Identification and validation of non-histone protein acetylation regulated by the DNA damage response

Abstract

Event Abstract Back to Event Identification and validation of non-histone protein acetylation regulated by the DNA damage response Judith Nicholson1, Junetha Syed Jabarulla1, Iolanda Vendrell1, 2, Xin Ying Yeo1, Roman Fischer2, Benedikt M. Kessler2 and Anne Kiltie1* 1 Department of Oncology, Medical Sciences Division, University of Oxford, United Kingdom 2 Nuffield Department of Medicine, Medical Sciences Division, University of Oxford, United Kingdom Background: The DNA Damage Response (DDR) is an attractive target for developing radiosensitising drugs, which can downregulate the efficiency of DNA repair in cancer cells induced by ionising radiation. Post-translational modifications of DNA repair proteins tightly coordinate the DDR events, among which lysine acetylation plays an important role in regulating histone and non-histone proteins in the DDR process. Our previous work on modulating acetylation with histone deacetylase inhibitor (HDACi) panobinostat in bladder cancer cell lines has shown to increase radiosensitivity by targeting DDR without any side effects to the normal tissue. In this study, we identified acetylation of DDR-associated non-histone proteins after treatment with panobinostat with the long-term goal of finding effective radiosensitisers with less systemic toxicity. Methods: Tandem mass spectrometry was used to identify potential DDR candidates that become acetylated following irradiation +/- panobinostat treatment. Acetylated protein enrichment was achieved by Immunoprecipitation. Radiosensitisation was determined by clonogenic assay, gamma H2AX levels by western blotting and interacting partners of the protein of interest by APEX-mediated proximity labelling. Results: In total, 228 proteins harbouring 657 unique acetylation sites with a subset related to DNA repair functions were identified. The acetylation of two hit DDR candidates sMEK1 and THRAP3 was validated. The clonogenic survival assay and the pattern of H2AX phosphorylation with acetyl-mimic and acetyl-dead constructs indicated the important role of the identified acetylation site in THRAP3 for radiosensitivity. Conclusion: Several non-histone proteins are regulated on a post-translational level by acetylation during the DNA damage response. The acetylated proteins identified in this dataset represent a complex sample of non-histone proteins, THRAP3 is one of which we validated as having a role in radiosensitisation via a specific acetyl site. Identifying the interacting partners of acetylated THRAP3 would help in understanding the biological significance of identified acetylation site with respect to DDR. Keywords: DNA repair protein, Proteomics, Acetylation, HDACi, THRAP3 Conference: Bladder Cancer Translational Research Meeting, London, United Kingdom, 29 Mar - 29 Mar, 2019. Presentation Type: Poster Topic: Optimisation of diagnostic pathways Citation: Nicholson J, Syed Jabarulla J, Vendrell I, Yeo X, Fischer R, Kessler BM and Kiltie A (2019). Identification and validation of non-histone protein acetylation regulated by the DNA damage response. Front. Oncol. Conference Abstract: Bladder Cancer Translational Research Meeting. doi: 10.3389/conf.fonc.2019.01.00017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 Mar 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Anne Kiltie, Department of Oncology, Medical Sciences Division, University of Oxford, Oxford, England, OX3 7DQ, United Kingdom, anne.kiltie@oncology.ox.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Google Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Google Scholar Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie PubMed Judith Nicholson Junetha Syed Jabarulla Iolanda Vendrell Xin Ying Yeo Roman Fischer Benedikt M Kessler Anne Kiltie Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.