Candida albicans SOD5: a novel copper-only superoxide dismutase

描述（由申请人提供）：在真核生物中，已知含铜和锌的超氧化物歧化酶（SOD 1）家族参与抗氧化防御和细胞信号传导。最近，Culotta实验室在真核生物中发现了一类新的SOD 1样分子，它们在没有锌离子的情况下发挥作用。这个家族的原型是白色念珠菌SOD 5，一种细胞外仅含铜的SOD，对真菌病原体的毒力至关重要。与SOD 1不同，SOD 5没有锌位点，并且由于不存在静电环VII而含有异常开放的铜位点。C.在白色念珠菌中，SOD 5可以快速地从金属的细胞外池获得其铜辅因子。SOD 5与经典SOD 1所显示的金属辅因子的这些独特特征可能代表了感染期间对宿主介导的铜和锌变化的适应。开始了解C.因此，我们将使用生物化学，光谱和细胞生物学方法的组合来探索SOD 5在没有锌的情况下运作的机制以及该酶在感染过程中如何与铜结合。目标1：为了确定SOD 5在没有锌金属离子辅因子的情况下如何发挥作用：对Cu-SOD 5的三维结构的分析揭示了与铜位点的氢键网络，该氢键网络可以取代该酶中的锌。该网络涉及保守的残基E110和D113，在初步研究中已被证明是重要的最大SOD 5活性。利用毕赤酵母分泌蛋白表达系统，我们将大量表达和纯化胞外SOD 5 E110和D113突变体。我们将表征其各自的金属结合能力，并获得动力学测量的催化脉冲辐解。这些研究将揭示E110和D113在Cu-SOD 5催化中的作用是否类似于锌在SOD 1中的作用。目标二。为了了解宿主铜在激活SOD 5以防御病原菌中的作用：C.白念珠菌依靠其动物宿主获得铜，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SOD 5被细胞外的铜迅速充电，它可以利用铜的爆发来为自己充电以进行抗氧化防御。通过结合多余的铜，SOD 5也可能有助于保护C。宿主介导的铜毒性引起的白色念珠菌。为了解决这个问题，我们将测试胞外SOD 5是否具有保护C的能力。在酵母培养物和巨噬细胞感染系统中，白色念珠菌与铜毒性无关。通过建立缺铜巨噬细胞，我们将测试SOD 5是否从C.白念珠菌是否携带来自巨噬细胞的铜，以及巨噬细胞铜池是否对感染期间杀死病原体很重要。总之，这些研究将增加我们在生物化学和细胞水平上对白色念珠菌SOD 5的基本理解，并可能最终导致针对新型仅含铜SOD的念珠菌病的新疗法的开发。白色念珠菌。

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.