Ferroportin and iron export from the macrophage

巨噬细胞的铁转运蛋白和铁输出

• 批准号: 7294079
• 负责人: Mitchell D Knutson
• 金额: $ 0.1万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294079
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; antisense nucleic acid; confocal scanning microscopy; densitometry; flow cytometry; fluorescence microscopy; immunofluorescence technique; iron metabolism; laboratory rabbit; laboratory rat; macrophage; membrane proteins; northern blottings; phagocytosis; protein structure function; protein transport; recombinant proteins; ultracentrifugation; western blottings

DESCRIPTION (provided by applicant): This application is for further training of the candidate, Mitchell Knutson, who has a Ph.D. in Nutrition and post-doctoral experience in the molecular biology of iron metabolism. Dr. Knutson's immediate career goal is to acquire new skills and knowledge that will enable him to study iron metabolism in macrophages. To accomplish this task, Dr. Knutson will be mentored by Dr. Lester Kobzik, an expert in macrophage biology at the Harvard School of Public Health (HSPH), and co-mentored by Dr. Marianne Wessling-Resnick, his current mentor at HSPH. The research proposal will investigate the function of the newly identified protein, ferroportin, FPN1 (also known as MTP1 or IREG1), in iron metabolism in the macrophage. The hypothesis to be tested is that FPN1 plays a role in iron export from the macrophage after phagocytosis of red blood cells. Erythrophagocytosis by macrophages, with the subsequent release of iron into the circulation, constitutes the largest flux of iron within the body. The mechanism for this, however, is unknown. To investigate the role of FPN1 in the macrophage, immunofluorescence experiments will determine the subcellular localization of this protein. Cytolocalization will be assessed before and after erythrophagocytosis. FPN1 mRNA and protein expression will be measured after erythrophagocytosis, and the changes will be compared to changes in rates of iron release, as measured by the efflux of 59Fe after phagocytosis of 59Fe-labeled erythrocytes. To test the hypothesis that FPN1 plays a role in iron release, efflux of erythrocyte-derived 59Fe will be measured after overexpressing FPN1 in macrophages using retroviral vector transduction, as well as after suppressing FPN1 using antisense techniques. The proximity of experts in macrophage biology, retroviral transduction, and antisense technology at HSPH, combined with the local expertise of investigators in the iron field, offers Dr. Knutson a highly suitable environment for learning the necessary skills required to carry out the proposed experiments. Successful completion of these experiments will contribute significantly to our understanding of iron metabolism in the macrophage and will enable Dr. Knutson to advance towards his long-term career goal of becoming an independent investigator and Assistant Professor of Nutrition. Moreover, a better understanding of iron release from the macrophage is of considerable clinical importance given the disturbances in macrophage iron metabolism characteristic of hereditary hemochromatosis and the anemia of chronic disease.

描述（由申请人提供）： 此申请旨在进一步培训候选人 Mitchell Knutson，他拥有博士学位。营养学博士学位和铁代谢分子生物学的博士后经验。克纳森博士的近期职业目标是获得新的技能和知识，使他能够研究巨噬细胞中的铁代谢。为了完成这项任务，Knutson 博士将得到哈佛大学公共卫生学院 (HSPH) 巨噬细胞生物学专家 Lester Kobzik 博士的指导，并由他在 HSPH 的现任导师 Marianne Wessling-Resnick 博士共同指导。该研究计划将研究新发现的蛋白质铁转运蛋白 FPN1（也称为 MTP1 或 IREG1）在巨噬细胞铁代谢中的功能。待检验的假设是 FPN1 在巨噬细胞吞噬红细胞后铁输出中发挥作用。巨噬细胞的红细胞吞噬作用，随后将铁释放到循环中，构成体内最大的铁通量。然而，其机制尚不清楚。为了研究 FPN1 在巨噬细胞中的作用，免疫荧光实验将确定该蛋白的亚细胞定位。将在吞噬红细胞之前和之后评估细胞定位。吞噬红细胞后将测量 FPN1 mRNA 和蛋白质表达，并将变化与铁释放率的变化进行比较，铁释放率的变化是通过吞噬 59Fe 标记的红细胞后 59Fe 的流出来测量的。为了检验 FPN1 在铁释放中发挥作用的假设，使用逆转录病毒载体转导在巨噬细胞中过度表达 FPN1 后，以及使用反义技术抑制 FPN1 后，将测量红细胞来源的 59Fe 的流出。 HSPH 拥有巨噬细胞生物学、逆转录病毒转导和反义技术方面的专家，再加上铁领域研究人员的当地专业知识，为 Knutson 博士提供了一个非常适合学习进行拟议实验所需的必要技能的环境。这些实验的成功完成将极大地促进我们对巨噬细胞中铁代谢的理解，并使克纳森博士能够朝着成为独立研究者和营养学助理教授的长期职业目标迈进。此外，考虑到遗传性血色素沉着症和慢性疾病贫血的巨噬细胞铁代谢紊乱特征，更好地了解巨噬细胞的铁释放具有相当大的临床重要性。