Functional Genomics of Heme Homeostasis by RNA Interference

描述（由申请人提供）：据估计，世界上60-80%的人口缺铁，使营养性缺铁成为最常见的营养障碍。大量证据表明，血红素是人体营养中重要的生物可利用铁源，但对血红素吸收和利用的途径知之甚少。血红素还作为蛋白质中的辅基，在许多不同的生物过程中发挥关键作用。由于血红素是细胞毒性和不溶性的，我们假设，特定的途径存在于细胞内的运输血红素从其网站的合成或进入细胞的各种细胞内的目的地。我们实验室的长期目标是鉴定分子并了解哺乳动物摄取，利用，运输和储存血红素的机制。C.秀丽线虫不能产生内源性血红素，但依赖于膳食血红素来维持代谢过程。像C。线虫，包括钩虫在内的许多寄生线虫缺乏血红素生物合成途径。寄生蠕虫从人类宿主体内释放血红素，导致或加剧缺铁。C.因此，秀丽隐杆线虫是鉴定血红素转运途径的独特遗传动物模型，因为它们允许对其他生物体中不允许的血红素水平进行外部控制。我们在C.在血红素应答基因（hrg-1）启动子的控制下表达绿色荧光蛋白（GFP）的线虫，该启动子对生物体血红素水平敏感。在我们的传感器菌株中，GFP荧光在低血红素下增加，并且当血红素浓度升高时减少。我们建议使用该菌株在一个功能反向遗传筛选使用RNA介导的干扰。大肠大肠杆菌微型饲养库，包括约400血红素反应基因鉴定的DNA微阵列，将用于暴露蠕虫的双链RNA和“敲低”的目标基因的传感器菌株。GFP荧光的变化将作为血红素水平和基因敲低的函数进行监测。与对照相比，改变GFP强度的克隆将被重新筛选以消除假阳性，并且从该筛选中鉴定的候选基因将通过DNA测序来确认。将使用定量实时PCR和RNA印迹分析表征从在不同水平的血红素中生长的蠕虫和饲喂的候选克隆中提取的RNA。将通过显微镜检查两种这样的候选蛋白的体内细胞定位;并且将使用荧光、放射性标记和毒性血红素类似物来确定它们的功能。 公共卫生相关性：在公共卫生相关性方面，本提案中描述的研究结果将为哺乳动物的血红素稳态提供新的机制见解。这些见解可能有助于开发基于血红素的营养干预措施来治疗人类铁缺乏症，并促进发现针对寄生蠕虫中血红素转运蛋白的药物，这些药物会加剧人类铁缺乏症

DESCRIPTION (provided by applicant): It is estimated that 60-80% of the world's population is iron-deficient, making nutritional iron deficiency the most common nutritional disorder. Considerable evidence suggests that heme is an essential bioavailable iron source in human nutrition, but the pathways for heme absorption and utilization are poorly understood. Hemes also serve as prosthetic groups in proteins which play key roles in numerous and diverse biological processes. Since hemes are cytotoxic and insoluble, we hypothesize that specific pathways exist within cells for trafficking hemes from their site of synthesis or entry into the cell to various intracellular destinations. The long term goals of our lab are to identify the molecules and understand the mechanisms of heme uptake, utilization, transport, and storage in mammals. C. elegans cannot produce endogenous heme but depends on dietary heme to sustain metabolic processes. Like C. elegans, many parasitic nematodes including hookworms lack the heme biosynthetic pathway. Parasitic worms scavenge heme from their human host, which causes or exacerbates iron deficiency. C. elegans, therefore, represents a unique genetic animal model to identify heme transport pathways because they allow external control of heme levels not permissible in other organisms. We have synthesized a transgenic "heme-sensor" strain in C. elegans expressing the green fluorescent protein (GFP) under the control of a heme-responsive gene (hrg-1) promoter, which is sensitive to organismal heme levels. In our sensor strain, GFP fluorescence increases under low heme and decreases when the concentration of heme is elevated. We propose to use this strain in a functional reverse genetic screen using RNA-mediated interference. An E. coli mini feeding library, comprising ~400 heme responsive genes identified from DNA microarrays, will be used to expose worms to double-stranded RNA and "knock-down" target genes in the sensor strain. Changes in GFP fluorescence will be monitored as a function of heme levels and gene knock-downs. Clones that alter GFP intensity, as compared to controls, will be re-screened to eliminate false positives, and candidate genes identified from this screen will be confirmed by DNA sequencing. RNA extracted from worms grown in varying levels of heme and fed candidate clones will be characterized using quantitative real-time PCR and RNA blot analysis. The in vivo cellular localization of two such candidate proteins will be examined by microscopy; and their function(s) will be determined using fluorescent, radiolabeled, and toxic heme analogs. PUBLIC HEALTH RELEVANCE: In terms of public health relevance, results from the studies described in this proposal will provide new mechanistic insights into heme homeostasis in mammals. These insights may aid in the development of heme-based nutritional interventions for human iron deficiency and facilitate discovery of drugs that target heme transporters in parasitic worms, which exacerbate human iron deficiency