Mechanisms and Regulation of Peroxiredoxins

 说明(申请人提供)：过氧化氢是人类免疫系统用来杀死传染性生物的一种毒素。现在人们普遍认为它也是一种常见的第二信使，由NADPH氧化酶故意产生，作为对人类健康至关重要的真核信号通路的一部分，如由细胞因子、许多生长因子和先天性免疫系统的Toll样受体触发的信号通路。在过去的20年里，部分由于这一提议的PI的工作，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(Prx)--已经从相对默默无闻变成了氧化还原生物学研究的一个主要焦点。在这二十年中，PI通过表征来自各种生物的PrX，特别是使用鼠伤寒沙门氏菌的过氧化还蛋白AhpC作为主要模型系统，在Prx酶学、生物物理表征和结构方面发展了专业知识。对Prxs的研究很重要，因为来自人类病原体的Prxs是抗生素开发的目标，也因为哺乳动物的Prxs参与调节关键的信号通路，PRX1基因敲除的小鼠在9个月大时会患上多种形式的癌症。2003年，我们发现在活性部位附近堆积的蛋白质片段的流动性是Prxs对过氧化氢介导的过氧化失活敏感性的关键决定因素，我们提出了这种对失活的敏感性将如何使生物体受益的“闸门假说”，其中过氧化氢被用作信号分子：Prxs的抗氧化特性将在正确的时间和地点被关闭，以允许过氧化氢的局部可控积累。从那时起，更多的翻译后修饰(PTM)被证明调节人类Prx的功能。鉴于Prx作为微生物致病因子、对抗氧化应激以及调节人类细胞生长和分化的重要性，我们建议在这里解决Prx研究中仍然存在最大悬而未决的问题的领域。在目标1中，我们将通过研究四种生理上相关的PTM对人PRX活性的生物物理和功能影响，加深我们对催化和对过氧化敏感性的关键决定因素的理解。在目标2中，我们开发了一种新的适用于核磁共振和结晶学研究的PRX‘模型系统’，它将提供前所未有的能力来测量动态特征并将其与结构和功能相关联。在目标3中，我们将推进对研究较少但通常是限速的Prx还原反应的知识，评估几个关键Prx的特异性和效率如何取决于寡聚体状态、修饰状态和第二个(拆分)半胱氨酸的位置。我们还将通过结晶学和/或核磁共振绘制相互作用界面。这些努力将涉及对Prx的功能和监管很重要的领域，尽管它们很重要，但尚未得到足够的重视，从而导致新的理解水平，通过这些水平可以预见与医学和生物相关的干预措施。

 DESCRIPTION (provided by applicant): Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms. It is now well accepted that it is also a common second messenger purposefully produced by NADPH oxidases as a part of eukaryotic signaling pathways crucial for human health such as those triggered by cytokines, many growth factors, and toll-like receptors of the innate immune system. Over the past 20 years, in part due to the work of the PIs of this proposal, a distinct, highly abundant family of peroxide-reducing enzymes, peroxiredoxins (Prxs), has gone from relative obscurity to become a major focus of redox biology research. Over these two decades, the PIs have developed expertise in Prx enzymology, biophysical characterization, and structure by characterizing Prxs from various organisms, especially using the peroxiredoxin AhpC of Salmonella typhimurium as a primary model system. Studies of Prxs are important both because Prxs from human pathogens are targets for antibiotic development, and because mammalian Prxs are involved in regulating key signaling pathways, with a Prx1 knockout mouse developing many forms of cancer by nine months of age. In 2003, we discovered that the mobility of protein segments packing near the active site is a key determinant of the sensitivity of Prxs to inactivation by peroxide-mediated hyperoxidation, and we proposed the "floodgate hypothesis" for how this sensitivity to inactivation would benefit organisms, like humans, where hydrogen peroxide is used as a signaling molecule: the antioxidant properties of the Prxs would be switched off at the right times and places to allow for a controlled local accumulation of peroxide. Since that time, additional posttranslational modifications (PTMs) have been shown to regulate the function of human Prxs. Given the importance of Prxs as microbial pathogenicity factors, for combating oxidative stress, and for regulating cell growth and differentiation in human cells, we propose here to address areas of Prx research where the biggest open questions remain. In Aim 1, we will deepen our understanding of key determinants of catalysis and of sensitivity toward hyperoxidation by investigating the biophysical and functional effects of four physiologically relevant PTMs on human Prx activity. In Aim 2, we develop a new Prx `model system' suitable for both NMR and crystallographic studies that will provide an unprecedented ability to measure and correlate dynamic features with structure and function. In Aim 3, we will advance knowledge of the poorly studied, but often rate-limiting, Prx reduction reaction, evaluating for several key Prxs how specificity and efficiency depend on oligomeric state, modification status, and location of a second (resolving) cysteine. We will also map out interaction interfaces by crystallography and/or NMR. These efforts will address areas important to the function and regulation of Prxs which have not yet received sufficient attention in spite of their importance, leading to a new level of understanding through which medically-and biologically-relevant interventions could be envisioned.