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

描述(由申请人提供)：多铜氧化酶(MCOs)将氧的4e-还原为2H2O与1电子供体的4个等价物的氧化结合在一起。这些普遍存在的酶的一个亚家族对FeII具有特异性，这使得它们对各自生物体中的铁稳态是必不可少的。我们的假设是，这些铁氧合酶的功能是通过将它们的FeIII产物引导到其铁代谢途径的下游伙伴。本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体的结构和功能。这项工作中涉及的两个基本问题是：1)什么结构基序赋予MCO以FeII为底物的这种专一性；2)Fet3p铁氧基酶反应是如何从动力学和物理上耦合到Ftr1p对FeIII的膜渗透的。在本申请中，我们提出了三个具体目标。在目标I中，我们将检验我们的假设，即什么结构基序定义一个铁氧基酶，特别是一个队列的羧酸侧链最大化三个因素，决定从FeII的e转移：FeII结合，FeII氧化还原电位和电子基质耦合的FeII和类型1 CUII(T1Cu)在铁氧化物酶。我们将根据我们的设计原则，通过相互转化漆酶和亚铁酸酶来实现这一点。此外，我们将检验我们的假设，即另一组羧酸侧链稳定了氧上不断增加的负电荷，因为它在三核簇合物(TNC)上分2，2e-步还原。最后，我们将检验我们的假设，即与TNC上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T1Cu的电子转移。在目的II中，我们建议检验我们的假设，即莱茵衣藻中的Fox1是一种人铜蓝蛋白样亚铁氧化酶。我们将对Fox1蛋白进行表征，证明它具有铁氧合酶特异性的基序，支持FeII氧化和FeIII在CrPM中的Fox1，Ftr1复合体中的运输我们将构建Fox1和Ftr1的突变体，我们预测这些突变体将对FeIII-螯合剂作为代谢物陷阱在两种蛋白质之间的铁吸收运输中起到敏感作用；我们已经在SC Fet3，Ftr1p复合体中使用了这一经典的通道测试。我们认为，Fox1和Ftr1之间的铁在铬中的转运为推测HCP和转铁蛋白之间的FeIII转运提供了一个现实的模型。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相同的PM Fet，FTR高亲和力铁摄取复合体。我们将通过这些络合物在原位和在SC中以重组形式定量测定59Fe的吸收动力学。针对Ca和CN蛋白中特定的铁氧合酶和铁转运残基，我们将检验我们的假设，即这些突变体表现出SC同源物所表现出的通道缺陷。我们认为，在体外和体内，表达这些通道突变的菌株将显示出较低的毒力。最后，使用这些对螯合剂敏感的克隆，将挖掘NCI多样性集合，以寻找在这些真菌病原体中具有抑制Fet、FTR铁吸收的潜在化合物。与公共健康相关：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，以管理其基本营养物质铁的新陈代谢。从面包酵母到人类病原体白念珠菌和新生葡萄球菌等真菌都使用这些酶来获得它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是如何工作的快照，然后利用这一知识来阻止它们对铁的运输，以此来抑制致病真菌在其他方面健康和免疫受损患者中的毒力。

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.