DNA damage induced phosphorylation and regulation of NF-kappaB
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
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Technical Summary
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.
Planned Impact
(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 NFkB 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.
(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 NFkB 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.
Organisations
People |
ORCID iD |
| Neil Perkins (Principal Investigator) |
Publications
Campbell AE
(2021)
Temporal modulation of the NF-?B RelA network in response to different types of DNA damage.
in The Biochemical journal
Cuevas-Ocaña S
(2023)
A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells
in International Journal of Molecular Sciences
| Description | Our recent project involved a collaboration between the Eyers lab (Liverpool) and Perkins lab (Newcastle). In the original proposal, Objectives 1 & 2 (Objective 1: 'DNA-damage-induced changes in PTM status of NF-?B'; Objective 2: 'Intact protein analysis - identification of NF-?B proteoforms') were to be performed in the Eyers lab on the proteomic analysis of post-translational modifications of the RelA NF-?B subunit. Whereas Objective 3, 'Roles and regulation of NF-?B PTMs', was work to be performed in the Perkins lab. To aid in objectives 1 & 2, protein samples were prepared in Newcastle and sent to Liverpool for analysis. The status of the work proposed in Objective 3 (the Perkins lab) is as follows. Objective 3.3 of our original award was critically dependent upon the provision of phospho-proteomic data generated in Objectives 1 & 2 from the Eyers lab in Liverpool. Unfortunately, this was delayed by almost 8 months due to personnel not being in post and additionally, the inevitable training associated with bringing new people onto the project. Consequently, there was a similar delay in our ability to functionally analyse DNA damage induced phosphorylation sites of RelA and so this work has not yet been published but is in the process of being written up. 3.1 siRNA kinome screen 3.2 Functional analysis of parallel signalling pathways The siRNA screen has been completed. We have identified a number of novel regulators of NF-?B activity. A problem we encountered here was the high rate of false positives from the initial screen, meaning that validation and analysis of true hits has been time consuming. The results of this screen are currently being prepared for publication. 3.3 Mutational analysis of the RelA/p65 subunit As proposed in our original application, we established the tetracycline (Tet) inducible T-Rex system (Thermo Fisher) as a strategy to investigate the functions of RelA phospho site mutants. This required the screening and creation of a 'parental' clone of U2OS cells, expressing the Tet receptor. This was then used to create a series of additional clones containing inducible wild type and mutant forms the RelA NF-kB subunit. These were all successfully created. Moreover, we established that we would simultaneously knockdown the endogenous RelA, removing one potential complication in terms of data analysis. A series of mutant forms of RelA were established based in information from the Eyers lab on sites of RelA induced phosphorylation following DNA damage with either of the cancer chemotherapeutic drugs etoposide or cisplatin. Our major findings from this study are that: • The RelA S203A mutation results in decreased cell viability following stimulation with etoposide or hydroxyurea (HU) • Induction of the RelA S45A mutation results in decreased activity and levels of the checkpoint kinase CHK1 following etoposide or cisplatin treatment. Although the T-Rex system allowed us to rapidly assess the effect of RelA phospho sites, it became clear during the period of this grant that the advent and adoption by the scientific community of genome engineering technology meant that this was no longer 'state of the art'. Therefore, we decided to invest time in developing CRISPR/Cas9 technology to engineer specific point mutations in target genes. This offers many advantages over the artificial expression of mutants, including analysis of proteins at their normal concentrations in cells while retaining expression from their endogenous gene promoters and enhancers. The process of optimisation of this procedure was performed by the postdoc on this grant, Dr George Schlossmacher. Although time consuming to optimise the protocol for this procedure, this has now been completed and we have a number of clones for the RelA S45A clone in the lab that are currently being analysed. We have been able to confirm that we see a similar effect on CHK1 activity when mutating endogenous RelA at S45 as we saw with the T-Rex over-expression system. We hope to write up this work for publication shortly. |
| Exploitation Route | This work will be of most use to the pharmaceutical and medical sectors since it provides a deeper understanding of regulation of the NF-kB transcription factor, known to play a critical role in many inflammatory diseases, cancer and ageing. |
| Sectors | Pharmaceuticals and Medical Biotechnology |