Candida albicans SOD5: a novel copper-only superoxide dismutase

描述(申请人提供)：在真核生物中，含铜和锌的超氧化物歧化酶家族(SOD1)被认为参与抗氧化防御和细胞信号转导。最近，库洛塔实验室在真核生物中发现了一类新的类似SOD1的分子，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SOD5，一种细胞外仅含铜的超氧化物歧化酶，对这种真菌病原体的毒力是必不可少的。与SOD1不同的是，SOD5没有锌中心，由于没有静电环VII，它含有一个异常开放的铜中心。当从白色念珠菌分泌时，SOD5可以迅速从细胞外的金属池中获得铜辅助因子。SOD5与典型的SOD1在金属辅助因子中表现出的这些独特特征可能代表了对感染期间宿主介导的铜和锌变化的适应。为了开始了解白色念珠菌SOD5的新的金属生物学，我们将结合生化、光谱和细胞生物学的方法来探索SOD5在没有锌的情况下工作的机制，以及在感染过程中酶是如何带电的。目的1：在没有锌金属离子辅助因子的情况下，确定SOD5的功能：对铜-SOD5的三维结构的分析揭示了铜位上的氢键网络，该网络可能在该酶中替代锌。这个网络包括保守残基E110和D113，初步研究表明它们对SOD5的最大活性是重要的。利用巴斯德毕赤酵母分泌蛋白的酵母表达系统，我们将表达和纯化大量的胞外SOD5 E110和D113突变体。我们将表征它们各自的金属结合能力，并使用脉冲辐解获得催化的动力学测量。这些研究将揭示E110和D113在铜-SOD5催化中的作用是否类似于锌在SOD1中的作用。目的2.了解宿主铜在激活SOD5进行病原体防御中的作用：白色念珠菌依赖其动物宿主获取铜，一个有趣的来源是巨噬细胞的铜爆裂--一种通过铜中毒杀死病原体的防御策略。由于SOD5迅速充满细胞外的铜，它可能会利用铜爆发的机会为自己充电，以进行抗氧化防御。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中毒。为了解决这个问题，我们将测试细胞外SOD5是否有能力在酵母培养和巨噬细胞感染系统中保护白色念珠菌免受铜中毒。通过创造铜缺乏的巨噬细胞，我们将测试白色念珠菌分泌的SOD5是否充满了巨噬细胞中的铜，以及这一巨噬细胞铜池是否对感染期间的病原体杀灭至关重要。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，并最终可能导致针对白色念珠菌的新型纯铜SOD的白念珠菌病的新疗法的开发。

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.