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)作为一种生物可利用的铁源在人类营养中的重要作用，但目前对血红素的吸收和利用途径尚不清楚。从细胞生物学的角度来看，血红素是许多重要生物学过程的修复基团，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分子，由于其内在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双层扩散，而且通过特定的细胞内途径进行主动运输。我们建议使用秀丽隐杆线虫的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，因为这种动物是已知的唯一不能合成血红素眼球的遗传动物模型，尽管需要血红素来维持代谢过程。由于线虫缺乏制造血红素的能力，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流动的能力。因此，线虫是一种专性的血红素营养缺陷体，将是研究膳食中的血红素吸收以及细胞内运输血红素以运输、封存和并入关键的血红素蛋白的良好动物模型。通过对从正向遗传筛查中分离的突变株的表型簇进行生化分析，对血红素水平和血红素蛋白活性进行分析，评估活性并使用血红素类似物跟踪活体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的分布，将绘制线虫中血红素运输的细胞路径图。突变基因的分子同一性将通过将突变基因归类到遗传互补群中，利用SNPs定位突变基因，通过RNA干扰和基因拯救来定位突变基因，通过对候选基因进行测序来确定分子损伤。从我们的DMA微阵列中发现的线虫中受血红素调控的基因，将通过实时荧光PCR和RNA印迹分析定量验证基因亚集，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来确定这些基因产物对血红素的响应的时间和空间表达。这些研究的结果将为真核生物中的血红素动态平衡提供新的机制见解，并可能有助于开发基于血红素的针对人类铁缺乏的营养干预措施，以及针对加剧铁缺乏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.