Mechanistic Studies of Alkyl Hydroperoxide Reductase and Related Redox Proteins

说明(申请人提供)：过氧化氢是一种被人类免疫系统用来杀死感染性生物的毒素，越来越多的证据表明，它也是真核信号中常见的第二信使。在人类中，细胞因子、生长因子和先天免疫系统的Toll样受体被认为是通过过氧化氢传递信号的。长期以来，过氧化氢酶和谷胱甘肽过氧化物酶一直被认为是细胞中降解过氧化氢的主要酶，然而在过去的几年里，一个独特的、高度丰富的过氧化氢还原酶家族--过氧化还蛋白(Prxs)已经从相对鲜为人知的水平成为氧化还原生物学研究的主要焦点。多年来，真核生物Prxs的过氧化物酶活性一直被忽视，因为那些在真核生物中高表达的Prxs很容易被过氧化氢灭活。我们在超过15年的病原菌(例如鼠伤寒沙门氏菌AhpC)中鉴定Prx的PRX酶学方面积累了专业知识。这些Prx是抗生素开发的目标，因为它们在保护细菌免受人类免疫系统的攻击方面起到了作用。2003年，我们对鼠伤寒沙门氏菌AhpC的结构和功能研究导致我们发现了对过氧化的敏感性的结构基础，这种结构基础对于在所有真核生物中高度表达的PrX的子集是保守的(这是支持本论文的结构假说的基础，该假说规定，在活性位点附近堆积的近端二级结构的移动性是PrXs对过氧化的敏感性和PrXs作为抗氧化剂的能力的关键决定因素)。我们进一步提出了“闸门假说”，即这种对失活的敏感性如何在过氧化氢被用作信号分子的生物体中实际上是有益的，以便在适当的条件下可以关闭Prxs的抗氧化特性，以允许过氧化氢水平的可控爆发。鉴于Prxs在病原体防御和人类细胞中对抗氧化应激和细胞调节的重要性，我们建议在这里通过研究几个精心选择的蛋白质和突变体的构象“迁移率”来确定对过度氧化的敏感性和有效抗氧化功能的关键决定因素；还将研究这些蛋白质的催化循环和失活途径中的相关速率常数(目标1)。在目标2中，我们将确定Prxs高度保守的活性部位周围的结构特征，这些结构特征对于不同的过氧化氢底物的结合和还原非常重要。在目标3中，我们将通过基于细胞的PRX功能研究，确定Prx对周转过程中过氧化氢失活的敏感性(“闸门”)是否对调节细胞信号事件期间产生的过氧化氢水平至关重要。 与公共健康相关：氧化损伤被认为在衰老、癌症的发展和许多退行性疾病中都是重要的。此外，控制增殖、分化和凋亡的细胞信号过程的损害与许多疾病状态有关。因此，加强对Prxs及其在细胞信号和抗氧化保护中所起作用的了解将对预防人类疾病具有重要意义。此外，Prxs在保护人类病原体免受免疫系统杀害方面的作用使Prxs成为开发新的治疗药物以对抗传染病的靶点。

DESCRIPTION (provided by applicant): Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms, and increasing evidence shows that it is also a common second messenger in eukaryotic signaling. In humans, cytokines, growth factors and toll-like receptors of the innate immune system are thought to signal via hydrogen peroxide. Catalase and glutathione peroxidase have long been viewed as the major enzymes degrading peroxide in cells, however, over the past few years, a distinct, highly abundant family of peroxide- reducing enzymes, peroxiredoxins (Prxs), have moved from relative obscurity to become a major focus of redox biology research. The peroxidase activity of eukaryotic Prxs was overlooked for many years, because those Prxs that are highly expressed in eukaryotes are easily inactivated by peroxide. We have developed expertise in Prx enzymology over more than 15 years of characterizing of Prxs from pathogenic bacteria (e.g. Salmonella typhimurium AhpC). These Prxs are targets for antibiotic development because of the role they play in protecting the bacteria from the human immune system. In 2003, our structural and functional studies on S. typhimurium AhpC led us to discover the structural basis for the sensitivity toward peroxides that is conserved for a subset of Prxs that are highly expressed across all eukarya (this is the basis for the structural hypothesis that underlies the present grant, which dictates that the mobility of proximal secondary structures packing near the active site is a key determinant of the sensitivity of Prxs to overoxidation by peroxides and of the ability of Prxs to act as antioxidants). We further proposed the "floodgate hypothesis" for how this sensitivity to inactivation would actually be beneficial in organisms where hydrogen peroxide is being used as a signaling molecule, so that the antioxidant properties of the Prxs could be switched off under appropriate conditions to allow for a controlled burst in peroxide levels. Given the importance of Prxs both in pathogen defense and in human cells for combating oxidative stress and for cellular regulation, we propose here to identify the key determinants of sensitivity toward overoxidation and of efficient antioxidant function by investigating the conformational "mobility" of a few carefully chosen proteins and mutants; relevant rates constants within the catalytic cycle and inactivation pathways for these proteins will also be examined (Aim 1). In Aim 2, we will identify structural features around the highly conserved active site of Prxs which are important for binding and reduction of distinct hydroperoxide substrates. In Aim 3, we will determine whether or not the sensitivity of Prxs toward inactivation by peroxides during turnover (the "floodgate") is critical to modulating the levels of H2O2 generated during cell signaling events through cell-based studies of Prx functions. PUBLIC HEALTH RELEVANCE: Oxidative damage is thought to be important in aging, in the development of cancer and in many degenerative diseases. Moreover, impairments in cell signaling processes controlling proliferation, differentiation and apoptosis are associated with many disease states. An enhanced understanding of Prxs and the roles they play in both cell signaling and antioxidant protection will thus have important implications for the prevention of human diseases. In addition, the role of Prxs in protecting human pathogens against killing by the immune system implicates Prxs as targets for the development of new therapeutic agents to combat infectious diseases.