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.

Grant Number: 5R35GM142505-05
NIH Institute/Center: NIH
Principal Investigator: George Burslem