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

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.

This research aims to elucidate new signalling mechanisms that mediate the effects of ROS on cells. It will test the hypothesis that the ROS-induced formation of disulphide-complexes with peroxiredoxins (Prx) is a redox-regulated modification that regulates the activity of target proteins. Amongst the proteins we have identified form disulphide complexes with the S. pombe Prx, Tpx1, are multiple components of a conserved MAPK signalling pathway (the p38-related Sty1 MAPK and the MAPK activated kinase Srk1) and the cAMP-activated protein kinase (Pka1). These kinases play vital roles in coordinating cell growth and division, stress resistance and survival in response to various environmental and metabolic stimuli in fungi, plants and animals. However, the mechanism/s by which their activity is regulated in response to many of these stimuli are poorly understood. We will use a range of genetic tools to establish how the formation of disulphide complexes with peroxiredoxins affects the cellular functions of Sty1, Srk1 and Pka1. We will determine how 'peroxiredoxinylation' affects their kinase activity, and whether these effects are mediated by effects on stability, localisation or protein-protein interactions. For instance, we will explore the possibility, that, by forming peroxide-induced disulphide complexes with multiple signalling proteins, Prx provide a scaffold, facilitating enhanced signal transduction. Thus, we aim to establish a paradigm, applicable to the regulation of other signalling pathways. Indeed, the final goal is to apply what we have learned to assess the contribution that 'peroxiredoxinylation', and the ability of Prx to oligomerise, make to the functions of the equivalent Prx, PRDX-2, in C. elegans and to the effects of caloric/dietary restriction on ageing in both model systems.

Our research will establish fundamental mechanisms by which cells sense and respond to rises in hydrogen peroxide, to which cells are exposed following immune cell attack but also as a result of changes in metabolic activity or following irradiation. As such, this programme will benefit the academic research community in a number of medically and commercially important areas (cancer, ageing and age-associated diseases, research into anti-fungals and diseases involving inflammation) and also be of broad popular interest. We anticipate this research will give rise to open access high quality publications in widely-read journals. These will impact on the academic community, and more broadly, as detailed below: [1] The identification of the mechanism/s by which formation of disulphide complexes between conserved cysteines in the p38/JNK-related MAPK Sty1 and peroxiredoxin, Tpx1, specifically regulate the activity of Sty1 in coordinating cell growth, division and survival will establish a paradigm for redox-signalling. It will also impact those studying ways to specifically target the function of p38 and related kinases in the treatment of cancer and fungal disease. [2] The identification of how oxidation, via the formation of disulphides with Prx, affects the anti-mitotic activity of a conserved MAPK-activated kinase Srk1(MK2). MK2 activity is established as a therapeutic target e.g. for increasing the effectiveness of irradiation in cancer cell killing (e.g. Dietlein et al 2015 Cell) and recently shown to play important roles in muscle regeneration(Hausburg et al 2015 Elife), autophagy (Wei et al 2015 Elife) and senescence(Herranz et al 2015 Nat Cell Biol). Therefore publication of a new mechanism by which Srk1/MK2 is regulated is likely to impact on many fields. [3] Further publication/s revealing how Prx regulate cAMP-dependent kinase activity (Pka1), and additional H2O2 sensor proteins (identified by pilot studies), and the roles that Prx oligomers play in these regulatory mechanisms will impact on multiple fields. The mutations described in these papers, that give rise to altered function, will also interest structural biologists and facilitate future drug discovery projects. [4] Elucidating how redox-changes in response to caloric restriction are sensed will impact on the ageing field as it strives to take basic studies of the biology of ageing in models, such as quiescent yeast and C. elegans (used here), closer towards therapeutic goals. Our research may have commercial impacts:[5] by identifying ways to use/target the identified H2O2-signalling mechanisms to optimise growth. [6] Moreover, we envisage the knowledge from our studies of Prx-fused proteins and oligomers could be exploited in a future biotechnological or synthetic biology application. For instance, similar approaches to explore the function of reversible covalent modification of proteins by the small ubiquitin-like modifier SUMO, led to fusion to a SUMO tag being successfully exploited commercially as a method for purification of proteins. https://www.lucigen.com/Expresso-SUMO-Cloning-Expression-Systems/#subcat-tabs4. The ability of Prx a to form decameric 'doughnut-like' and other higher molecular structures, including self-assembling nanotubes (Phillips et al 2014 Biomacromolecules) supports our hypothesis that Prx may act as scaffolds for assembly of signalling protein complexes and may lead to potential applications. The impact of this research will be maximised by: (i)Dissemination of research at international and national scientific meetings (see travel), (ii) invited seminars/plenary talks and (iii)protection of intellectual property (iv) press releases and (v) public engagement activities (see pathways to impact for details). The RA's training in redox-signalling, molecular and cell biology techniques in multiple systems will make them highly employable in public and private research sectors and increase capacity in growing areas.