Redox Regulation of Cysteine-Dependent Peroxidases and Signal Transduction Pathways

摘要 虽然过氧化氢长期以来被认为是一种被人类免疫系统用来杀死 感染性有机体，直到最近才被广泛接受，它是 真核生物，对生长因子、细胞因子和免疫系统效应物作出反应并促进或 调节下游的信号转导通路。通过在一定程度上由 PI是一种依赖半胱氨酸的过氧化氢还原酶家族，称为过氧化还蛋白(Prxs) 从相对默默无闻到得到广泛认可，不仅仅是作为主要的氧化剂去除之一 系统在几乎所有的生物体中，但也作为细胞信号通路的关键调节器。皮普尔的研究成果 来自各种生物体的Prx的酶学、生物物理属性和结构对 了解这一高度丰富的酶家族的机制和调节。2003年，Pi Poole和 合作者安迪·卡普拉斯发表了一篇科学论文，在论文中，PRX敏感性的结构决定因素 对过氧化氢介导的活性部位半胱氨酸的过氧化作用进行了鉴定。这导致了我们的建议 “闸门假说”解释了这种由过氧化氢介导的“关闭开关”的潜在好处； 过氧化氢水平开始上升的条件(如NADPH氧化酶激活)，Prx失活将促进 过氧化氢在源头附近的局部积累，允许替代蛋白质靶标的氧化。Dr。 普尔还一直站在开发化学工具以评估细胞中蛋白质氧化的前沿，通过 以过氧化氢氧化的直接产物--磺酸(R-SOH)为靶标。这些探测器现在是 商业上可用的，并已被研究氧化还原调节和信号传递的研究人员广泛使用 以较高的时空精度评估蛋白质氧化。皮普尔的实验室使用这些工具来证明 癌症相关生长因子引起内化受体近端蛋白质氧化的“热点”， 为闸门假说提供了支持。这里提出的未来研究将建立在我们现有的基础上 优势和协作。具体地说，我们建议调查额外的翻译后的影响 对Prx结构和活性的修饰，包括硝化、乙酰化和磷酸化。我们还将 研究硫氧还蛋白调节和被人类Prxs调节的机制。该界面 另一个领域是Prx的功能与涉及蛋白质氧化的信号转导通路的调节 有显著的差距；我们计划跟进我们的数据，表明Prx失活和过氧化氢上升 水平是细胞周期调控的关键。最后，我们目前正在合作研究的一个新领域 与Sharon Campbell一起的是KRAS致癌G12C突变体的氧化敏感性，它具有 有可能严重限制最近开发的治疗剂的有效性。这些努力将解决 对Prx功能和蛋白质氧化很重要的区域，导致了对这些区域的新的理解 可以设想与医学和生物相关的干预措施。

SUMMARY While hydrogen peroxide has long been understood as a toxin used by the human immune system to kill infectious organisms, only recently has it become well accepted that it serves as a second messenger in eukaryotes, produced in response to growth factors, cytokines and immune system effectors and promoting or modulating downstream signal transduction pathways. Through insights contributed in part by the work of the PI, a family of cysteine-dependent, peroxide-reducing enzymes known as the peroxiredoxins (Prxs) have also emerged from relative obscurity to become widely recognized not just as one of the primary oxidant removal systems in almost all organisms, but also as key modulators of cell signaling pathways. PI Poole's work on the enzymology, biophysical attributes and structures of Prxs from a variety of organisms has contributed greatly to understanding the mechanism and regulation of this highly abundant family of enzymes. In 2003, PI Poole and collaborator Andy Karplus published a Science paper in which structural determinants of the sensitivity of Prxs toward peroxide-mediated hyperoxidation of the active site cysteine were identified. This led to our proposal of the “floodgate hypothesis” explaining the potential benefits of such a peroxide-mediated “off switch”; under conditions where peroxide levels begin to rise (e.g. NADPH oxidase activation), Prx inactivation would promote the local accumulation of peroxide near the source, allowing for the oxidation of alternative protein targets. Dr. Poole has also been at the forefront of developing chemical tools to evaluate protein oxidation in cells through targeting sulfenic acid (R-SOH), the direct product of peroxide-mediated oxidation. These probes are now commercially available and have been used widely by researchers studying redox regulation and signaling to evaluate protein oxidation with high spatiotemporal precision. PI Poole's lab used these tools to show that cancer-associated growth factors elicit “hot spots” of protein oxidation proximal to the internalized receptors, providing support for the floodgate hypothesis. Future studies proposed here will build upon our existing strengths and collaborations. Specifically, we propose to investigate the effects of additional posttranslational modifications, including nitration, acetylation and phosphorylation, on Prx structure and activity. We will also investigate the mechanism by which thioredoxin can regulate and be regulated by human Prxs. The interface of Prx function with the regulation of signal transduction pathways involving protein oxidation is another area with significant gaps; we plan to follow up on our data suggesting that Prx inactivation and rising peroxide levels are key to cell cycle regulation. Finally, a new area that we are currently investigating in collaboration with Sharon Campbell is the oxidation sensitivity of the cancer-causing G12C mutant of KRAS, which has the potential to severely limit the effectiveness of recently developed therapeutic agents. These efforts will address areas important to Prx function and protein oxidation, leading to a new level of understanding through which medically-and biologically-relevant interventions could be envisioned.