Ferroportin and iron export from the macrophage

描述(由申请人提供)： 这份申请是为了进一步培训候选人Mitchell Knutson，他拥有营养学博士学位和铁代谢分子生物学的博士后经验。克努森博士目前的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，克努森博士将接受哈佛大学公共卫生学院(HSPH)巨噬细胞生物学专家莱斯特·科布齐克博士的指导，并由他目前在HSPH的导师玛丽安·韦斯林-雷斯尼克博士共同指导。这项研究计划将调查新发现的蛋白质，铁蛋白，FPN1(也称为MTP1或IREG1)，在巨噬细胞铁代谢中的功能。需要检验的假设是，FPN1在吞噬红细胞后从巨噬细胞中排出铁的过程中发挥作用。巨噬细胞吞噬红细胞，随后将铁释放到循环中，构成了体内最大的铁通量。然而，这其中的机制尚不清楚。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。红细胞吞噬前后将评估细胞定位。红细胞吞噬后将测量FPN1的mRNA和蛋白表达，并将这些变化与铁释放速率的变化进行比较，这是通过59Fe标记的红细胞吞噬后59Fe的外流来测量的。为了验证FPN1在铁释放中起作用的假设，我们将通过逆转录病毒载体转导在巨噬细胞中过表达FPN1以及使用反义技术抑制FPN1后，测量红细胞来源的59Fe的外流。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在当地的专业知识，这为Knutson博士提供了一个非常适合学习进行拟议实验所需技能的环境。这些实验的成功完成将大大有助于我们了解巨噬细胞中的铁代谢，并将使Knutson博士能够朝着他成为一名独立研究员和营养学助理教授的长期职业目标前进。此外，考虑到遗传性血色素沉着症和慢性病贫血等巨噬细胞铁代谢的紊乱，更好地了解巨噬细胞铁的释放具有重要的临床意义。

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.