Editing Acetylation and Protein Homeostasis

Abstract Protein homeostasis is crucial to maintain healthy cells and is predominantly controlled by the ubiquitin proteasome system (UPS) whereby proteins are tagged with ubiquitin, via a cascade of 3 enzymes, resulting in recognition by the proteasome and subsequent degradation. While some proteins are constitutively recognized and degraded by this system, others are marked as substrates for the UPS by post-translational modifications such as phosphorylation. Recently, acetylation of non-histone proteins has emerged as an important mechanism of regulation for the ubiquitin-proteasome system, particularly at the level of E3 ligase substrate recognition. Leveraging our expertise of the ubiquitin proteasome and protein-protein interactions we propose to elucidate the molecular mechanisms and biological pathways resulting in acetylation driven modulation of protein homeostasis (Project 1). Additionally, building on our previous work with proteolysis targeting chimera, we will develop heterobifunctional approaches to modulate protein acetylation states as a novel mechanism to control protein homeostasis for both the study of this fundamental biological regulation and as a potential therapeutic approach (Project 2). In Project 1, we will identify and characterize proteins with stability regulated at the level of post-translational acetylation. Using proteomics experiments paired with RNA-Seq we will generate a database of proteins with intracellular levels directly controlled by p300 driven acetylation, not altered at the level of transcription. Furthermore, we will characterize the molecular recognition of acetyl degron substrates by the relevant E3 ligases using biophysical, biochemical and structural approaches, revealing unique insights into this mechanism of protein homeostasis. In Project 2, we will develop heterobifunctional compounds which recruit an acetyltransferase or deacetylase to a neo-substrate. Building on the concept of chemically induced post- translational modifications, exemplified by proteolysis targeting chimera, we will identify the (de)acetylation machinery most amenable to this approach via chemical biology approaches before designing and synthesising compounds to edit acetylation in native systems.Together these projects provide insights into basic biological processes regulating protein stability and a novel chemical biology approach to modify them.