Ferroxidase (Fet3) and Permease (Ftr1) in Iron Uptake

铁吸收中的铁氧化酶 (Fet3) 和通透酶 (Ftr1)

• 批准号: 7597057
• 负责人: DANIEL J. KOSMAN
• 金额: $ 31.05万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7597057
• 关键词: Address; Aerobiosis; Affinity; Archives; Binding; Biology; Candida albicans; Cell membrane; Ceruloplasmin; Charge; Chelating Agents; Chlamydomonas reinhardtii; Collection; Complex; Coupled; Coupling; Cryptococcus neoformans; Databases; Defect; Dioxygen; Electron Transport; Electronics; Electrons; Environment; Enzymes; Eukaryota; Exhibits; Family; Generations; Genome; Goals; Homeostasis; Homologous Gene; Human; Hydrolysis; Immunocompromised Host; In Situ; In Vitro; Ions; Iron; Kinetics; Knowledge; Laccase; Maps; Membrane; Metabolic Pathway; Metabolism; Metals; Mining; Modeling; Nutrient; Organism; Oxidases; Oxidation-Reduction; Oxygen; Pathway interactions; Physiology; Play; Positioning Attribute; Process; Protein Conformation; Proteins; Protons; Reaction; Reactive Oxygen Species; Recombinants; Reducing Agents; Research; Resistance; Role; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Shipping; Ships; Side; Solvents; Specificity; Stream; Structure; Structure-Activity Relationship; System; Testing; Time; Transferrin; Virulence; Work; Yeasts; autooxidation; base; carboxylate; cohort; copper oxidase; cytotoxicity; design; electron donor; fitness; fungus; in vivo; inhibitor/antagonist; insight; mutant; oxidation; pathogen; permease; prevent; programs; public health relevance; repository; trafficking; uptake

DESCRIPTION (provided by applicant): Multicopper oxidases (MCOs) couple the 4e- reduction of dioxygen to 2H2O with the oxidation of 4 equivalents of a 1-electron donor. A sub-family of these ubiquitous enzymes possesses specificity towards FeII that makes them essential to iron homeostasis in their respective organisms. Our hypothesis is that these ferroxidases function by channeling their FeIII product to a down-stream partner in their iron metabolic pathways. This program has delineated the structure and function in the Fet3p, Ftr1p high-affinity Fe-uptake complex in the Saccharomyces cerevisiae (Sc) plasma membrane (PM). The two fundamental questions addressed in this work are: 1) what structural motifs confer on an MCO this specificity for FeII as substrate; and 2) how is the Fet3p ferroxidase reaction coupled kinetically and physically to the membrane permeation of FeIII by Ftr1p. In this application we propose three specific aims. In Aim I we will test our hypothesis of what structural motifs define a ferroxidase, specifically that a cohort of carboxylate side chains maximize three factors that determine e- transfer from FeII: FeII binding, FeII redox potential and electronic matrix coupling of the FeII and the type 1 CuII (T1Cu) in the ferroxidase. We will do this by interconverting laccase and ferroxidase enzymes based on our design principles. In addition, we will test our hypothesis that another cohort of carboxylate side chains stabilizes the increasing negative charge on dioxygen as it is reduced in 2, 2e- steps at the trinuclear cluster (TNC). Last we will test our hypothesis that the coordination changes associated with O2 reduction at the TNC trigger e- transfer from the T1 Cu via the canonical MCO His-Cys-His motif that connects the two. In Aim II we propose to test our hypothesis that Fox1 in Chlamydomonas reinhardtii (Cr) is a human ceruloplasmin-like ferroxidase. We will characterize the Fox1 protein, demonstrating that it has the ferroxidase-specificity motifs that support both FeII oxidation and FeIII trafficking in a Fox1, Ftr1 complex in the Cr PM. We will construct mutants of both Fox1 and Ftr1 that we predict will be sensitive to a FeIII-chelator acting as a metabolite trap in Fe-trafficking between the two proteins in Fe-uptake; we have used this classic test of channeling in the Sc Fet3, Ftr1p complex. We propose that the Fe-trafficking between Fox1 and Ftr1 in Cr provides a realistic model of the putative FeIII-trafficking between hCp and transferrin. In Aim III, we will test our hypothesis that the two human fungal pathogens, Candida albicans and Cryptococcus neoformans express an equivalent PM Fet, Ftr high-affinity Fe-uptake complex. We will quantify the 59Fe-uptake kinetics via these complexes both in situ and in recombinant form in Sc. Targeting specific ferroxidase and Fe-trafficking residues in the Ca and Cn proteins, we will test our hypothesis that these mutants exhibit the channeling defect exhibited by the Sc homologues. We propose that strains expressing these channeling mutants will exhibit a reduced virulence in vitro and in vivo. Last, using these chelator-sensitive clones, the NCI diversity collection will be mined for compounds with the potential as inhibitors of Fet, Ftr Fe-uptake in these fungal pathogens. PUBLIC HEALTH RELEVANCE: All oxygen-utilizing organisms from fungi to humans require the activity of a copper oxidase enzyme - a multicopper oxidase - to manage their metabolism of the essential nutrient, iron. Fungi from baker's yeast to the human pathogens C. albicans and C. neoformans use these enzymes to acquire the iron they need to thrive and survive. The goal of this research is to take a snap-shot of how these enzymes work and then to use this knowledge to block their trafficking of iron as way of suppressing the virulence of pathogenic fungi in both otherwise healthy and immunocompromised patients.

描述（由申请人提供）：多铜氧化酶（MCO）将双氧的 4e-还原成 2H2O 与 4 当量的 1-电子供体的氧化结合起来。这些普遍存在的酶的一个亚家族对 FeII 具有特异性，这使得它们对于各自生物体中的铁稳态至关重要。我们的假设是，这些亚铁氧化酶通过将其 FeIII 产物输送至铁代谢途径的下游伙伴来发挥作用。该程序描述了酿酒酵母 (Sc) 质膜 (PM) 中 Fet3p、Ftr1p 高亲和力铁吸收复合物的结构和功能。这项工作解决的两个基本问题是：1）什么结构基序赋予 MCO 这种以 FeII 作为底物的特异性； 2) Fet3p 亚铁氧化酶反应如何与 Ftr1p 对 FeIII 的膜渗透进行动力学和物理耦合。在此应用中，我们提出了三个具体目标。在目标 I 中，我们将测试我们的假设，即哪些结构基序定义了亚铁氧化酶，特别是一组羧酸侧链最大化了决定 FeII 电子转移的三个因素：FeII 结合、FeII 氧化还原电位以及 FeII 和铁氧化酶的电子基质耦合。亚铁氧化酶中的 1 型 CuII (T1Cu)。我们将根据我们的设计原理，通过漆酶和亚铁氧化酶的相互转化来实现这一点。此外，我们将测试我们的假设，即另一组羧酸侧链稳定双氧上增加的负电荷，因为它在三核簇 (TNC) 上以 2, 2e- 步骤减少。最后，我们将测试我们的假设，即 TNC 处与 O2 还原相关的协调变化会通过连接两者的典型 MCO His-Cys-His 基序触发来自 T1 Cu 的电子转移。在目标 II 中，我们建议检验我们的假设，即莱茵衣藻 (Cr) 中的 Fox1 是一种人类铜蓝蛋白样亚铁氧化酶。我们将表征 Fox1 蛋白，证明它具有亚铁氧化酶特异性基序，支持 Cr PM 中 Fox1、Ftr1 复合物中 FeII 氧化和 FeIII 运输。我们将构建 Fox1 和 Ftr1 的突变体，我们预测它们将对 FeIII 螯合剂敏感，该螯合剂充当铁吸收中两种蛋白质之间铁运输的代谢物陷阱；我们在 Sc Fet3、Ftr1p 复合体中使用了这种经典的通道测试。我们认为 Cr 中 Fox1 和 Ftr1 之间的 Fe 转运提供了 hCp 和转铁蛋白之间推定的 FeIII 转运的现实模型。在目标 III 中，我们将检验我们的假设，即两种人类真菌病原体白色念珠菌和新生隐球菌表达等效的 PM Fet、Ftr 高亲和力铁吸收复合物。我们将通过这些复合物在 Sc 中原位和重组形式量化 59Fe 吸收动力学。针对 Ca 和 Cn 蛋白中的特定亚铁氧化酶和 Fe 运输残基，我们将检验我们的假设，即这些突变体表现出 Sc 同源物表现出的通道缺陷。我们认为表达这些通道突变体的菌株在体外和体内表现出毒力降低。最后，使用这些螯合剂敏感克隆，NCI 多样性集合将挖掘有潜力作为这些真菌病原体中 Fet、Ftr Fe 吸收抑制剂的化合物。公共健康相关性：从真菌到人类的所有耗氧生物体都需要铜氧化酶（一种多铜氧化酶）的活性来管理其必需营养素铁的代谢。从面包酵母到人类病原体白色念珠菌和新型隐球菌的真菌都使用这些酶来获取它们生长和生存所需的铁。这项研究的目的是了解这些酶的工作原理，然后利用这些知识来阻止它们的铁运输，从而抑制健康和免疫功能低下患者中病原真菌的毒力。