Mechanism of Ferroportin Ubiquitination and its Therapeutic Potential in Anemia of Chronic Disease

海普西丁-铁门蛋白信号通路在许多铁代谢紊乱中起着重要作用，包括铁超载综合征(血色素沉着症)和贫血。慢性病贫血是第二种最常见的贫血形式，与感染、恶性肿瘤和炎症有关。慢性病贫血的特征是血浆铁浓度低，尽管骨髓、肝脏和脾中储存了足够的铁。在这些情况下，炎性细胞因子诱导肝脏激素海普西丁的表达。海普西丁翻译后调节铁出口蛋白铁蛋白的细胞表面表达。铁转运蛋白是唯一的膜通道，能够从细胞内输出铁，从而调节循环铁量。海普西丁与铁蛋白结合，诱导其泛素化、内化和溶酶体降解。其结果是，可用于发育红细胞的铁的含量减少。本研究的目的是确定海普西丁诱导的铁蛋白泛素化所涉及的酶，并确定抑制铁蛋白泛素化是否为治疗慢性病贫血的一种潜在的新疗法。融合绿色荧光蛋白(GFP)的可诱导表达铁门蛋白的细胞系将被用于筛选商业上可用的siRNA文库，该文库针对可能参与泛素向铁门蛋白转移的个别酶。在确定了参与铁蛋白降解的泛素结合和连接酶之后，将研究siRNA介导的这些酶的敲除在松节油诱导的慢性病小鼠贫血发生中的作用。宿主实验室已经建立了一株表达可诱导的铁孔蛋白-绿色荧光蛋白的细胞系，并能够通过BMP处理来诱导海普西丁的表达。这使得在更多的生理条件下分析铁蛋白的降解成为可能。此外，东道主实验室的负责人布洛赫博士在铁稳态领域和慢性病小鼠模型的使用方面拥有丰富的经验。拟议的研究计划将通过阐明铁转运蛋白泛素化和降解的机制，增加我们对铁稳态的理解。预计这些研究的结果将允许开发新的方法来治疗铁限制障碍患者。

The hepcidin-ferroportin signaling pathway has an important role in many disorders of iron metabolism, including iron overload syndromes (hemochromatosis) and anemia. Anemia of chronic disease is the second most common form of anemia and is present in conditions associated with infection, malignancy and inflammation. Anemia of chronic disease is characterized by low plasma iron concentrations despite sufficient stores of iron in the bone marrow, liver and spleen. In patients with these conditions, inflammatory cytokines induce expression of the hepatic hormone hepcidin. Hepcidin post-translationally regulates the cell surface expression of the iron exporter ferroportin. Ferroportin is the only membrane channel that is able to export iron from inside cells and thereby regulates the amount of circulating iron. Hepcidin binds to ferroportin and induces its ubiquitination, internalization and lysosomal degradation. As a result, the amount of iron that is available to developing red blood cells decreases. The objectives of the proposed research are to identify the enzymes involved in hepcidin- induced ferroportin ubiquitination and to determine whether inhibition of ferroportin ubiquitination is a potential novel therapy for the treatment of anemia of chronic disease. A cell line that inducible expresses ferroportin fused to green fluorescent protein (GFP) will be used to screen a commercially-available siRNA library that targets individual enzymes that are potentially involved in the transfer of ubiquitin to ferroportin. After identifying the ubiquitin conjugating and ligating enzymes that are involved in the degradation of ferroportin, the effect of siRNA-mediated knockdown of these enzymes on the development of turpentine-induced anemia of chronic disease in mice will be investigated. The host laboratory has established a cell line that expresses inducible ferroportin-GFP and is able to induce the expression of hepcidin by BMP treatment. This allows the analysis of ferroportin degradation under more physiological conditions. In addition, the chief of the host laboratory, Dr. Bloch, has extensive experience in the field of iron homeostasis and the use of murine models of chronic disease. The proposed research program will increase our understanding of iron homeostasis by elucidating the mechanism of ferroportin ubiquitination and degradation. It is anticipated that the results of these studies will permit the development of novel approaches to treat patients with iron-restrictive disorders.