Identification and validation of non-histone protein acetylation regulated by the DNA damage response
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1
Department of Oncology, Medical Sciences Division, University of Oxford, United Kingdom
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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
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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