The Biological Role of Heme in Human Nutrition

描述（由申请人提供）：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障碍。虽然大量的实验和营养数据支持血红素（铁-原卟啉IX）作为人体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基，在真核生物中，它是在线粒体基质中合成的。由于血红素是一种疏水性分子，并且由于其固有的过氧化物酶活性而具有细胞毒性，因此我们假设血红素不仅通过脂质双层扩散，而且通过特定的细胞内途径积极转运。我们建议使用动物模型，秀丽隐杆线虫，以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遗传动物模型，不能合成血红素眼no^o，尽管需要血红素来维持代谢过程。自从C.线虫缺乏制造血红素的能力，但它为我们提供了一个没有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，C.线虫是专性血红素营养缺陷型，将是研究膳食血红素吸收以及细胞内血红素运输、螯合和掺入关键血红素蛋白的极好动物模型。血红素转运的细胞途径将在C.通过表征从正向遗传筛选中分离的突变体的表型簇，通过生化测定血红素水平和血红素蛋白活性，评估存活力和用血红素类似物跟踪活动物中的血红素运输，以及使用电子显微镜在超微结构水平上确定血红素分布，来鉴定线虫。突变基因的分子身份将通过以下方式确定：将突变体分类到遗传互补组中，使用SNP对突变基因进行作图，通过RNA干扰和基因拯救定位突变基因，以及对候选基因进行测序以确定分子损伤。C.从我们的DMA微阵列中鉴定的线虫的特征在于通过实时PCR和RNA印迹分析定量验证基因的子集，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体来确定这些基因产物响应血红素的时间和空间表达。这些研究的结果将为真核生物中血红素稳态提供新的机制见解，并可能有助于开发基于血红素的营养干预措施，用于人类铁缺乏症，以及用于加剧铁缺乏症的人类蠕虫感染的潜在药物靶点。

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.