Membrane proteins and iron delivery to cells

膜蛋白和铁输送至细胞

• 批准号: 7244743
• 负责人: THOMAS WALZ
• 金额: $ 33.8万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244743
• 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

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.

摘要 我们的长期目标是了解向细胞输送铁的结构基础。尤其是我们 我将研究以下三种蛋白质的结构：(/)转铁蛋白(Tf)-转铁蛋白受体(Tfr) 很复杂。我们已经使用TFR的可溶性构建物确定了Tf-TFR复合体的结构 无柄区域的外域。由此得到的结构强烈地表明TFR茎参与了 在转铁蛋白结合中。现在我们将在茎存在的情况下确定复合体的结构并执行 功能研究阐明TFR茎对受体N-末端叶释放铁的影响 绑定Tf。(//)二价金属离子转运蛋白1(DMT1)。从Tf-TFR复合体释放的铁是 通过DMT1跨内膜运输，同样的蛋白质介导铁的摄取来自 肠腔通过十二指肠肠上皮细胞的顶面。DMT1基因突变导致严重 低色素性小细胞性贫血和铁超载。我们已经表达了DMT1直系同源基因的毫克量 来自E.co//。我们正在使用这种蛋白质来产生适合电子的二维(2D)晶体 晶体结构测定。同时，我们将尝试生长三维(3D)晶体 用于X射线晶体结构测定和其他细菌同系物的结构研究 以及人类DMT1。(嗨)铁-磷灰石。另一种铁的运输者，铁蛋白，将铁出口到世界各地。 十二指肠肠上皮细胞的基底侧膜进入血液循环。铁转运蛋白基因突变导致IV型 血色素沉着症，又称铁色素沉着病。我们将表达人铁蛋白的2D和稍后的 3D结晶试验，以确定其结构的电子或X射线结晶学。到时候我们会的 用多肽激素海普西丁修饰铁门蛋白2D晶体以阐明结合作用。 相关性 许多蛋白质依赖铁作为氧化还原反应或配体配位的辅助因素，使铁成为 基本要素。亚铁(Fe2+)和铁离子(Fe3+)之间的简单转化具有重要意义 然而，对活细胞来说是危险的，因为它会导致羟基自由基的形成，羟基自由基是 对蛋白质、核酸和脂质的氧化损伤。此外，在生理条件下，铁离子 形成一种高度不溶的氢氧化物络合物，因此尽管铁含量丰富，但很难获得 细胞。毒性和不溶性迫使高度复杂的机器进化以获取， 储存和分发铁。这些机器的故障会导致缺铁症或 铁超载疾病。