Candida albicans SOD5: a novel copper-only superoxide dismutase

描述（由申请人提供）：在真核生物中，已知含有铜和锌的超氧化物歧化酶（SOD1）家族参与抗氧化防御和细胞信号传导。最近，库洛塔实验室在真核生物中发现了一类新的sod1样分子，它们在没有锌离子的情况下起作用。该家族的原型是白色念珠菌SOD5，这是一种细胞外纯铜SOD，对真菌病原体的毒力至关重要。与SOD1不同的是，SOD5没有锌位点，由于缺乏静电环路VII， SOD5含有一个异常开放的铜位点。当从白色念珠菌分泌时，SOD5可以从细胞外的金属池中迅速获得铜辅助因子。SOD5与典型SOD1在金属辅助因子中表现出的这些独特特征可能代表了感染期间宿主介导的铜和锌变化的适应性。为了开始了解白色念珠菌SOD5的新型金属生物学，我们将采用生物化学、光谱和细胞生物学相结合的方法来探索SOD5在没有锌的情况下运作的机制，以及在感染过程中该酶是如何被铜带电的。目的1：为了确定SOD5在没有锌金属离子辅助因子的情况下是如何起作用的：对Cu-SOD5的三维结构的分析揭示了铜位点的氢键网络，可以替代该酶中的锌。该网络涉及保守残基E110和D113，在初步研究中已被证明对最大SOD5活性很重要。利用毕赤酵母分泌蛋白表达系统，我们将表达和纯化大量的细胞外SOD5 E110和D113突变体。我们将描述它们各自的金属结合能力，并使用脉冲辐射分解获得催化的动力学测量。这些研究将揭示E110和D113在Cu-SOD5催化中的作用是否类似于锌在SOD1中的作用。目标2。为了了解宿主铜在SOD5激活中对病原体防御的作用：白色念珠菌依赖其动物宿主获取铜，其中一个有趣的来源是巨噬细胞的“铜爆发”——一种通过铜毒性杀死病原体的防御策略。由于SOD5被细胞外的铜迅速充电，它可以利用铜的爆发给自己充电来进行抗氧化防御。通过结合过量的铜，SOD5也可能有助于保护白色念珠菌免受宿主介导的铜毒性。为了解决这个问题，我们将在酵母培养物和巨噬细胞感染系统中测试细胞外SOD5是否具有保护白色念珠菌免受铜毒性的能力。通过创建缺铜巨噬细胞，我们将测试白色念珠菌分泌的SOD5是否携带巨噬细胞中的铜，以及巨噬细胞中的铜池是否对感染期间的病原体杀死很重要。总之，这些研究将增加我们在生化和细胞水平上对白色念珠菌SOD5的基本了解，并可能最终导致针对白色念珠菌新型纯铜SOD5的新疗法的发展。

DESCRIPTION (provided by applicant): In eukaryotes, the family of copper and zinc containing superoxide dismutases (SOD1) are known to participate in anti-oxidant defense and cell signaling. Very recently, the Culotta laboratory has uncovered a new class of SOD1-like molecules in eukaryotes that function without a zinc ion. The prototype of this family is Candida albicans SOD5, an extracellular copper-only SOD that is essential for virulence of the fungal pathogen. Unlike SOD1, SOD5 has no zinc site and contains an unusually open copper site due to absence of an electrostatic loop VII. When secreted from C. albicans, SOD5 can rapidly acquire its copper co-factor from extracellular pools of the metal. These unique features in metal co-factors displayed by SOD5 vs the canonical SOD1 may represent adaptations to host-mediated changes in copper and zinc during infection. To begin to understand the novel metallobiology of C. albicans SOD5, we shall use a combination of biochemical, spectroscopic and cell biology approaches to explore mechanisms by which SOD5 operates without zinc and how the enzyme is charged with copper during infection. Aim 1: To determine how SOD5 functions without a zinc metal ion cofactor: Analysis of the three dimensional structure of Cu-SOD5 has revealed a hydrogen bond network to the copper site that may substitute for zinc in this enzyme. This network involves conserved residues E110 and D113 that in preliminary studies have been shown to be important for maximal SOD5 activity. Using a Pichia pastoris yeast expression system for secretory proteins, we will express and purify large quantities of extracellular SOD5 E110 and D113 mutants. We will characterize their respective metal binding capabilities and obtain kinetic measurements of catalysis using pulse radiolysis. These studies will reveal whether the role of E110 and D113 in Cu-SOD5 catalysis is analogous to the role of zinc in SOD1. Aim 2. To understand the role of host copper in the activation of SOD5 for pathogen defense: C. albicans relies on its animal host for acquiring copper and one intriguing source is the "copper burst" of macrophages - a defense strategy to kill pathogens through copper toxicity. Since SOD5 is rapidly charged with extracellular copper, it may take advantage of the copper burst to charge itself for anti-oxidant defense. By binding excess copper, SOD5 might also help protect C. albicans from host-mediated copper toxicity. To address this, we will test whether extracellular SOD5 has the capacity to protect C. albicans from copper toxicity in yeast cultures and in macrophage infection systems. By creating copper deficient macrophages, we will test whether SOD5 secreted from C. albicans is charged with copper from the macrophage, and whether this pool of macrophage copper is important for pathogen killing during infection. Together, these studies will increase our basic understanding of Candida albicans SOD5 at both the biochemical and cellular levels and may ultimately lead to the development of new therapies for candidiasis directed at the novel copper-only SODs of C. albicans.