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; 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，已知含有超氧化物歧化酶（SOD1）的铜和锌家族参与抗氧化剂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SOD1样分子，这些分子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，这是一种仅细胞外铜的草皮，对于真菌病原体的毒力至关重要。与SOD1不同，SOD5没有锌位点，并且由于缺乏静电环VII而包含一个异常开放的铜位点。当从白色念珠菌分泌时，SOD5可以从金属的细胞外池中迅速获取其铜的副因素。这些独特的特征由SOD5与SOD5显示的金属辅助因素相比，规范SOD1可能代表了感染过程中宿主介导的铜和锌变化的适应。为了开始理解白色念珠菌SOD5的新型级化生物学，我们将使用生化，光谱和细胞生物学方法的组合来探索SOD5在没有锌的情况下运行的机制以及在感染过程中如何用铜收取酶的酶。目的1：确定SOD5在没有锌金属离子辅因子的情况下的功能：对Cu-Sod5的三维结构的分析已揭示了向铜位的氢键网络，该网络可能代替该酶中的锌。该网络涉及保守的残基E110和D113，在初步研究中已被证明对最大SOD5活性很重要。我们将使用用于分泌蛋白的Pichia Pastoris酵母表达系统，我们将表达和纯化大量细胞外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进行催化的动力学测量。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。目的2。要了解宿主铜在SOD5激活病原体防御中的作用：白色念珠菌依靠其动物宿主获取铜和一个有趣的来源是巨噬细胞的“铜爆发”，这是一种通过铜毒性杀死病原体的防御策略。由于SOD5迅速充满了细胞外铜，因此它可能利用铜爆发来充电以进行抗氧化防御。通过结合多余的铜，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是否具有保护白色念珠菌免受酵母培养物和巨噬细胞感染系统中铜毒性的能力。通过创建铜缺乏巨噬细胞，我们将测试从白色念珠菌分泌的SOD5是否被控巨噬细胞中的铜，以及这种巨噬细胞铜池是否对于感染过程中的病原体杀死很重要。总之，这些研究将增加我们对白色念珠菌SOD5在生化和细胞水平上的基本理解，并最终可能导致针对白色念珠菌仅铜的新型念珠菌病的新疗法发展。