Mechanistic Studies of Alkyl Hydroperoxide Reductase and Related Redox Proteins

描述（由申请人提供）：过氧化氢是人体免疫系统用来杀死感染性生物的一种毒素，越来越多的证据表明它也是真核生物信号传导中常见的第二信使。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发出信号的。过氧化氢酶和谷胱甘肽过氧化物酶长期以来被认为是细胞中降解过氧化物的主要酶，然而，在过去的几年里，一个独特的，高度丰富的过氧化物还原酶家族，过氧化物还毒素（Prxs），已经从相对默默无闻变成了氧化还原生物学研究的主要焦点。真核Prxs的过氧化物酶活性多年来一直被忽视，因为那些在真核生物中高表达的Prxs很容易被过氧化物灭活。我们在Prx酶学方面拥有超过15年的专业知识，可以从致病菌（如鼠伤寒沙门氏菌AhpC）中鉴定Prx。这些Prxs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侵害方面发挥着作用。2003年，我们对鼠伤寒沙门氏菌AhpC的结构和功能研究使我们发现了对过氧化物敏感的结构基础，这是Prxs在所有真核生物中高度表达的一个子集（这是目前资助的结构假设的基础）。这表明靠近活性位点的近端二级结构的流动性是Prxs对过氧化物过度氧化的敏感性和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在过氧化氢被用作信号分子的生物体中实际上是有益的，因此Prxs的抗氧化特性可以在适当的条件下被关闭，从而允许过氧化氢水平的受控爆发。考虑到Prxs在病原体防御和人类细胞对抗氧化应激和细胞调节中的重要性，我们建议通过研究一些精心选择的蛋白质和突变体的构象“流动性”来确定对过度氧化敏感性和有效抗氧化功能的关键决定因素；这些蛋白质的催化循环和失活途径中的相关速率常数也将被检查（目的1）。在目标2中，我们将确定Prxs高度保守的活性位点周围的结构特征，这对于不同的过氧化氢底物的结合和还原是重要的。在Aim 3中，我们将通过基于细胞的Prx功能研究，确定Prxs在细胞转换过程中对过氧化物失活的敏感性（“闸门”）是否对调节细胞信号事件中产生的H2O2水平至关重要。

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.