Membrane Transport Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd., Herston, Brisbane, QLD 4006, Australia.
Am J Physiol Cell Physiol. 2010 Jan;298(1):C75-84. doi: 10.1152/ajpcell.00621.2008. Epub 2009 Oct 21.
Ferroportin disease is a heterogeneous iron release disorder resulting from mutations in the ferroportin gene. Ferroportin protein is a multitransmembrane domain iron transporter, responsible for iron export from cells, which, in turn, is regulated by the peptide hormone hepcidin. Mutations in the ferroportin gene may affect either regulation of the protein's transporter function or the ability of hepcidin to regulate iron efflux. We have used a combination of functional analysis of epitope-tagged ferroportin variants coupled with theoretical modeling to dissect the relationship between ferroportin mutations and their cognate phenotypes. Myc epitope-tagged human ferroportin expression constructs were transfected into Caco-2 intestinal cells and protein localization analyzed by immunofluorescence microscopy and colocalization with organelle markers. The effect of mutations on iron efflux was assessed by costaining with anti-ferritin antibodies and immunoblotting to quantitate cellular expression of ferritin and transferrin receptor 1. Wild-type ferroportin localized mainly to the cell surface and intracellular structures. All ferroportin disease-causing mutations studied had no effect on localization at the cell surface. N144H, N144T, and S338R mutant ferroportin retained the ability to transport iron. In contrast, A77D, V162Delta, and L170F mutants were iron transport defective. Surface staining experiments showed that both ends of the protein were located inside the cell. These data were used as the basis for theoretical modeling of the ferroportin molecule. The model predicted phenotypic clustering of mutations with gain-of-function variants associated with a hypothetical channel through the axis of ferroportin. Conversely, loss-of-function variants were located at the membrane/cytoplasm interface.
铁蛋白病是一种铁释放紊乱的异质性疾病，由铁蛋白基因的突变引起。铁蛋白蛋白是一种多跨膜结构域铁转运体，负责细胞内铁的输出，而铁蛋白则受肽激素铁调素的调节。铁蛋白基因的突变可能影响蛋白转运功能的调节或铁调素调节铁外排的能力。我们结合了对表位标记的铁蛋白变体的功能分析和理论建模，以剖析铁蛋白突变与其表型的关系。将 Myc 表位标记的人铁蛋白表达构建体转染到 Caco-2 肠细胞中，并通过免疫荧光显微镜和与细胞器标记物的共定位分析蛋白质定位。通过与抗铁蛋白抗体共染色并用免疫印迹定量细胞内铁蛋白和转铁蛋白受体 1 的表达来评估突变对铁外排的影响。野生型铁蛋白主要定位于细胞表面和细胞内结构。研究的所有铁蛋白病致病突变对细胞表面的定位均无影响。N144H、N144T 和 S338R 突变铁蛋白仍具有转运铁的能力。相比之下，A77D、V162Delta 和 L170F 突变体则铁转运功能缺陷。表面染色实验表明，蛋白质的两端都位于细胞内。这些数据被用作铁蛋白分子理论建模的基础。该模型预测突变的表型聚类与具有功能获得的变体相关，这些变体与铁蛋白轴上的假想通道相关。相反，功能丧失的变体位于膜/细胞质界面。
