Date of Award
Doctor of Philosophy (PhD)
Acetylation is one of several post-translational modifications (PTMs) that occurs on histones to regulate chromatin dynamics and function in DNA transcription, replication, repair, and other DNA-templated activities, and is carried out by histone acetyltransferases (HATs). Of the multiple HATs that have been identified to date, the most studied have been classified into five subfamilies based on sequence homology and substrate acetylation properties. This work is focused on the MYST family (named for founding members MOZ, YBF2/SAS3, SAS2, and Tip60) of acetyltransferases, and more specifically, hMOF (human males absent on the first). Despite being the largest, most diverse known family of HATs, the MYST proteins have not been as well studied as other families. The MYST family has been linked with a variety of diseases including Alzheimer’s disease, diabetes, and cancer, and some efforts have been made to develop inhibitors of these proteins. The existing inhibitors for the MYST family, however, are lacking in either potency, selectivity, or favorable pharmacokinetic properties. The first part of this thesis will describe our efforts to identify potent, selective hMOF inhibitors using a high throughput screening campaign. Although this project ultimately uncovered no hMOF inhibitors, it revealed major hurdles that may be encountered when studying the MYST proteins that will be outlined in detail.
The second part of this thesis will discuss autoacetylation, which is a mechanism of regulation for acetyltransferases. The MYST family is autoacetylated at an active site lysine residue to facilitate substrate lysine binding and acetylation, however the mechanism and regulation of this autoacetylation are not perfectly understood. Here, we will describe a molecular investigation of Lys-274 autoacetylation of hMOF. These studies revealed that substitutions of Lys-274 are able to bind cofactor but are destabilized, and are catalytically inactive for histone H4 peptide lysine acetylation, stemming from a disordering of the residue 274-harboring α2-β7 loop. We also provide evidence that a catalytically inactive C316S/E350Q mutant and a K268M mutant can still undergo K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF Lys-274 autoacetylation in hMOF stability and cognate substrate acetylation and argue that binding of AcCoA to hMOF likely drives Lys-274 autoacetylation for subsequent cognate substrate acetylation.