Mechanisms and Regulation of Peroxiredoxins

 描述（由申请方提供）：过氧化氢是一种由人体免疫系统用于杀死感染性生物的毒素。现在公认的是，它也是一种常见的第二信使，由NADPH氧化酶有目的地产生，作为对人类健康至关重要的真核细胞信号传导途径的一部分，例如由细胞因子、许多生长因子和先天免疫系统的toll样受体触发的信号传导途径。在过去的20年里，部分由于这项提案的PI的工作，一个独特的，高度丰富的过氧化物还原酶家族，过氧化物还原酶（Prxs），已经从相对默默无闻成为氧化还原生物学研究的主要焦点。在这二十年中，PI通过表征来自各种生物体的Prx，特别是使用鼠伤寒沙门氏菌的过氧化物酶AhpC作为主要模型系统，开发了Prx酶学，生物物理表征和结构方面的专业知识。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，并且因为哺乳动物Prxs参与调节关键信号通路，Prx 1敲除小鼠在9个月大时会发展出多种形式的癌症。在2003年，我们发现在活性位点附近包装的蛋白质片段的流动性是Prxs对过氧化物介导的过氧化作用失活的敏感性的关键决定因素，我们提出了“闸门假说”，说明这种失活的敏感性如何使生物体受益，如人类，其中过氧化氢被用作信号分子：Prxs的抗氧化性能将在适当的时间和地点被关闭，以允许过氧化物的受控局部积累。从那时起，额外的翻译后修饰（PTM）已被证明可以调节人Prx的功能。考虑到Prxs作为微生物致病因子、对抗氧化应激以及调节人类细胞的细胞生长和分化的重要性，我们在这里建议解决Prx研究中仍然存在的最大开放问题。在目标1中，我们将通过研究四种生理相关PTM对人类Prx活性的生物物理和功能影响，加深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Prx“模型系统”，适用于NMR和晶体学研究，这将提供前所未有的能力来测量和关联动态功能与结构和功能。在目标3中，我们将进一步了解研究不足，但往往限速，Prx还原反应，评估几个关键Prx的特异性和效率如何取决于寡聚状态，修饰状态和第二个（解析）半胱氨酸的位置。我们还将通过晶体学和/或NMR绘制出相互作用界面。这些努力将解决对Prxs的功能和调节很重要的领域，尽管它们很重要，但尚未得到足够的关注，从而使人们对医学和生物学相关干预措施的理解达到一个新的水平。

 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.