Ferroportin and APP: Regulation of Iron Trafficking at the Blood-Brain Barrier

铁转运蛋白和 APP：血脑屏障铁运输的调节

• 批准号: 9540089
• 负责人: DANIEL J. KOSMAN
• 金额: $ 39.25万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9540089
• 关键词: APLP1 gene; APLP2 gene; Alzheimer' s Disease; Amino Acids; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Amyloid deposition; Astrocytes; Binding; Binding Proteins; Biological Models; Biophysics; Blood - brain barrier anatomy; Blood capillaries; Brain; Capillary Endothelial Cell; Cell membrane; Cells; Cerebral Amyloid Angiopathy; Chimeric Proteins; Clinical Data; Copper; Cultured Cells; Data; Disease; Elements; Exhibits; Extravasation; Ferritin; Fluorescence Resonance Energy Transfer; Functional disorder; Human; Hypoxia; Iron; Knockout Mice; Link; Mammalian Cell; Maps; Modeling; Molecular; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neurons; Oxidation-Reduction; Oxidative Stress; Pathology; Peptides; Pericytes; Permeability; Pharmacology; Physiological; Play; Production; Protein Biosynthesis; Proteins; Proteomics; Publishing; Reactive Oxygen Species; Recombinants; Regulation; Regulon; Research; Role; Structure; Testing; Trace metal; Transcript; Transition Elements; Two-Hybrid System Techniques; Work; Yeasts; amyloid precursor protein processing; amyloidogenesis; beta-site APP cleaving enzyme 1; cell type; cerebral capillary; cerebral microvasculature; crosslink; design; formycin triphosphate; genetic approach; hepcidin; imaging potential; insight; interstitial; iron metabolism; metal transporting protein 1; neurovascular unit; novel; prevent; protein B; protein metabolism; protein protein interaction; response; secretase; stem; success; synthetic peptide; trafficking

Redox active first row transition metals such as iron and copper have been implicated in the pathophysiology of amyliodogenic diseases; these correlations are poorly understood at the level of mechanism. We also have limited understanding of the physiologic role for which the amyloid precursor protein (APP) has been selected. However, the fact that the APP transcript contains an iron response binding protein (IRBP) stem-loop structure and like ferritin, APP protein synthesis directly correlates with cell iron content indicating that APP is part of the “iron regulon.” This iron regulation of APP remains an unknown with respect to both iron metabolism and amyloidogenesis; here we propose to test a specific premise with respect to a physiologic function of APP and its secretase-dependent products. First, published work from our lab provides a clue as to the connection between APP and iron metabolism. We have identified a unique function exhibited by a 22-amino acid residue peptide element at the N-terminus of the BC helical bundle contained in the E2 domain in APP. Using recombinant BC or a synthetic peptide (residues 327-348 within BC), we show that this sequence tethers APP to and stabilizes ferroportin (Fpn) in the plasma membrane. Fpn is the sole mammalian iron exporter; by stabilizing Fpn in the PM, sAPP stimulates cell iron-efflux. We have designated this species FTP for Ferroportin Targeting Peptide. In binding to Fpn, sAPP/FTP acts as antagonist of the key regulator of Fpn function, hepcidin (Hepc) indicating that sAPP is a key regulator of iron efflux from any mammalian cell that expresses this amyliodogenic protein or shares an interstitial space that includes another cell type that does. Although the effect of sAPP on Fpn plasma membrane display is now established, the molecular basis of this activity and its physiologic function remain significant unknowns, significant because of: 1) the key role APP and its products play in Alzheimer's disease and in cerebral amyloid angiopathy, CAA; 2) the implied role that iron plays in this neurodegenerative disorder; and 3) the potential that FTP or its synthetic congeners may have as (a) pharmacologic regulator of iron metabolism. The key element of this Application is our blood-brain barrier transwell model system composed of brain capillary endothelial cells, astrocytes and neurons, i.e, the cells of the neurovascular unit. Our premise is that: 1) iron regulates the expression of APP so as to modulate the level of iron in the brain's abluminal space; 2) iron also modulates the processing of APP to Aß; and 3) Aß in turn serves as an iron scavenger and by this mechanism is cytoprotective. We will test this premise by a combination of biophysical structure-function, cell biologic and genetic approaches including the use of primary cells cultured from strategically chosen mice KO lines. This research plan will: 1) provide novel insight into the mechanism of the Fpn/APP interaction and its role in cellular trafficking of iron; 2) test our premise that mismanagement of this iron leads to increased permeability in our BBB model; and 3) test our premise that Aß is a key player in preventing this capillary leakage by suppressing iron's activity as a potent pro-oxidant.

具有氧化还原活性的第一行过渡金属如铁和铜与病理生理学有关 淀粉样碘生成性疾病;这些相关性在机制水平上知之甚少。我们也有 对选择淀粉样前体蛋白（APP）的生理作用的理解有限。 然而，APP转录物含有铁反应结合蛋白（IRBP）茎环结构的事实， 和铁蛋白一样，APP蛋白质合成与细胞铁含量直接相关，表明APP是铁蛋白的一部分。 “铁调节子”APP的这种铁调节在铁代谢和铁代谢方面仍然是未知的。 淀粉样蛋白生成;在这里，我们建议测试一个特定的前提，就生理功能的APP和 分泌酶依赖性产物。首先，我们实验室发表的工作提供了一个线索， APP和铁代谢之间的联系我们已经确定了一个22个氨基酸残基的独特功能， APP中E2结构域所含BC螺旋束N末端的肽元件。使用 重组人β B-C或合成肽（β B-C内的残基327-348），我们表明，该序列连接 APP在质膜上并稳定膜铁转运蛋白（Fpn）。FPN是唯一的哺乳动物铁输出者; 稳定PM中的Fpn，sAPP刺激细胞铁流出。我们已经指定这个物种FTP为 铁转运蛋白靶向肽。在与Fpn的结合中，sAPP/FTP充当Fpn关键调节因子的拮抗剂 铁调素（Hepc）的功能，表明sAPP是铁从任何哺乳动物细胞流出的关键调节因子， 表达这种淀粉样碘生成蛋白或共享包括另一种细胞类型的间隙空间。 虽然现在已经确定了sAPP对Fpn质膜展示的影响，但这种影响的分子基础仍然是未知的。 活性及其生理功能仍然是重要的未知数，重要的原因是：1）APP的关键作用 及其产物在阿尔茨海默病和脑淀粉样血管病中的作用; 2） 铁在这种神经退行性疾病中起作用; 3）FTP或其合成同系物可能 作为铁代谢的药物调节剂。这个应用程序的关键要素是我们的血脑 由脑毛细血管内皮细胞、星形胶质细胞和神经元组成的屏障transwell模型系统，即 神经血管单位的细胞。我们的前提是：1）铁调节APP的表达，从而调节 铁在脑腔外空间中的水平; 2）铁也调节APP到ApoE的加工;和3）ApoE 反过来作为铁清除剂，并通过这种机制具有细胞保护作用。我们将通过一个 生物物理结构-功能、细胞生物学和遗传学方法的组合，包括使用初级 从策略性选择的小鼠KO系培养的细胞。本研究计划将：1）提供新的见解， Fpn/APP相互作用的机制及其在铁的细胞运输中的作用; 2）测试我们的前提， 这种铁的管理不当导致我们的BBB模型中的渗透性增加;以及3）测试我们的前提，即 是通过抑制铁作为一种有效的促氧化剂的活性来防止毛细血管渗漏的关键人物。