Functional Genomics of Heme Homeostasis by RNA Interference

描述（由申请人提供）：据估计，全球60-80%的人口缺铁，使营养性缺铁成为最常见的营养失调。大量证据表明，血红素是人类营养中必不可少的生物可利用铁来源，但血红素吸收和利用的途径尚不清楚。血红素还作为蛋白质的修复基团，在许多不同的生物过程中起着关键作用。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位点或进入细胞到细胞内的各种目的地。我们实验室的长期目标是鉴定血红素分子并了解哺乳动物血红素摄取、利用、运输和储存的机制。秀丽隐杆线虫不能产生内源性血红素，但依靠膳食血红素维持代谢过程。与秀丽隐杆线虫一样，包括钩虫在内的许多寄生线虫缺乏血红素生物合成途径。寄生虫从人类宿主体内清除血红素，从而导致或加剧缺铁。因此，秀丽隐杆线虫代表了一种独特的遗传动物模型来识别血红素运输途径，因为它们允许外部控制血红素水平，这在其他生物中是不允许的。在血红素反应基因（hrg-1）启动子的控制下，我们在秀丽隐杆线虫中合成了一株表达绿色荧光蛋白（GFP）的转基因“血红素传感器”菌株，该菌株对机体血红素水平敏感。在我们的传感器菌株中，GFP荧光在低血红素下增加，当血红素浓度升高时减少。我们建议使用rna介导的干扰将该菌株用于功能性反向遗传筛选。由DNA微阵列鉴定的约400个血红素反应基因组成的大肠杆菌迷你喂养文库将用于将蠕虫暴露于双链RNA并“敲除”传感器菌株中的目标基因。GFP荧光的变化将作为血红素水平和基因敲除的功能进行监测。与对照相比，改变GFP强度的克隆将重新筛选以消除假阳性，并通过DNA测序确认从该筛选中鉴定出的候选基因。从不同血红素水平生长的蠕虫和饲喂的候选克隆中提取的RNA将使用定量实时PCR和RNA印迹分析进行表征。两种候选蛋白的体内细胞定位将通过显微镜检查；它们的功能将通过荧光、放射性标记和有毒血红素类似物来确定。

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