The Biological Role of Heme in Human Nutrition

描述（申请人提供）：世界上五分之四的人可能缺铁，使营养性缺铁成为最常见的营养失调。虽然大量的实验和营养数据支持血红素（铁原卟啉IX）作为人类营养中铁的生物可利用来源的重要作用，但血红素吸收和利用的途径目前尚不清楚。从细胞生物学的角度来看，血红素是许多重要生物过程的辅助基团，在真核生物中，血红素是在线粒体基质中合成的。由于血红素是一种疏水分子，并且由于其固有的过氧化物酶活性而具有细胞毒性，我们假设血红素不仅通过脂质双分子层扩散，而且通过特定的细胞内途径积极运输。我们建议使用秀丽隐杆线虫动物模型来确定血红素运输的细胞途径和介导血红素运输的分子，因为这种动物是唯一已知的无法合成血红素的遗传动物模型，尽管需要血红素来维持代谢过程。由于秀丽隐杆线虫缺乏制造血红素的能力，它为我们提供了一个缺乏内源性血红素的干净的遗传背景，以及从外部控制血红素代谢通量的能力。因此，秀丽隐杆线虫是一种专性血红素营养不良动物，将成为研究膳食血红素吸收以及细胞内血红素运输、隔离和纳入关键血红蛋白的良好动物模型。通过生物化学分析血红素水平和血红蛋白活性，评估血红素类似物在活体动物中的生存能力和追踪血红素运输，以及使用电子显微镜在超微结构水平上确定血红素分布，来表征从前向遗传筛选中分离的突变体的表型簇，从而绘制秀丽隐杆线虫血红素运输的细胞途径。突变基因的分子特性将通过以下方法确定：在基因互补组中对突变进行分类，使用snp绘制突变基因，通过RNA干扰和基因拯救定位突变基因，以及通过测序候选基因来确定分子病变。通过DMA微阵列鉴定的秀丽隐杆线虫血红素调控基因，将通过实时PCR和RNA印迹分析定量验证基因亚群，通过RNA干扰确定候选基因的功能，并通过合成转录和翻译报告基因融合来确定这些基因产物在血红素响应下的时空表达。这些研究的结果将为真核生物血红素稳态提供新的机制见解，并可能有助于开发以血红素为基础的人类缺铁营养干预措施，以及潜在的药物靶点，以治疗加剧缺铁的人类蠕虫感染。

DESCRIPTION (provided by applicant): Four out of five people in the world may be iron-deficient, making nutritional iron deficiency the most common nutritional disorder. Although considerable experimental and nutritional data support an essential role for heme (iron-protoporphyrin IX) as a bioavailable source of iron in human nutrition, the pathways for heme absorption and utilization are currently unknown. From a cell biological perspective, heme is the prosthetic group for many important biological processes, and in eukaryotes it is synthesized in the mitochondrial matrix. Because heme is a hydrophobic molecule and is cytotoxic due to its intrinsic peroxidase activity, we hypothesize that heme does not merely diffuse through lipid bilayers but is actively transported via specific intracellular pathways. We propose to use the animal model, Caenorhabditis elegans, to identify the cellular pathways for heme transport and the molecules mediating heme trafficking, because this animal is the only known genetic animal model that is unable to synthesize heme eye no^o albeit requiring heme to sustain metabolic processes. Since C. elegans lacks the ability to make heme, it provides us with a clean genetic background devoid of endogenous heme, and the capacity to externally control the metabolic flux of heme. Thus, C. elegans is an obligate heme auxotroph and will be an excellent animal model to study dietary heme absorption as well as intracellular heme trafficking for transport, sequestration and incorporation into critical hemoproteins. The cellular pathways for heme transport will be mapped in C. elegans by characterizing phenoclusters of mutants isolated from forward genetic screens by biochemically assaying for heme levels and hemoprotein activity, evaluating viability and tracking heme transport in live animals with heme analogs, and determining heme distribution at the ultrastructural level using electron microscopy. The molecular identities of the mutated genes will be determined by categorizing mutants in genetic complementation groups, mapping the mutated genes using SNPs, localizing the mutant gene by RNA interference and gene rescue, and by sequencing candidate genes to determine the molecular lesion. The heme regulated genes in C. elegans, identified from our DMA microarrays, will be characterized by quantitatively validating subsets of genes by real-time PCR and RNA blot analysis, determining the function of the candidate genes by RNA interference, and defining the temporal and spatial expression of these gene products in response to heme by synthesizing transcriptional and translational reporter gene fusions. The results from these studies will provide new mechanistic insights into heme homeostasis in eukaryotes and may aid in the development of heme-based nutritional interventions for human iron deficiency, and potential drug targets for human helminthic infections that exacerbate iron deficiency.