable iron, but the genes and pathways responsible for heme transport and utilization in human enterocytes remain elusive. In humans, greater than 60% of total body iron is present as heme in hemoglobin. Iron from heme is recycled by phagocytosis of senescent red blood cells and proteolytic digestion of hemoglobin in macrophages. Similar to enterocytes, the pathways for heme transport and utilization across the phagolysosomal membrane in macrophages are unknown. Since heme is a hydrophobic, cytotoxic macrocycle, we assert that heme does not passively diffuse through membranes but is actively transported via specific intra- and inter-cellular pathways that comprise heme uptake, trafficking, and sequestration. We have demonstrated that the roundworm Caenorhabditis elegans is an excellent animal model to identify heme transport pathways because it synthesizes a large number of hemoproteins with human homologs but does not synthesize heme de novo. Worms require dietary heme for growth and reproduction. Thus, the worm model provides a clean genetic background devoid of endogenous heme and the ability to externally manipulate the metabolic flux of intracellular heme. Utilizing C. elegans as a genetic animal model of heme auxotrophy, we identified HRG-1, the first eukaryotic heme importer/ transporter (Nature 2008). HRG-1 is a permease that is conserved in humans and binds and transports heme. The worm and human HRG-1 proteins co-localize to the endo-lysosomal compartment. Knockdown of hrg-1 in zebrafish causes hydrocephalus, yolk tube defects, and anemia - phenotypes that are fully rescued by worm HRG-1. These studies established a conserved model for cellular heme transport and validated C. elegans as a bona fide model for the identification of heme homeostasis pathways. The studies in this proposal are designed to elucidate the precise mechanisms of heme transport by HRG-1 at the molecular, cellular, and organismal levels. We seek to test the hypothesis that heme transport mediated by HRG-1 is an integral component of the heme homeostasis regulatory network in animals. We will utilize a structure-function approach to identify the functional elements of HRG-1 responsible for heme transport, a cell biological approach to establish HRG-1 as the vesicular transporter for heme-iron recycling, and a systems approach to identify the complex regulatory circuit which integrates HRG-1 with organismal heme trafficking. Our goal is to obtain a comprehensive understanding of the pathways which mediate heme homeostasis in mammals that have, heretofore, remained poorly understood. 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 heme is transported for the synthesis of proteins containing iron and heme will permit the design of synthetic heme-based "nutraceuticals" specifically targeted to iron-deficient individuals including pregnant mothers and infants. Identifying the molecular basis of heme-iron recycling to produce hemoglobin will lead to new strategies for the synthesis of artificial blood.