Cuproproteins for Redox Biology

用于氧化还原生物学的铜蛋白

• 批准号: 10569650
• 负责人: Valeria C Culotta
• 金额: $ 41.43万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569650
• 关键词: Active Sites; Aerobic; Animals; Antioxidants; Binding; Biochemical; Biology; Biophysics; Candida albicans; Cells; Cellular biology; Copper; Cu-Superoxide Dismutase; Cuprozinc Superoxide Dismutase; Enzymes; Equilibrium; Eukaryota; Family; Genes; Goals; Growth; Human; Hydrogen Peroxide; Metabolism; Metals; Modeling; NADPH Oxidase; Natural Products; Nature; Organism; Oxidation-Reduction; Pathogenesis; Pathogenicity; Pathway interactions; Proteins; Reactive Oxygen Species; Regenerative capacity; Signal Transduction; Signaling Molecule; Site; Solvents; Source; Superoxide Dismutase; Superoxides; System; Tandem Repeat Sequences; Tissues; Variant; Yeasts; antioxidant enzyme; cofactor; design; extracellular; fungus; metallicity; pathogen; pathogenic fungus; polypeptide; rho GTP-Binding Proteins; structural biology; weapons

All aerobic organisms continuously generate reactive oxygen species (ROS) such as superoxide anion and H2O2 as natural products of metabolism. ROS are potentially damaging to biomolecules, but can also be exploited by cells as weapons for attacking pathogens and as molecules for signaling. To balance the beneficial and potentially harmful effects of ROS, aerobic organisms are armed with a suite of anti-oxidant enzymes, and in eukaryotes, the only enzyme for superoxide is the superoxide dismutase (SOD). SODs use a metal co-factor such as Cu to catalyze at extraordinary rates, the disproportination of superoxide to O2 and H2O2. Until recently, the bimetallic Cu/Zn SOD was believed to be the only Cu SOD for eukaryotes, but in 2014, our lab discovered a new class of SODs that cannot bind Zn and lack sequences to cover the active site, hence, a Cu-only SOD with a highly unusual solvent exposed Cu co-factor. Cu-only SODs are wide-spread throughout the fungal kingdom as the sole extracellular SOD for fungi. In animals, the Cu-only SOD gene underwent twice duplication, resulting in 4x tandem repeats of Cu-only SODs on a single polypeptide we call CSRP (Cu-only SOD repeat protein). What sets the Cu-only SOD family apart from other SODs is their restricted localization and unusual open active site. All Cu-only SODs and CSRP molecules are predicted to be extracellular and remarkably, we find that Cu-only SODs do not acquire their Cu-cofactor from intracellular metal pools, unlike other eukaryotic secreted cuproproteins. Instead, Cu-only SODs are activated outside the cell by extracellular Cu. We hypothesize this new family of cuproproteins evolved to function exclusively outside the cell in extracellular redox biology. Here we combine biophysical, structural biology, and cell biology approaches to examine how the Cu site of Cu-only SODs is fine-tuned to capture Cu and not other metals outside the cell, and how the enzyme functions with extracellular superoxide. In eukaryotes, the primary source of extracellular superoxide is the NADPH oxidase (NOX), typically activated by Rho GTPases to produce ROS for signaling. Recently, we uncovered a surprising NOX - Cu-only SOD partnership in a unicellular fungal pathogen that represents a remarkably simple and unique form of Rho GTPase control of ROS. We will elucidate the mechanism underlying this redumentary system for ROS signaling and define how pathogenic yeasts can use Cu-only SODs and ROS to signal polarized growth. CSRP may likewise function in pathways involving NOX and ROS signaling. Using a vertebrate model, we find CSRP most abundantly expressed in tissues with a high capacity for regeneration by signaling through ROS. Our goals are to uncover the biochemical activities of CSRP and elucidate its function in possible relationship to NOX in tissues with high regenerative capacity. It is remarkable that nature has designed two distinct variations of Cu-containing SODs: the Cu/Zn versus the Cu-only. Our studies promise to uncover how the Cu-only SODs are specialized to operate outside the cell in redox and/or metallobiology.

所有需氧生物体持续产生活性氧物质（ROS），例如超氧阴离子和超氧阴离子。 H2 O2是自然代谢产物。ROS对生物分子具有潜在的破坏性，但也可能是 被细胞用作攻击病原体的武器和信号分子。平衡 有益的和潜在的有害影响的活性氧，有氧生物武装与一套抗氧化剂 在真核生物中，超氧化物的唯一酶是超氧化物歧化酶（SOD）。SODs使用 金属辅因子如Cu以非常高的速率催化，超氧化物歧化为O2， 过氧化氢直到最近，Cu/Zn SOD被认为是真核生物中唯一的Cu SOD，但在 2014年，我们的实验室发现了一类新的SOD，它们不能结合Zn，并且缺乏覆盖活性位点的序列， 因此，具有高度不寻常溶剂的仅含Cu的SOD暴露Cu辅因子。仅含Cu的SOD广泛分布 作为真菌唯一的胞外SOD。在动物中，只含铜的SOD基因 进行了两次重复，导致在一个单一的多肽上只有Cu的SOD的4x串联重复，我们称之为 CSRP（Cu-only SOD repeat protein）。使仅含Cu的SOD家族与其他SOD区别开来的是它们的 局限的定位和不寻常的开放活性位点。所有仅含Cu的SOD和CSRP分子都被预测为 值得注意的是，我们发现仅含铜的SOD不从细胞内获得其铜辅因子， 金属池，不像其他真核分泌铜蛋白。相反，仅Cu的SOD在细胞外被激活。 细胞外Cu。我们假设这个新的铜蛋白家族进化成专门功能 在细胞外氧化还原生物学中。在这里，我们结合联合收割机生物物理学，结构生物学，和细胞生物学 研究仅含Cu的SOD的Cu位点如何被微调以捕获Cu而不是其他金属的方法 以及酶如何与细胞外超氧化物一起发挥作用。在真核生物中， 细胞外超氧化物的主要来源是NADPH氧化酶（NOX），它通常被Rho GTP酶激活产生ROS 用于信号。最近，我们在一种单细胞真菌中发现了一种令人惊讶的NOX-Cu-only SOD伙伴关系， 这种病原体代表了一种非常简单和独特的Rho GT3控制ROS的形式。我们将 阐明这种ROS信号传导冗余系统的机制并定义致病性如何 酵母可以使用仅含Cu的SOD和ROS来发出极化生长的信号。CSRP可能同样在以下途径中起作用： 涉及NOX和ROS信号传导。使用脊椎动物模型，我们发现CSRP在哺乳动物中表达最丰富。 组织具有通过ROS信号传导的高再生能力。我们的目标是揭露 CSRP的生物化学活性，并阐明其功能可能与组织中的NOX高 再生能力值得注意的是，自然界设计了两种不同的含Cu SOD变体： Cu/Zn对比仅Cu。我们的研究有望揭示只有Cu的SOD是如何专门用于 在氧化还原和/或金属生物学中在细胞外操作。