Functional Genomics of Heme Homeostasis by RNA Interference

描述(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一种重要的生物可利用铁源，但人们对其吸收和利用的途径知之甚少。血红素还可以作为蛋白质中的修复基团，在众多不同的生物过程中发挥关键作用。由于血红素具有细胞毒性和不溶性，我们假设细胞内存在特定的途径，将血红素从其合成部位或进入细胞内运输到不同的细胞内目的地。我们实验室的长期目标是识别这些分子，并了解哺乳动物对血红素的吸收、利用、运输和储存的机制。线虫不能产生内源血红素，但依赖于饮食中的血红素来维持代谢过程。像线虫一样，包括钩虫在内的许多寄生线虫缺乏血红素生物合成途径。寄生蠕虫从它们的人类宿主中清除血红素，这会导致或加剧铁缺乏。因此，线虫代表了一种独特的动物遗传模型来识别血红素的运输途径，因为它们允许外部控制其他生物体中不允许的血红素水平。我们已经在线虫中合成了一种在血红素反应基因(HRG-1)启动子控制下表达绿色荧光蛋白(GFP)的转基因“血红素传感器”菌株，该启动子对生物体内的血红素水平敏感。在我们的传感器菌株中，GFP荧光在低血红素浓度下增加，随着血红素浓度的增加而减少。我们建议将该菌株用于使用RNA介导的干扰的功能性反向遗传筛查。一个包含从DNA微阵列中鉴定出的约400个血红素反应基因的大肠杆菌微型饲喂文库，将被用来将蠕虫暴露于双链RNA和传感器菌株中的“击倒”目标基因。绿色荧光蛋白荧光的变化将作为血红素水平和基因敲除的函数进行监测。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，从该筛选中确定的候选基因将通过DNA测序得到确认。从生长在不同水平的血红素中的蠕虫和喂养的候选克隆中提取的RNA将使用实时定量PCR和RNA印迹分析进行表征。两个这样的候选蛋白的体内细胞定位将通过显微镜检查；它们的功能(S)将使用荧光、放射性标记和有毒的血红素类似物来确定。 公共卫生相关性：就公共卫生相关性而言，本提案中描述的研究结果将为哺乳动物体内的血红素动态平衡提供新的机械性见解。这些见解可能有助于开发以血红素为基础的针对人类铁缺乏的营养干预措施，并有助于发现针对寄生虫中的血红素转运体的药物，这些寄生虫会加剧人类的铁缺乏。

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