DNA damage induced phosphorylation and regulation of NF-kappaB

To respond to threats from the environment or infectious agents, organisms 'activate' specialized groups of proteins that regulate the response of different genes, thus allowing cells of the body to adapt and survive, or fight the infection. Among these types of proteins, the Nuclear Factor kappa B (NF-kB) family is of particular importance as a regulator of the immune, inflammatory and stress responses. Because of the central role it plays, NF-kB can respond to a large number of different stimuli that include bacteria, viruses, inflammatory proteins and cell stresses such as DNA damage. Although the effect of DNA damage is not studied to the same extent as the response due to infection and inflammation, it is of great importance and can occur through exposure to environmental genotoxins. Many common cancer therapies that rely on DNA damage as their mechanism of killing tumour cells also active this protein family. Moreover, the production of reactive oxygen species (ROS) during inflammation or as a result of ageing can also lead to DNA damage and trigger 'activation' of NF-kB.
The consequences of NF-kB activation in response to these stimuli can vary enormously, depending on the type of stimulation, the cell type in which it is occurring and the presence of other proteins activated at the same time. These differences manifest themselves as activation or repression of different genes by NF-kB, which can vary according to the context. Thus the effect of activating NF-kB on the cell and the organism will also vary depending on the type of stimulus. These differences include effects on cell proliferation, survival, production of inflammatory proteins and generation of ROS. This is also true of DNA damage, where different types of DNA damage and DNA damaging agent can result in very different effects on NF-kB function; this in turn differs from NF-kB activated in response to inflammatory stimuli. To a large extent, these differences can be explained by modifications to the protein structure through addition of small functional chemical groups, such as phosphate, a process termed post-translational modification (PTM). Such PTM is a form of code that rapidly regulates protein function in cells.

Preliminary data from the Eyers group, looking at the effect of the cytokine TNF on the NF-kB protein RelA, has indicated that the level and complexity of PTMs on this protein is much greater than previous thought. In this proposal we will extend this analysis and use state of the art techniques to characterise the multiple PTMs that occur in response to different types of DNA damage. This analysis will include not only the identification of the sites of modification but also how they change during time and whether they occur simultaneously on the same molecules or are present separately on different molecules (generating a mix of differently modified proteins). In parallel with the analysis in the Eyers group, the Perkins group will determine the function and importance of these PTMs. This includes the identification of the enzymes, called kinases that regulate the addition of phosphate groups to different positions in the NF-kB protein. Furthermore, based the analysis in the Eyers lab, amino acids in the NF-kB protein RelA will be mutated such that it can no longer be modified in this manner, thus allowing the importance of these modifications on the regulation of the DNA-damage-induced response of NF-kB to be assessed.

These experiments will give new insights into the regulation of NF-kB by DNA damage during inflammation, ageing and cancer chemotherapy. They will also provide a template for future analysis to investigate other aspects of NF-kB activity (different stimuli, context and activation pathways) while also serving more generally as a relevant example of a stimulus responsive network.

The NF-kB transcription factors play essential roles in the physiology and stress responses of multicellular organisms. Although NF-kB activation is frequently associated with inflammation and the immune response, it also plays a critical role in the cellular response to DNA damage. This is most often studied in the context of cancer, where its activation can either inhibit or promote the effect of many chemotherapeutic drugs. However, this is also a physiologically important process, relevant to the functions of NF-kB associated with inflammation and aging, where, for example, the production of reactive oxygen species can result in DNA damage. While much has been done to start to unravel the 'nuclear-to cytoplasmic' signalling of NF-kB in response to DNA damage, it remains to be defined how the transcriptional functions of the different NF-kB dimers is specifically regulated by these types of stimuli. Numerous papers from the Perkins lab and others have demonstrated that DNA damage-induced NF-kB is functionally distinct to that found following exposure to inflammatory cytokines or engagement of Toll-like receptors. These different effects appear to be mediated, to a significant extent, by differential post-translational modifications (PTMs) of NF-kB subunits. This proposal aims to exploit state-of-the art mass spectrometric techniques to investigate DNA-damage-induced changes in post-translational modification status of the NF-kB transcription factors. Furthermore, we will elucidate how these PTMs are regulated over time, discern mechanism of regulation and evaluate how changes in these modification fingerprints correlate with NF-kB activity and transcriptional function. The experiments performed here will provide a template for future analysis to investigate other aspects of NF-kB signalling (different stimuli, context and activation pathways) while also serving more generally as a relevant example of a stimulus responsive signalling and transcription network.

(1) Academic Researchers (1-4 yrs): This project will have a major impact on academic researchers by revealing the complexity of NF-kB transcriptional regulation by PTMs as well as the experimental tools required to investigate it. These experiments will provide a template for how future studies to determine these effects can be performed. Although challenging, the 'top down' proteomics studies in particular will represent a paradigm shift in signalling analysis as no systematic study of a signalling system at the level of the intact protein components has been performed previously. As there are many similarities between this pathway of NF-kB regulation and parallel signalling pathways, this research will be relevant to researchers worldwide working in the related fields of signal responsive transcriptional networks, of which there are many.
(2) Clinical researchers and clinicians (3-10 yrs): NF-kB signalling affects many processes central to the health and wellbeing of humans and animals, particularly inflammatory diseases associated with ageing, such as arthritis and cancer. The data generated in this proposal, although very much at the level of fundamental research, will impact our understanding of these processes and so be of interest to clinicians. We will gain insights into ability of NF-kB to affect the response to cancer chemotherapeutic drugs. Understanding how PTMs affect these processes could, for example, suggest beneficial combinatorial therapies between traditional DNA damage inducing drugs and kinase inhibitors. Alternatively, this research could lead to the development of biomarkers to allow an assessment of NF-kB activity and function in patients before and after therapy.
(3) The pharmaceutical and biotech industry (3-10 yrs). In recent years there has been great interest in targeting the NF-kB pathway to treat a range of inflammatory disease and cancer. Indeed, many existing drugs, such as glucocorticoids or cancer chemotherapeutics, already do this, either directly or indirectly. Frequently this effect has only been discovered at a later point, after the drug entered clinical use. A recent focus has been on targeting IKKb, the primary kinase regulating the classical NF-kB pathway. However, total inhibition of NF-kB through this pathway can potentially lead to many side effects. The work in this proposal will lead to additional mechanisms of targeting NF-kB transcriptional activity in cells, through modulation of the parallel signalling pathways that control NF-kB PTMs. This strategy provides an alternative and attractive mechanism to inhibit the pathological affects of NF-kB activity, without the side effects of abolishing NF-kB activity entirely.
(4) Staff employed on the project (1-4 yrs): The collaborative and multi-disciplinary nature of this project means that both PDRAs will receive training in a broad range of experimental techniques. The Newcastle PDRA will be trained in the analysis and interpretation of MS data, while the Liverpool PDRA will receive further training in the biochemical/cell biology based approaches. Both PDRAs will contribute to the writing of research papers and will be given the opportunity to present their data (either oral or poster) at conferences. The PDRAs will be carefully coached in presenting research at the project meetings in Liverpool/Newcastle, and at staff development seminars in the respective universities. Furthermore, it is anticipated that nearing the end of the grant the Liverpool PDRA, Dr. Lanucara, should be at a position to apply for an independent fellowship; training will therefore be given in grant writing and lab management, enabling him to make a valuable and practical contribution to the growth of UK science.
(5) The general public (>10 yrs): In the long term, the general public will benefit from this research through the development and better application of new and existing therapies for the wide range of NF-kB associated diseases.