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: 8055270
• 负责人: DANIEL J. KOSMAN
• 金额: $ 31万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055270
• 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; Health; 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; Plants; 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; Thermodynamics; 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; 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）：多型氧化酶（MCOS）将二氧化合物的4E降低与2H2O的降低，并氧化为1-电子供体的4次等效物。这些无处不在的酶的亚家族对FEII具有特异性，这使其对于各自生物体中的铁稳态至关重要。我们的假设是，这些铁氧化酶通过将FEIII产物引导到铁代谢途径中的下游伴侣来发挥作用。该程序描述了酿酒酵母（SC）质膜（PM）中FET3P FTR1P高亲和力Fe摄取复合物中的结构和功能。这项工作中提出的两个基本问题是：1）在MCO上赋予FEII作为基板的MCO的结构图案； 2）FTR1P的FET3P铁氧化酶反应如何在运动和物理上耦合到FEIII的膜渗透。在此应用程序中，我们提出了三个具体目标。在目的中，我们将检验我们的假设，即哪些结构基序是定义的铁氧化酶，具体是，一系列羧酸盐侧链链最大化了三个因素，这些因素从FEII：FEII结合，FEII氧化还原电位和FEII和1型CUII型（T1CU）的E-FEII结合，FEII氧化还原电位和电子基质耦合。我们将根据我们的设计原理将漆酶和铁氧化酶酶互换来做到这一点。此外，我们将测试我们的假设，即另一个羧酸盐侧链稳定在二恶英上的负电荷增加，因为在三核簇（TNC）的2、2e-台阶减少时，它会稳定下来。最后，我们将测试我们的假设，即与TNC触发在T1 Cu的o2降低相关的协调变化通过与两者连接的规范MCO His-cys Tobif相关。在AIM II中，我们提出了我们的假设，即Chlamydomonas Reinhardtii（CR）中的FOX1是人类ceruloplasmin样的铁氧化酶。我们将表征FOX1蛋白，表明它具有支持FEII氧化和FEIII的FEX1中FOX1，FTR1复合物中的FEII氧化和FEIII运输的基序。我们将构建FOX1和FTR1的突变体，我们预计将对Feiii-Chelator敏感，该志偶会在Fe-uptake中充当Fe-Fe-traupticking的代谢物陷阱。我们已经在SC FET3（FTR1P复合物）中使用了这种频道的经典测试。我们建议，CR中FOX1和FTR1之间的Fe贩运者为HCP和转铁蛋白之间的推定Feiii贩运提供了现实的模型。在AIM 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孢子虫都表达了同等的PM FET，FTR高亲和力的Fe-Euttake复合物。我们将通过原位和SC中的重组形式来量化59fe-uteake动力学。我们将在CA和CN蛋白中靶向特定的铁氧化酶和Fe贩运残基，我们将测试我们的假设，即这些突变体表现出SC同源物表现出的通道缺陷。我们建议表达这些通道突变体的菌株在体外和体内表现出降低的毒力。最后，使用这些螯合剂敏感的克隆，将开采NCI多样性的收集，用于具有FET抑制剂，FTR FE摄取的潜力，在这些真菌病原体中。公共卫生相关性：从真菌到人类的所有耗氧生物都​​需要一种铜氧化酶的活性（一种多型氧化酶）来管理其基本营养剂铁的代谢。从贝克的酵母到人类病原体的真菌C. bichans和Neoformans使用这些酶来获取其成长和生存所需的铁。这项研究的目的是拍摄这些酶的工作原理，然后利用这些知识来阻止其对铁的贩运，以抑制在其他健康和免疫受损的患者中抑制致病真菌的毒力。