Candida albicans SOD5: a novel copper-only superoxide dismutase

白色念珠菌 SOD5：一种新型纯铜超氧化物歧化酶

• 批准号: 8782888
• 负责人: Ryan Loren Peterson
• 金额: $ 5.2万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782888
• 关键词: Active Sites; Address; Alleles; Animals; Antioxidants; Binding; Biochemical; Biochemistry; Biocide; Buffers; Candida albicans; Candidiasis; Catalysis; Cell Line; Cell Wall; Cellular biology; Charge; Coculture Techniques; Copper; Cuprozinc Superoxide Dismutase; Development; Electrostatics; Enzyme Kinetics; Enzymes; Eukaryota; Family; Growth; Human; Hydrogen Bonding; Infection; Ions; Kinetics; Laboratories; Lead; Ligands; Measurement; Mediating; Metals; Modeling; Mutate; Natural Immunity; Oxidative Stress; Pathogenesis; Pichia; Play; Positioning Attribute; Production; Property; Proteins; Pulse Radiolysis; Recombinants; Reporting; Resistance; Role; Signal Transduction; Site; Source; Superoxide Dismutase; System; Testing; Toxic effect; Virulence; Work; Yeasts; Zinc; cofactor; copper histidine; enzyme activity; extracellular; insight; killings; macrophage; mutant; novel; pathogen; prototype; public health relevance; research study; secretory protein; three dimensional structure

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.

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（SOD1）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型 SOD1 样分子，无需锌离子即可发挥作用。该家族的原型是白色念珠菌 SOD5，这是一种细胞外纯铜 SOD，对于真菌病原体的毒力至关重要。与 SOD1 不同，SOD5 没有锌位点，并且由于不存在静电环 VII 而包含异常开放的铜位点。当白色念珠菌分泌时，SOD5 可以快速从细胞外金属库中获取其铜辅因子。 SOD5 与经典 SOD1 相比所表现出的金属辅因子的这些独特特征可能代表了感染过程中对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 的新型金属生物学，我们将结合使用生化、光谱和细胞生物学方法来探索 SOD5 在没有锌的情况下发挥作用的机制以及该酶在感染过程中如何带上铜。目标 1：确定 SOD5 在没有锌金属离子辅因子的情况下如何发挥作用：对 Cu-SOD5 三维结构的分析揭示了铜位点的氢键网络，该氢键网络可能会替代该酶中的锌。该网络涉及保守残基 E110 和 D113，初步研究已证明这两个残基对于最大 SOD5 活性非常重要。使用毕赤酵母分泌蛋白表达系统，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，并使用脉冲辐射分解获得催化动力学测量。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. 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“铜爆发”——一种通过铜毒性杀死病原体的防御策略。由于 SOD5 可以快速地用细胞外铜充电，因此它可以利用铜的爆发来给自身充电以进行抗氧化防御。通过结合过量的铜，SOD5 还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，我们将测试细胞外 ​​SOD5 是否有能力保护白色念珠菌免受酵母培养物和巨噬细胞感染系统中的铜毒性。通过创建缺铜的巨噬细胞，我们将测试白色念珠菌分泌的 SOD5 是否带有来自巨噬细胞的铜，以及巨噬细胞铜池是否对于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 SOD 的念珠菌病新疗法的开发。