Molecular Mechanisms of Intestinal Metal Ion Transport During Iron Deficiency

说明(由申请人提供)：由于不存在活跃的排泄系统，膳食铁在牛仔中的吸收决定了全身铁的水平。因此，必须严格控制这一过程，以避免组织铁蓄积(例如遗传性血色素沉着症)或缺乏(例如慢性炎症贫血)的不良后果。在过去的十年里，我们一直在研究肠道上皮细胞(IECS)中铁运输的分子方面，长期目标是开发药物或饮食治疗来调节人类对铁的吸收。虽然已经确定了铁的进口商和出口商，但关于铁在IECS之间的流动和出口到流通中的具体细节(这是限速步骤)缺乏了解。我们做了一个新的观察，即啮齿类动物的缺铁激活了与铜相关的过程。在肠细胞中，随着细胞内铜水平的升高，铜转运酶(ATP7A)和铜结合蛋白(金属硫蛋白)表达上调。这些观察提供了对体内铜水平(在缺铁和铁超负荷减少期间增加)和控制肠道铁运输之间的关系的机械性洞察。基于这些发现，确定涉及铁通量控制的铜特定机制是当务之急。从肠细胞输出的亚铁(Fe2+)在功能上与一个氧化步骤偶联，这是铁(Fe3+)与间质液中的转铁蛋白(Tf)结合所必需的。一种膜结合的多铜铁氧化物酶(FOX)，赫菲斯汀(Heph)，可能介导这一步骤。然而，HephKO小鼠是存活的，肠道铁转运只有部分减弱，这表明还有其他狐狸存在。我们最近发现，肠上皮细胞有两种截然不同的新型胞液铁氧合酶(FOX)，一种是一种未被发现的可溶性HEPH(SHeph)，另一种称为细胞FOX。我们推测胞内FOX参与了跨IECS的铁跨细胞转运。在HephKO小鼠中的研究表明，这两种蛋白对胞浆Fox活性有贡献(sHeph~35-40%；cell FOX~60-65%)。另一种循环狐，铜蓝蛋白(CP)，也可能参与IECS的铁出口。目前的提议将通过检验以下中心假设来阐明肠道铁转运的具体机制细节：肠细胞中新的多铜FOX(sHeph和cell FOX)和血液中的CP在控制IECS中铁的输出方面起着不可或缺的作用。我们还将确定这些蛋白质生物合成的铜输送机制(S)，可能涉及肠道ATP7A。在本申请中概述的综合方法，使用哺乳动物铁转运的独特体内和体外模型， 我将首先破译(在目标1中)ATP7A在将铜输送到sHEH以介导铁外流和在增强肝脏铜负荷方面的作用。目标2将确定新的可溶性FOX在跨细胞铁途径中的作用(S)，而目标3将阐明CP在铁吸收中的作用，并将确定缺铁时促进Holo-CP产生的机制。总体而言，该项目将通过揭示铁运输的新机制细节来推动铁生物学领域的发展，并可能为开发调节铁吸收的分子方法提供机会。

DESCRIPTION (provided by applicant): Absorption of dietary iron in the doudenum determines overall body iron levels as no active excretory systems exist. As such, this process must be tightly controlled to avoid the adverse consequences of tissue iron accumulation (e.g. in hereditary hemochromatosis) or deficiency (e.g. in anemia of chronic inflammation). We have been investigating molecular aspects of iron transport across intestinal epithelial cells (IECs) fo the past decade, with a long-term goal of developing drugs or dietary treatments to modulate iron absorption in humans. Although iron importers and exporters have been identified, a paucity of knowledge exists regarding the specific details of iron movement across IECs and export into the circulation (which is the rate-limiting step). We made the novel observation that copper-related processes are activated by iron deprivation of rodents. In enterocytes, a copper transporting ATPase (Atp7a) and a copper-binding protein (metallothionein) were upregulated in the setting of increased intracellular copper levels. These observations provided mechanistic insight into the relationship between body copper levels (which increase during iron deficiency & decrease in iron overload) and control of intestinal iron transport. Based upon these findings, identification of copper-specific mechanisms involved in control of iron flux was an imperative. Ferrous iron (Fe2+) export from enterocytes is functionally coupled to an oxidation step which is required for iron (Fe3+) binding to transferrin (Tf) in the interstitial fluid. A membrane-bound, multi-copper ferroxidase (FOX), Hephaestin (Heph), may mediate this step. However, Heph KO mice are viable and intestinal iron transport is only partially attenuated, suggesting that other FOXs exist. We recently discovered that enterocytes have two, distinct novel cyosolic ferroxidases (FOXs), one being an undiscovered, soluble form of Heph (sHeph) and the other termed cytoFOX. We postulate that cytosolic FOXs participate in transcytosis of iron across IECs. Studies in Heph KO mice demonstrated that both proteins contribute to cytosolic FOX activity (sHeph ~35-40%; cytoFOX ~60-65%). Another, circulating FOX, ceruloplasmin (Cp), may also participate in Fe export from IECs. The current proposal will elucidate specific, mechanistic details of intestinal Fe transport by testing the central hypothesis that novel multi-copper FOXs (sHeph and cytoFOX) in enterocytes and Cp in blood play integral roles in control of iron export from IECs. We will also define the mechanism(s) of Cu delivery for the biosynthesis of these proteins, likely involving intestinal Atp7a. The integrative approach outlined in this application, using unique in vivo and in vitro models of mammalian iron transport, will first decipher (in Aim 1) the role of Atp7a in delivering copper to sHeph to mediate iron efflx and in potentiating hepatic copper loading. Aim 2 will define the role(s) of novel soluble FOXs in the transcytotic iron pathway, while Aim 3 will clarify the role of Cp in iron absorption and will determine the mechanism of enhanced production of holo-Cp during iron deficiency. Overall, this project will advance the field of iron biology by revealing new mechanistic details of iron transport and may provide opportunities to develop molecular approaches to modulate iron absorption.