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Abstract
Post-translational modifications (PTMs) are important tasks given to enzymes that are responsible for adding specific moieties onto proteins and have been dubbed as writers in the epigenetic code. Of the modifications, lysine acetylation is a common and vital modification that regulates many processes that occur in the cell. Histone acetyltransferases (HATs) are writers whose canonical function is to transfer the acetyl moiety from acetyl CoA to the -amino group of lysine residues. This modification on histone and non-histone proteins has also been linked to a number of diseases. Despite the many efforts to discover HAT inhibitors, currently, there are no FDA (Food and Drug Administration) approved drugs that target this type of enzyme. To aid in the discovery of drugs, that target HATs, the goal of this work is to investigate new assays and catalytic functions of HATs. More specifically, the focus will be on HAT1 because this enzyme has been associated with various devastating diseases such as cardiovascular disease and cancer; however, it is poorly studied in comparison to the other HATs. This work will aim to develop a biochemical assay that can detect HAT activity and is compatible with high throughput screening, as well as fill in the gap in knowledge about novel cofactors and substrates of HAT1. Results show that the developed scintillation proximity assay (SPA) is efficient and suitable to detect HAT activity for inhibition screenings. Using the SPA, a potent inhibitor was identified for HAT1 that was competitive towards both substrate and cofactor. To expand our understanding of HAT1 activity, a novel crotonylation activity was also discovered to occur on and by this enzyme. Lastly, as a proof of concept, we showed that a rationally designed biorthogonal probe was able to label substrates of HAT1. Overall, this study was able to enhance our understanding of HAT1 activity and aided in the future development of therapeutics that target this enzyme.