MOLECULAR APPROACHES TO BASOLATERAL GI IRON TRANSPORT

描述（改编自应用程序） 铁是必不可少的正常代谢功能和干扰铁 体内平衡可能具有显著的临床后果。中的铁含量 身体在小肠的吸收点被控制，然而 铁穿过肠上皮的机制很差 明白在这个应用程序中，我们的目的是详细研究铁的机制， 运动的上皮细胞和进入身体通过调查 最近克隆的两个基因的生物学。其中之一，赫菲斯汀， 在上皮细胞的铁输出中起作用，而另一个，ireg 1， 在这一过程中发挥重要作用。我们假设 hephaestin和ireg 1相互作用形成复合物， 肠上皮细胞的基底外侧膜，并将其递送至 脱铁转铁蛋白这一假设将通过解决几个关键问题来检验。 问题：（1）hephaestin和ireg 1是否共同定位和物理相互作用， 上皮细胞？(2)这种相互作用是否足以调节铁的运动 穿过细胞膜吗(3)这个综合体能直接或间接地 铁转铁蛋白？（4）铁转运是如何通过这些介导的？ 蛋白质调控？这些问题将通过开展平行的 使用肠组织和过表达hephaestin的细胞系的研究 和IREG 1以及研究动物模型中的铁转运。免疫显微镜检查， 共沉淀和酵母双杂交系统将被用来寻找 两种蛋白质之间的相互作用。Hephaestin和Ireg 1在铁代谢中的作用 运输将研究通过发展铁流出试验在哺乳动物和 酵母细胞，并通过在小鼠中靶向破坏ireg 1基因， 分析其表型。为了确定是否hephaestin/ireg 1复合物 将铁传递给脱铁转铁蛋白，我们将寻找铁与脱铁转铁蛋白之间的直接相互作用。 蛋白质，并将研究脱铁转铁蛋白的能力， 铁从肠组织释放和在重构铁流出系统中。 Ireg 1和Hephaestin的铁依赖性调节将在 细胞和动物模型以及对调节系统的详细分析 将被执行。在所有研究中，将使用定点诱变来 在分子水平上剖析相互作用。铁流出或转移步骤 肠道铁吸收的研究在过去已经证明是困难的， 在某些情况下，很可能是吸收的速度限制。 然而，hephaestin和ireg 1的鉴定提供了前所未有的 有机会在分子水平上研究这一过程， 它在人类铁代谢紊乱中的作用， 并提供了潜在的治疗靶点。

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.