MOLECULAR APPROACHES TO BASOLATERAL GI IRON TRANSPORT

说明(改编自应用程序) 铁是正常新陈代谢功能和铁的紊乱所必需的 动态平衡可能会产生重大的临床后果。人体内的铁含量 身体被控制在小肠的吸收点，但 铁穿过肠上皮的机制很差。 明白了。在这一应用中，我们的目标是详细研究铁的机理 通过研究上皮细胞外和体内的运动 最近克隆的两个基因的生物学。其中一种名为Hephaestin，已知 在上皮细胞的铁输出中起作用，而另一个基因，ireg1，是 建议在这一进程中发挥重要作用。我们假设 Hephastin和ireg1相互作用，形成一种复合体，将铁输送到 肠上皮细胞的基侧膜，并将其输送到 载脂蛋白。这一假设将通过解决以下几个关键问题来检验 问题：(1)Hephestn和ireg1是否共定位并在 上皮细胞？(2)这种相互作用足以调节铁的运动吗？ 跨细胞膜？(3)这种复合体能否直接或间接地传递 铁到载脂蛋白？以及(4)铁的转运是如何由这些物质来调节的 蛋白质受调控吗？这些问题将通过并行进行 利用肠道组织和细胞系高表达肝纤维化蛋白的研究 和ireg1，并研究动物模型中铁的运输。免疫显微镜检查， 将使用共沉淀和酵母双杂交系统来寻找 这两种蛋白质之间的相互作用。Hephestn和ireg1在铁中的作用 将通过发展哺乳动物体内的铁外流分析来研究转运 酵母细胞，以及通过靶向破坏小鼠和 对其表型进行分析。为了确定Hephaestin/ireg1复合体是否 将铁传递给载脂蛋白，我们将寻找 并将研究其对载脂蛋白的促进作用 从肠道组织和重建的铁外排系统中释放铁。 Ireg1和Hephaestin的铁依赖调节将在 细胞和动物模型以及调控系统的详细分析 将会被执行。在所有研究中，定点突变将被用于 在分子水平上剖析相互作用。铁外流或转移步骤 肠道铁吸收的研究在过去被证明是困难的，然而它 在某些情况下很可能是吸收的速度限制。 然而，对Hephaestin和ireg1的鉴定提供了一种前所未有的 有机会在分子水平上研究这一过程并评估 它在人类铁代谢紊乱中的作用，如遗传性 血色素沉着症，并提供潜在的治疗靶点。

DESCRIPTION (adapted from the application) Iron is essential for normal metabolic functions and disturbances of iron homeostasis can have significant clinical consequences. The amount of iron in the body is controlled at the point of absorption in the small intestine, yet the mechanism by which iron traverses the intestinal epithelium is poorly understood. In this application we aim to study in detail the mechanism of iron movement out of the epithelial cells and into the body by investigating the biology of two recently cloned genes. One of these, hephaestin, is known to play a role in iron export from the epithelium, while the other, ireg1, is proposed to play an important role in this process. We hypothesize that hephaestin and ireg1 interact to form a complex which transports iron across the basolateral membrane of intestinal enterocytes and delivers it to apotransferrin. This hypothesis will be tested by addressing several key questions: (1) Do hephaestin and ireg1 co-localise and physically interact in epithelia] cells? (2) Is this interaction sufficient to mediate iron movement across cellular membranes? (3) Can this complex directly or indirectly deliver iron to apotransferrin? and (4) How is the iron transport mediated by these proteins regulated? These issues will be addressed by conducting parallel studies using both intestinal tissue and cell lines overexpressing hephaestin and ireg1 and by studying iron transport in animal models. Immunomicroscopy, co-precipitation and the yeast two-hybrid system will be used to search for interactions between the two proteins. The role of hephaestin and ireg1 in iron transport will be studied by developing iron efflux assays in mammalian and yeast cells, and by the targeted disruption of the ireg1 gene in mice and the analysis of its phenotype. To determine whether the hephaestin/ireg1 complex delivers iron to apotransferrin, we will search for direct interactions between the proteins and will investigate the ability of apotransferrin to facilitate iron release from intestinal tissue and in a reconstituted iron efflux system. The iron-dependent regulation of ireg1 and hephaestin will be studied in cellular and animal models and a detailed analysis of the regulatory system will be carried out. In all studies, site directed mutagenesis will be used to dissect interactions at the molecular level. The iron efflux or transfer step of intestinal iron absorption has proved difficult to study in the past yet it is likely to be the rate-limiting for absorption under certain circumstances. However, the identification of hephaestin and ireg1 provides an unprecedented opportunity to investigate this process at the molecular level and to evaluate its role in human disorders of iron metabolism such as hereditary hemochromatosis and provides potential therapeutic targets.