Membrane proteins and iron delivery to cells

膜蛋白和铁输送至细胞

• 批准号: 7726873
• 负责人: THOMAS WALZ
• 金额: $ 34.74万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7726873
• 关键词: Apical; Binding; Blood Circulation; Carrier Proteins; Cells; Complex; Condition; Cryoelectron Microscopy; Crystallization; Detergents; Dietary Iron; Disease; Docking; Electron Microscopy; Electrons; Elements; Enterocytes; Escherichia coli; Evolution; Family; Goals; Helix (Snails); Hemochromatosis; Homologous Gene; Human; Hydroxide Ion; Hydroxides; Hydroxyl Radical; In Transferrin; Individual; Insecta; Integral Membrane Protein; Intestines; Ions; Iron; Iron Metabolism Disorders; Iron Overload; Knowledge; Lead; Life; Ligands; Lipids; Lobe; Lysosomes; Mammalian Cell; Maps; Measures; Mediating; Membrane; Membrane Proteins; Metals; Micelles; Modeling; Mutation; N-terminal; Nature; Nramp protein; Nucleic Acids; Orthologous Gene; Oxidation-Reduction; Phase; Physiological; Positioning Attribute; Process; Proteins; Protons; Reaction; Relative (related person); Research Personnel; Resolution; Roentgen Rays; SLC11A2 gene; Sampling; Signal Transduction; Source; Structure; Surface; Techniques; Testing; Toxic effect; Transferrin; Transferrin Receptor; X-Ray Crystallography; abstracting; apical membrane; base; basolateral membrane; density; divalent metal; ear helix; electron crystallography; hepcidin; improved; insight; interest; member; metal transporting protein 1; microcytic/hypochromic anemia; monolayer; numb protein; particle; peptide hormone; programs; protein structure function; receptor binding; two-dimensional; uptake; Apical; Binding; Blood Circulation; Carrier Proteins; Cells; Complex; Condition; Cryoelectron Microscopy; Crystallization; Detergents; Dietary Iron; Disease; Docking; Electron Microscopy; Electrons; Elements; Enterocytes; Escherichia coli; Evolution; Family; Goals; Helix (Snails); Hemochromatosis; Homologous Gene; Human; Hydroxide Ion; Hydroxides; Hydroxyl Radical; In Transferrin; Individual; Insecta; Integral Membrane Protein; Intestines; Ions; Iron; Iron Metabolism Disorders; Iron Overload; Knowledge; Lead; Life; Ligands; Lipids; Lobe; Lysosomes; Mammalian Cell; Maps; Measures; Mediating; Membrane; Membrane Proteins; Metals; Micelles; Modeling; Mutation; N-terminal; Nature; Nramp protein; Nucleic Acids; Orthologous Gene; Oxidation-Reduction; Phase; Physiological; Positioning Attribute; Process; Proteins; Protons; Reaction; Relative (related person); Research Personnel; Resolution; Roentgen Rays; SLC11A2 gene; Sampling; Signal Transduction; Source; Structure; Surface; Techniques; Testing; Toxic effect; Transferrin; Transferrin Receptor; X-Ray Crystallography; abstracting; apical membrane; base; basolateral membrane; density; divalent metal; ear helix; electron crystallography; hepcidin; improved; insight; interest; member; metal transporting protein 1; microcytic/hypochromic anemia; monolayer; numb protein; particle; peptide hormone; programs; protein structure function; receptor binding; two-dimensional; uptake

Abstract Our long-term objective is to understand the structural basis for the delivery of iron to cells. In particular we will study the structure of the following three proteins: (/) The transferrin (Tf)-transferrin receptor (TfR) complex. We have determined the structure of the Tf-TfR complex using a soluble construct of the TfR ectodomain lacking the stalk region. The resulting structure strongly suggests that the TfR stalk is involved in Tf binding. We will now determine the structure of the complex in the presence of the stalk and perform functional studies to elucidate the effect of the TfR stalk on iron release from the N-terminal lobe of receptor- bound Tf. (//) The divalent metal ion transporter-1 (DMT1). Iron released from the Tf-TfR complex is transported across the endosomal membrane by DMT1, the same protein that mediates iron uptake from the intestinal lumen through the apical surface of duodenal enterocytes. Mutations in DMT1 cause severe hypochromic microcytic anemia and iron overload. We have expressed mg amounts of the DMT1 ortholog from E. co//. We are using this protein to produce two-dimensional (2D) crystals suitable for electron crystallographic structure determination. In parallel, we will attempt to grow three-dimensional (3D) crystals for X-ray crystallographic structure determination and perform structural studies on other bacterial homologs as well as human DMT1. (Hi)Ferroportin. A second iron transporter, ferroportin, exports iron across the basolateral membrane of duodenal enterocytes to the circulation. Mutations in ferroportin cause type IV hemochromatosis, also known as ferroportin disease. We will express human ferroportin for 2D and later for 3D crystallization trials to determine its structure either by electron or X-ray crystallography. We will then decorate ferroportin2D crystals with the peptide hormone hepcidin to elucidate the binding interaction. Relevance Many proteins depend on iron as a co-factor for redox reactions or ligand coordination, making iron an essential element. The facile conversion between ferrous (Fe2+) and ferric iron (Fe3+) poses significant dangers to living cells, however, because it can lead to the formation of hydroxyl radicals, a major source for oxidative damage to proteins, nucleic acids and lipids. Moreover, under physiological conditions ferric iron forms a highly insoluble hydroxide complex, so that despite its abundance, iron is not easily accessible to cells. Toxicity and insolubility have forced the evolution of highly sophisticated machineries for acquiring, storing, and distributing iron. Malfunctioning of these machineries lead either to iron deficiency disorders or iron overload diseases.

抽象的 我们的长期目标是了解将铁递送到细胞的结构基础。特别是我们 将研究以下三种蛋白的结构：（/）转铁蛋白（TF） - 转铁蛋白受体（TFR） 复杂的。我们使用TFR的可溶构建体确定了TF-TFR复合物的结构 外生域缺乏茎区域。最终的结构强烈表明涉及TFR茎 在TF结合中。现在，我们将在茎的存在下确定复合物的结构 功能研究以阐明TFR茎对受体N端叶释放的影响 绑定的TF。 （//）二价金属离子转运蛋白1（DMT1）。从TF-TFR建筑群释放的铁是 通过DMT1在内体膜上运输，同样的蛋白质介导了铁的摄取 肠腔穿过十二指肠肠细胞的顶端表面。 DMT1突变导致严重 低色素微细胞贫血和铁超负荷。我们已经表达了MG数量的DMT1直系同源物 来自E. Co //。我们正在使用这种蛋白质产生适合电子的二维（2D）晶体 晶体结构的确定。同时，我们将尝试生长三维（3D）晶体 用于X射线晶体结构的确定并对其他细菌同源物进行结构研究 以及人类DMT1。 （嗨）铁蛋白。第二个铁运输蛋白的铁蛋白在整个 循环的十二指肠肠细胞的基底外侧膜。铁蛋白的突变导致IV型 血色素肿大，也称为铁蛋白疾病。我们将以2D和以后的人来表达人类铁蛋白 3D结晶试验通过电子或X射线晶体学确定其结构。然后我们会 用肽激素肝素来装饰铁蛋白2D晶体，以阐明结合相互作用。 关联 许多蛋白质依赖铁作为氧化还原反应或配体配位的共同因素，使铁成为 基本元素。亚铁（Fe2+）和铁铁（Fe3+）之间的便利转化构成显着的 但是，对活细胞的危险，因为它可以导致形成羟基自由基，这是 对蛋白质，核酸和脂质的氧化损伤。此外，在生理条件下铁铁 形成一种高度不溶性的氢氧化物络合物，因此尽管它丰富，但铁不容易到达 细胞。毒性和不溶性迫使高度复杂的机器的进化用于获取， 存储并分发铁。这些机器的故障导致铁缺乏障碍或 铁超负荷疾病。