Fluorescence-based screen to probe hepcidin-ferroportin interactions

基于荧光的筛选来探测铁调素-铁转运蛋白相互作用

• 批准号: 7169460
• 负责人: Marianne Wessling-Resnick
• 金额: $ 20.5万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-08 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169460
• 关键词: NIH Roadmap Initiative tag; biological signal transduction; dietary iron; drug discovery /isolation; ferriheme; fluorescent dye /probe; high throughput technology; inhibitor /antagonist; iron metabolism; macrophage; protein binding; protein protein interaction; protein structure function; protein transport; small molecule; technology /technique development

DESCRIPTION (provided by applicant): The final step of dietary iron assimilation - the exsorption of iron from the mucosa into circulation - involves the iron transport protein ferroportin. Macrophages of the reticuloendothelial system also play a major role in iron metabolism by recycling iron from damaged or senescent erythrocytes. Ferroportin also plays a major role in the release of iron from macrophages, and thus provides the major mechanism for the export of iron from the cells of our body. Body iron stores, hypoxia, the rate of erythropoiesis, pregnancy and inflammation all have dramatic effects on dietary iron assimilation and iron recycling by the reticuloendothelial system. How such factors influence the activity of ferroportin to modify iron export is an active area of interest in the field. The most important recent discovery has been the role of the liver-derived peptide hepcidin. Hepcidin is produced under iron- loading and inflammatory conditions to suppress dietary iron absorption and macrophage iron recycling, and its synthesis is diminished in response to iron deficiency, pregnancy or enhanced erythropoiesis to promote iron uptake from the diet and enhance iron recycling. This peptide therefore provides a direct link between the liver, bone marrow and intestine to adjust metabolism to meet the body's demand for iron. Hepcidin regulates ferroportin protein levels through binding interactions that induce its internalization and lysosomal degradation. The model that ferroportin is a receptor for hepcidin implicates a homeostatic mechanism that allows systemic regulation of intestinal iron absorption and macrophage iron recycling directly in response to the body's iron demands targeting ferroportin function. This model is borne out by clinical studies of hemochromatosis, an inherited disorder leading to abnormal iron accumulation in different tissues. This disease is associated with mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2 or FPN. Although the molecular pathogenesis has not been precisely elucidated in all cases, abnormal regulation of iron metabolism by hepcidin has been proposed as a common, unifying feature of the different forms of this disease. The specific aim of this R21 proposal is to develop a high throughput assay to screen for small molecule inhibitors of hepcidin-ferroportin binding interactions. Our strategy is to utilize Sfp-catalyzed site-specific protein labeling to establish a rapid, sensitive, and reproducible fluorescent-based screen for small molecules that block ligand-induced degradation of the hepcidin receptor, ferroportin. The project has broader applicability in the development of HTS to assay ligand-receptor interactions as well as representing a major advance towards the discovery of small molecule probes of iron transport.

描述（由申请人提供）：饮食中铁同化的最后一步 - 粘膜中铁的渗透到循环中 - 涉及铁转运蛋白铁蛋白。 网状内皮系统的巨噬细胞也通过从受损或衰老的红细胞中回收铁在铁代谢中起着重要作用。 铁托蛋白在从巨噬细胞中释放铁中也起着重要作用，因此为从人体细胞中出口铁提供了主要机制。 人体铁储存，缺氧，红细胞生成率，妊娠和炎症的速度对网状内皮系统对饮食中的铁同化和铁回收产生了巨大影响。 这些因素如何影响铁托蛋白改变铁出口的活性是该领域感兴趣的活跃领域。 最近最重要的发现是肝脏衍生的肽肝素的作用。 肝素是在铁负荷和炎症条件下产生的，以抑制饮食中的铁吸收和巨噬细胞铁回收，并且由于铁缺乏症，妊娠或增强的红细胞生成而减少了其合成，以促进饮食和增强铁回收的饮食中的铁。 因此，该肽提供了肝，骨髓和肠之间的直接联系，以调节新陈代谢以满足人体对铁的需求。 肝素通过结合相互作用来调节铁蛋白蛋白水平，从而诱导其内在化和溶酶体降解。 铁托蛋白是肝素的受体的模型暗示了一种稳态机制，该机制允许全身调节肠道铁吸收和巨噬细胞铁回收，直接响应人体的铁需求，以靶向铁杆素功能。 该模型由血色素症的临床研究揭示，这是一种遗传性疾病，导致了不同组织中铁的异常积累。 该疾病与编码HFE，肝素，血果蛋白，转铁蛋白受体-2或FPN的基因突变有关。 尽管在所有情况下尚未精确阐明分子发病机理，但肝素对铁代谢的异常调节被认为是这种疾病的不同形式的常见，统一的特征。 该R21提案的具体目的是开发高吞吐量测定法，以筛选肝素 - 铁蛋白结合相互作用的小分子抑制剂。 我们的策略是利用SFP催化的位点特异性蛋白标记来建立一个快速，敏感且可重现的基于荧光的筛选，以阻止配体诱导的肝素受体降解的小分子，即肝素受体。 该项目在HTS的开发中具有更广泛的适用性，以分析配体 - 受体相互作用，并代表了在发现铁转运小分子探针方面的重大进步。