Cuproproteins for Redox Biology

所有好氧生物不断产生活性氧物种(ROS)，如超氧阴离子和 作为新陈代谢的天然产物的过氧化氢。ROS对生物分子有潜在的破坏作用，但也可能 被细胞用作攻击病原体的武器和发出信号的分子。为了平衡 ROS的有益和潜在有害影响，好氧生物配备了一套抗氧化剂 在真核生物中，产生超氧化物的唯一酶是超氧化物歧化酶(SOD)。Sod使用一种 金属辅因子，如铜，以非常快的速度催化，将超氧化物分解为O2和 过氧化氢。直到最近，双金属铜/锌超氧化物歧化酶被认为是真核生物中唯一的铜超氧化物歧化酶，但在 2014年，我们的实验室发现了一类新的超氧化物歧化酶，它不能结合锌，并且缺乏覆盖活性部位的序列， 因此，只含铜的超氧化物歧化酶与一种非常不寻常的溶剂暴露了铜的辅助因子。仅限CU的SoD分布广泛 在整个真菌王国，作为真菌唯一的胞外超氧化物歧化酶。在动物中，仅含铜的超氧化物歧化酶基因 经过两次重复，在我们称为单一多肽的单一多肽上产生了4倍的仅含铜的超氧化物歧化酶串联重复 CSRP(纯铜超氧化物歧化酶重复蛋白)。仅含铜的超氧化物歧化酶家族区别于其他超氧化物歧化酶的是他们的 受限的本地化和异常开放的活动站点。所有只含铜的超氧化物歧化酶和CSRP分子预计都会 值得注意的是，我们发现仅含铜的超氧化物歧化酶并不是从细胞内获得铜辅助因子 金属池，不同于其他真核生物分泌的CuProtein。相反，仅含铜的SoD在外部激活 细胞的胞外铜。我们假设这个新的CuProtein家族进化为具有独有功能 胞外氧化还原生物学中的细胞外。这里我们结合了生物物理学、结构生物学和细胞生物学。 研究只含铜的SOD的铜位如何微调以捕获铜而不是其他金属的方法 细胞外，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，最主要的来源 细胞外超氧化物是NADPH氧化酶(NOX)，通常由Rho GTP酶激活以产生ROS 用来发信号。最近，我们在一种单细胞真菌中发现了一种令人惊讶的只有NOX-铜的超氧化物歧化酶伙伴关系 病原体代表了Rho GTP酶控制ROS的一种非常简单和独特的形式。我们会 阐明ROS信号转导系统的潜在机制，并确定致病机理 酵母菌可以使用仅含铜的SoD和ROS来传递极化生长的信号。CSRP可能同样在通路中发挥作用 涉及NOX和ROS信号。使用脊椎动物模型，我们发现CSRP在 具有高再生能力的组织，通过ROS信号传递。我们的目标是发现 CSRP的生化活性及其在高血压病组织中与NOX的可能关系 再生能力。值得注意的是，大自然设计了两种截然不同的含铜超氧化物歧化酶： 铜/锌与只含铜的对照。我们的研究承诺揭示只含铜的超氧化物歧化酶是如何专门化的 在氧化还原和/或金属生物学的细胞外操作。

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.