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: 7825303
• 负责人: DANIEL J. KOSMAN
• 金额: $ 31.17万
• 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/7825303
• 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）将分子氧的4 e-还原为2 H2O与4当量的1-电子供体的氧化偶联。这些普遍存在的酶的一个亚家族对Fe II具有特异性，这使得它们对各自生物体中的铁稳态至关重要。我们的假设是，这些铁氧化酶的功能，引导他们的Fe III产品的下游合作伙伴在其铁代谢途径。该程序描述了酿酒酵母（Saccharomycescerevisiae，Sc）质膜（PM）中Fet 3 p，Ftr 1 p高亲和铁吸收复合物的结构和功能。在这项工作中解决的两个基本问题是：1）什么样的结构基序赋予MCO这种特异性FeII作为底物;和2）如何是Fet 3 p铁氧化酶反应耦合动力学和物理膜渗透FeIII Ftr 1 p。在本申请中，我们提出了三个具体目标。在目的I中，我们将测试我们的假设，即什么样的结构基序定义了铁氧化酶，具体地说，一组羧酸酯侧链最大化决定从Fe II的电子转移的三个因素：Fe II结合，Fe II氧化还原电位和Fe II与铁氧化酶中的1型Cu II（T1 Cu）的电子矩阵耦合。我们将根据我们的设计原则，通过相互转化漆酶和铁氧化酶来实现这一点。此外，我们将测试我们的假设，即另一组羧酸酯侧链稳定了分子氧上不断增加的负电荷，因为它在三核簇（TNC）中以2，2 e-步骤被还原。最后，我们将测试我们的假设，即与TNC处的O2还原相关的配位变化通过连接两者的典型MCO His-Cys-His基序触发从T1 Cu的电子转移。在目的II中，我们建议测试我们的假设，即Fox 1在莱茵衣藻（铬）是一个人类铜蓝蛋白样铁氧化酶。我们将表征Fox 1蛋白，证明它具有铁氧化酶特异性基序，支持Fe II氧化和Fe III贩运Fox 1，Ftr 1复合物中的铬PM。我们将构建Fox 1和Ftr 1的突变体，我们预测它们将对FeIII螯合剂敏感，FeIII螯合剂在Fe摄取中充当两种蛋白质之间Fe运输的代谢物陷阱;我们已经使用了Sc Fet 3，Ftr 1 p复合物中通道的经典测试。我们建议，铁之间的Fox 1和Ftr 1在铬贩运提供了一个现实的模型，推定的铁III之间的运输hCp和转铁蛋白。在目的III中，我们将测试我们的假设，即两种人类真菌病原体，白色念珠菌和新型隐球菌表达一个等效的PM Fet，Ftr高亲和力铁摄取复合物。我们将通过这些复合物在原位和重组的形式在Sc.靶向特定的铁氧化酶和Fe-贩运残基的Ca和Cn蛋白定量的59 Fe-摄取动力学，我们将测试我们的假设，这些突变体表现出通道缺陷所表现出的Sc同系物。我们建议，表达这些通道突变体的菌株将表现出降低的毒力在体外和体内。最后，使用这些螯合剂敏感的克隆，NCI多样性收集将被挖掘的化合物与潜在的抑制剂Fet，Ftr铁吸收在这些真菌病原体。公共卫生相关性：从真菌到人类，所有利用氧气的生物都需要铜氧化酶（一种多铜氧化酶）的活性来管理其必需营养素铁的代谢。从面包酵母菌到人类病原体C.白色念珠菌和C.新生儿利用这些酶来获得他们茁壮成长和生存所需的铁。这项研究的目标是拍摄这些酶如何工作的快照，然后利用这些知识来阻止它们运输铁，以抑制致病真菌在其他健康和免疫功能低下患者中的毒力。