中国科学院昆明动物研究所机构知识库

铁是人体的必需微量元素。铁参与多种生理代谢过程，如氧化代谢、红细胞 生成、免疫应答等。但过量的铁具有细胞毒性，细胞内外的铁离子浓度通过调节 转入和运出得以平衡。转铁蛋白（ferroportin, FPN）是目前哺乳细胞中唯一已知 的铁转出转运体。转铁蛋白功能异常会引发转铁蛋白病或 4 型血色病。然而膜蛋 白表达和纯化的困难使得执行精确的生物化学和生物物理实验十分困难，进而阻 碍了人们对转铁蛋白分子水平上的铁转运和调控机制的理解。尽管细菌同源体 BbFPN 的结构得到了解析，但由于 BbFPN 和人源转铁蛋白只有 24%的一致性和 40%的相似性，BbFPN 能提供的人源转铁蛋白（Human ferroportin, hFPN）结构 信息十分有限。而在缺乏模板情况下提出的 hFPN 分子结构模型需要确切的实验 检验。 真核转铁蛋白的高保守性为通过哺乳类转铁蛋白研究人源转铁蛋白提供了 合理性。在早期蛋白表达筛选的基础上，本论文展示了异源表达和纯化菲律宾眼 镜猴（Tarsius syrichta）的转铁蛋白（Monkey ferroportin，mFPN）的过程，表明 了纯化的 mFPN 蛋白对二价铁和钴离子的有结合和转运活性。进一步的研究表 明 mFPN 对二价离子的亲和性在 pH6.0 时基本丧失，说明离子的结合位点有可 能是由酸性氨基酸侧链组成。 用 mFPN 作为模型，本论文研究了两个人类致病性遗传突变体 N174I 和 D181V的功能。我表达纯化了突变体并测量它们对底物转运活性及结合亲和力。 实验发现 N174I 突变体在转运速率和底物结合亲和力上都出现显著性降低，这说 明它可能是直接影响底物的结合。D181V 转运活性出现显著降低，但底物结合亲 和力却没有降低，这意味着该突变可能通过影响 FPN 构象变化来阻碍转运。以 上实验结果从分子水平上阐释了这两种突变引起疾病的发病机理。 用细菌同源体 BbFPN 的结构作为模板，发现 mFPN 上 S35、D39、H43、 T150、D181、E219、D325 和 R466 有可能会跟底物结合位点有关。进一步对这 些位点进行突变，表达纯化突变体并测量它们对底物转运活性及结合亲和力。实 摘 要 II 验发现这些突变对底物的亲和力都不发生变化，但是大部分的突变体降低底物的 转运速度。这些实验结果说明细菌同源体 BbFPN 的结构跟哺乳类 FPN 的结构可 能会有较大的差异。 本研究从分子层面上加深了我们对 FPN 转运底物的理解，为铁代谢调控提 供了思路。

Iron is an essential microelement in human. Iron is crucial in physiological processes such as oxidative metabolism, erythropoiesis and immune responses. However, excessive iron is cytotoxic and the intracellular concentration of iron is balanced by adjusting its import and export. The solute carrier family 40 member 1 (SLC40A1), also known as ferroportin (FPN), is the only known iron exporter in mammals. Although it is known that dysfunction of FPN causes the ferroportin disease or type 4 hemochronatosis with symptoms such as liver damage, arrhythmias, and diabetes, we have limited knowledge of how FPN recognizes and transports iron and how the iron transport is regulated. This knowledge gap is mainly due to difficulties in obtain large amount of pure FPN for precise biochemical and biophysical characterizations. Structures of a bacterial iron transporter, BbFPN, was recently resolved, but due to the low homology between the bacterial and human FPN proteins, knowledge gained from structural and functional studies on the bacterial transporter may not apply to human FPN. The FPNs are highly conserved in mammals, and in this thesis I show for the first time large-scale expression and purification of a FPN from Tarsius syrichta, commonly known as tarsier monkey, mFPN. I then implemented a biding assay to measure ion affinity on mFPN and a transport assay to measure ion transport for mFPN reconstituted in proteoliposmes. I demonstrated that the purified mFPN protein binds to and transports divalent cations such as Fe2+ and Co2+. Further studies showed that mFPN loses its affinity to divalent cations at pH 6.0, suggesting that the metal binding site is composed of negatively charged residues. Since mFPN is 93% identical to human FPN, I studied two human mutations N174I and D181V that cause ferroportin diseases on the mFPN. I expressed and purified the mutant proteins and examined their binding and transport activities. I found that the N174I mutant has a significantly reduced binding affinity and a lower rate of Abstract IV transport, indicating that it may be directly involved in substrate binding. The D181V mutant has a lowered transport activity but its affinity to divalent cation is similar to that of the wild type, suggesting that the mutation affects conformational changes required for substrate transport. These results provide a molecular level understanding for how the two mutations cause diseases in human. I then examined if the bacterial BbFPN structure can predict where the iron binding site is on mammalian FPNs. Using the structure of BbFPN as a template, I found that residues S35, D39, H43, T150, D181, E219, D325 and R466 on the mammalian mFPN may be close to the iron binding site. I made mutations to these residues, one at a time, expressed and purified mutant proteins, and measured their binding and transport activities. I found that all of the mutants have a neglible effect on divalent ion binding but most of them have an effect on the rate of ion transport. These results indicate that the substrate binding site predicted by the bacterial BpFPN structure may not reflect the binding site on mammalian FPNs. This research increased our understanding of mammalian ferroportin functions at the molecular level and provided a solid basis for future structural and functional studies.