Molecular Mechanisms of Intestinal Metal Ion Transport During Iron Deficiency

缺铁期间肠道金属离子转运的分子机制

• 批准号: 8813554
• 负责人: James F. Collins
• 金额: $ 32.63万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8813554
• 关键词: ATP phosphohydrolase; Anabolism; Anemia; Attenuated; Binding; Biology; Blood; Blood Circulation; Cells; Ceruloplasmin; Chronic; Complement; Copper; Coupled; Diet; Dietary Iron; Endosomes; Enterocytes; Enzymes; Epithelial Cells; Goals; Health; HepG2; Hepatic; Hepatocyte; Hereditary hemochromatosis; Human; Human Pathology; In Transferrin; Inflammation; Intercellular Fluid; Intestines; Ion Transport; Iron; Iron Overload; Knowledge; Liver; Location; Mediating; Membrane; Metallothionein; Metals; Molecular; Movement; Mus; Mutation; Nature; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Process; Production; Protein Biosynthesis; Proteins; Rattus; Regulation; Rodent; Role; System; Testing; Tissues; Vesicle; absorption; adverse outcome; base; copper-binding protein; copper-transporting ATPase; deprivation; in vitro Model; in vivo; insight; iron deficiency; metal transporting protein 1; mutant; novel; oxidation; research study; transcytosis

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.

描述（由申请人提供）：由于不存在活跃的排泄系统，十二指肠中膳食铁的吸收决定了总体铁水平。因此，必须严格控制这一过程，以避免组织铁积累（例如遗传性血色素沉着症）或缺乏（例如慢性炎症性贫血）的不良后果。过去十年来，我们一直在研究铁跨肠上皮细胞 (IEC) 转运的分子方面，长期目标是开发药物或饮食疗法来调节人类铁的吸收。尽管已经确定了铁进口商和出口商，但对于铁通过 IEC 运输和出口到流通领域（这是限速步骤）的具体细节仍知之甚少。我们进行了新的观察，即啮齿动物缺铁会激活与铜相关的过程。在肠细胞中，铜转运 ATP 酶 (Atp7a) 和铜结合蛋白（金属硫蛋白）在细胞内铜水平增加的情况下上调。这些观察结果为了解体内铜水平（铁缺乏时增加和铁过载减少）与肠道铁转运控制之间的关系提供了机制见解。基于这些发现，确定涉及铁通量控制的铜特异性机制势在必行。从肠细胞输出的二价铁 (Fe2+) 在功能上与氧化步骤耦合，这是铁 (Fe3+) 与间质液中的转铁蛋白 (Tf) 结合所需的。膜结合的多铜亚铁氧化酶 (FOX)、赫菲斯汀 (Heph) 可能介导此步骤。然而，Heph KO 小鼠仍能存活，并且肠道铁转运仅部分减弱，表明存在其他 FOX。我们最近发现肠上皮细胞有两种不同的新型胞质亚铁氧化酶 (FOX)，一种是尚未发现的可溶形式的 Heph (sHeph)，另一种称为 cytoFOX。我们假设胞质 FOX 参与跨 IEC 的铁转胞吞作用。对 Heph KO 小鼠的研究表明，这两种蛋白都有助于细胞质 FOX 活性（sHeph ~35-40%；cytoFOX ~60-65%）。另一种循环 FOX，铜蓝蛋白 (Cp)，也可能参与 IEC 的铁输出。目前的提案将通过测试中心假设来阐明肠道铁转运的具体机制细节，即肠细胞中的新型多铜 FOX（sHeph 和 cytoFOX）和血液中的 Cp 在控制 IEC 的铁输出中发挥着不可或缺的作用。我们还将定义这些蛋白质生物合成的 Cu 输送机制，可能涉及肠道 Atp7a。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， 将首先破译（在目标 1 中）Atp7a 在将铜输送至 sHeph 以介导铁 efflx 和增强肝铜负荷方面的作用。目标 2 将定义新型可溶性 FOX 在转细胞铁途径中的作用，而目标 3 将阐明 Cp 在铁吸收中的作用，并将确定铁缺乏期间增强 Holo-Cp 产生的机制。总体而言，该项目将通过揭示铁转运的新机制细节来推进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。