Peroxiredoxinylation; a new post-translational modification promoting redox signal transduction?

所有使用氧气的生物体都不可避免地会遇到由大量外源性和内源性来源产生的活性氧（ROS）。ROS可能具有极强的破坏性，因此高度保守的机制已经进化来防止这种损伤。在酵母、植物和动物细胞中，这些包括激活保守的信号传导途径，如p38/JNK MAPK途径，其启动各种反应，包括增加保护性蛋白的水平。然而，ROS在这些生物体中也具有许多其他信号传导功能;通过影响细胞生长、分裂、分化和迁移来促进伤口愈合、根尖生长、癌细胞运动等。此外，虽然氧化损伤是许多疾病和衰老的一般标志，但ROS的小幅增加实际上对于饮食限制/线粒体活性变化的促长寿效应至关重要。这一发现挑战了ROS导致衰老的理论，并导致了目前的观点，即局部ROS信号能够防止衰老和年龄相关疾病。然而，尽管涉及ROS信号的重要生物过程的数量不断增加，但我们对这些ROS信号如何被实际感知和传输的理解仍然存在很大的差距。例如，在大多数情况下，ROS调节的信号蛋白的身份以及ROS调节其活性的机制都是未知的。这项工作将解决这些问题，利用酵母的公认优势（粟酒裂殖酵母）和显微镜下的线虫（秀丽隐杆线虫）作为研究参与调节细胞分裂、ROS应答和衰老的机制的强大模型系统。申请人先前的工作已经确立了普遍存在的过氧化物反应蛋白，2-Cys过氧化物氧化还原蛋白（Prx），在酵母和蠕虫中促进ROS信号传导和长寿。重要的是，ROS信号传导和抗衰老功能已被发现在哺乳动物和植物中由Prx共享，这说明了在这些模型中研究用于鉴定保守的ROS信号传导和促长寿机制的价值。然而，潜在的许多ROS信号和促长寿功能的Prx的机制尚未被发现。试点数据：我们的方法，以确定ROS调节的靶蛋白已经确定了一些蛋白质，形成ROS诱导的二硫键结合的复合物与Prx。这些Prx复合蛋白包括p38相关MAPK信号通路的多个组分，其在协调对环境/代谢刺激的反应中起关键作用。在这里，我们将测试的假设，这些可逆的化学键的形成与Prx，或“peroxiredoxinylation”，代表了一种新的蛋白质修饰，调节这些蛋白质，从而介导许多的ROS的生理效应。通过确定Prx如何调节这些信号蛋白，我们希望建立一个新的范例，ROS信号如何被转导到细胞反应。我们将使用酵母和蠕虫含有针对特定活动的突变版本，以确定哪些Prx的功能需要其与其他蛋白质形成二硫键复合物（或“peroxiredoxinylate”）的能力，或聚集在一起形成可堆叠的甜甜圈状环结构。例如，我们将确定是否“peroxiredoxinylation”或Prx形成这些环结构的能力对Prx的抗衰老功能很重要。这对于理解我们已经确定的新机制如何有助于ROS的一些有益作用，例如对衰老的作用，将是非常重要的。事实上，我们希望这项工作能够填补我们目前对ROS如何影响许多生物反应的理解中的一个重大空白。

All oxygen-using organisms inevitably encounter reactive oxygen species (ROS), produced by a multitude of exogenous and endogenous sources. ROS can be extremely damaging, hence highly conserved mechanisms have evolved to prevent this damage. In yeast, plants and animal cells, these include activating conserved signalling pathways, such as p38/JNK MAPK pathways, that initiate a variety of responses, including increasing the levels of protective proteins. However, ROS have many other signalling functions in these organisms too; promoting wound healing, root tip growth, movement of cancer cells etc through effects on cell growth, division, differentiation and migration. Moreover, although oxidative damage is a general hallmark of many diseases and ageing, small increases in ROS are actually essential for the pro-longevity effects of dietary restriction/changes in mitochondrial activity. This discovery has challenged the theory that ROS cause ageing and led to the current view that localised ROS signals are able to protect against ageing and age-associated diseases. Nevertheless, despite the ever-increasing number of important biological processes in which ROS signals are implicated, there remain big gaps in our understanding of how these ROS signals are actually sensed and transmitted. For instance, in most cases, the identity of the ROS-regulated signalling proteins, and the mechanisms by which ROS regulate their activity are both unknown. This work will address these questions, exploiting the well-established advantages of yeast (Schizosaccharomyces pombe) and microscopic nematode worms (Caenorhabditis elegans) as powerful model systems for studying the mechanisms involved in regulating cell division, ROS responses and ageing.The applicant's previous work has established important roles for ubiquitous, peroxide-reactive proteins, 2-Cys peroxiredoxins (Prx), in promoting ROS-signalling and longevity in yeast and worms. Importantly, ROS-signalling and anti-ageing functions have been found to be shared by Prx in mammals and plants, illustrating the value of studies in these models for identifying conserved ROS-signalling and pro-longevity mechanisms. However, the mechanisms underlying many of the ROS-signalling and pro-longevity functions of Prx have yet to be uncovered. Pilot data: Our approach to identify ROS-regulated target proteins has identified a number of proteins that form ROS-induced disulphide-bonded complexes with Prx. These Prx-complexed proteins include multiple components of a p38-related MAPK signalling pathway, that plays a key role in coordinating responses to environmental/metabolic stimuli. Here we will test the hypothesis that the formation of these reversible chemical bonds with a Prx, or 'peroxiredoxinylation', represents a new protein modification, regulating these proteins and thus mediating many of the physiological effects of ROS. By establishing how Prx regulates these signalling proteins, we hope to establish a new paradigm for how ROS signals are transduced into cell responses. We will use yeast and worms containing mutant versions targeting specific activities to determine which of Prx's functions require its ability to form disulphide complexes with (or 'peroxiredoxinylate') other proteins, or to come together to form stackable, doughnut-like ring structures. For instance, we will determine whether 'peroxiredoxinylation' or the ability of Prx to form these ring structures is important for Prx's anti-ageing function. This will be important for understanding how the new mechanisms we have identified contribute to some of the beneficial effects of ROS e.g.on ageing. Indeed, we expect this work to fill a significant gap in our current understanding of how ROS effect many biological responses.