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Biochemical, biophysical, and cellular investigations of the interactions of transferrin receptor with transferrin and the hereditary hemochromatosis protein, HFE

机译：转铁蛋白受体与转铁蛋白和遗传性血色素沉着蛋白HFE相互作用的生化，生物物理和细胞研究

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摘要

Hereditary hemochromatosis (HH) is a prevalent genetic disorder that results in the daily excess absorption of dietary iron. If untreated this disease leads to systemic organ failure and death. HH is caused by mutations to the gene coding for a protein called HFE, a type I transmembrane glycoprotein with a demonstrated role in regulating cellular iron homeostasis. HFE binds to the cell-surface receptor transferrin receptor (TfR), a dimeric type II transmembrane glycoprotein responsible for iron uptake into most mammalian cell types. TfR binds iron-loaded transferrin (Fe-Tf) from the blood and transports it to acidic recycling endosomes where iron is released from Fe-Tf in a TfR-facilitated process. Iron-free transferrin (apo-Tf) remains bound to TfR and is recycled to the cell surface, where apo-Tf rapidly dissociates from TfR upon exposure to the basic pH of blood. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This reduction could result from direct competition between HFE and Fe-Tf for receptor binding sites, from negative cooperativity, or both. We sought to understand the mechanism of HFE, Fe-Tf, and apo-Tf binding by TfR to help define HFE's role in iron homeostasis. We determined the binding constants for HFE, Fe-Tf, and apo-Tf to an extensive set of site-directed TfR mutants and discovered that HFE and Tf bind to an overlapping site on TfR, indicating the two proteins compete with each other for receptor binding. The mutagenesis results also identified differences in the contact points between TfR and the two forms of Tf, Fe-Tf and apo-Tf. By combining the mutations that are required for apo-Tf, but not Fe-Tf, binding we find that a highly conserved hydrophobic patch on the TfR surface is required for the receptor-mediated stimulation of iron release from Fe-Tf. From these data we propose a structure-based model for the mechanism of TfR-assisted iron release.To explore the mechanism of the HFE-induced affinity reduction for Fe-Tf binding by TfR, we engineered a heterodimeric TfR (hdTfR) that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Competition binding experiments using hdTfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of HFE's effects on iron homeostasis result from competition with Fe-Tf for TfR binding. Using transfected cell lines we show that HFE is dependent on its interactions with TfR for transport to endosomal compartments and that competition with extracellular Fe-Tf can alter HFE trafficking patterns. These data suggest that HFE's role in iron homeostasis is as a sensor of body iron status.
机译：遗传性血色素沉着病（HH）是一种普遍的遗传疾病，导致每日过量摄取膳食铁。如果不加以治疗，这种疾病会导致全身器官衰竭甚至死亡。 HH是由编码称为HFE的蛋白质的基因的突变引起的，HFE是一种I型跨膜糖蛋白，具有调节细胞铁稳态的作用。 HFE与细胞表面受体转铁蛋白受体（TfR）结合，TfR是负责大多数哺乳动物细胞类型摄取铁的二聚体II型跨膜糖蛋白。 TfR结合血液中载铁的转铁蛋白（Fe-Tf），并将其转运至酸性循环内体，在此过程中，铁通过TfR促进的过程从Fe-Tf释放出来。无铁转铁蛋白（apo-Tf）仍然与TfR结合，并循环到细胞表面，在暴露于血液的基本pH值时，apo-Tf会从TfR迅速解离。 HFE和Fe-Tf可以同时与TfR结合形成三元复合物，但是HFE与TfR的结合降低了Fe-Tf / TfR相互作用的表观亲和力。这种减少可能是由于HFE和Fe-Tf之间直接竞争受体结合位点，负协同作用或两者兼而有之。我们试图了解通过TfR结合HFE，Fe-Tf和apo-Tf的机制，以帮助定义HFE在铁稳态中的作用。我们确定了HFE，Fe-Tf和apo-Tf与大量定点TfR突变体的结合常数，并发现HFE和Tf与TfR上的重叠位点结合，表明这两种蛋白彼此竞争受体捆绑。诱变结果还确定了TfR与Tf的两种形式Fe-Tf和apo-Tf之间的接触点差异。通过结合载脂蛋白-Tf而不是Fe-Tf所需的突变，结合我们发现，受体介导的刺激铁从Fe-Tf释放所需的TfR表面上高度保守的疏水性斑块是必需的。从这些数据中，我们提出了基于结构的TfR辅助铁释放机理的模型。为探讨HFE诱导的TfR与Fe-Tf结合的亲和力降低的机理，我们设计了包含突变的异二聚TfR（hdTfR）。这样一条TfR链仅结合HFE，而另一条仅结合Fe-Tf。使用hdTfR的竞争结合实验表明，TfR在异向配体结合中不显示协同作用，这表明HFE对铁稳态的部分或全部影响是由于与Fe-Tf竞争TfR结合而引起的。使用转染的细胞系，我们表明HFE依赖于其与TfR的相互作用才能转运至内体区室，并且与细胞外Fe-Tf的竞争可以改变HFE的运输方式。这些数据表明，HFE在铁稳态中的作用是人体铁状态的传感器。

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  • 作者

    Giannetti Anthony Michael;

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  • 年度 2004
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  • 原文格式 PDF
  • 正文语种 {"code":"en","name":"English","id":9}
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