Long-chain lysine fatty acylation : a chemical proteomics study

Lysine fatty acylation is a class of post translational modifications, including acetylation, propionylation, butyrylation, myristoylation and palmitoylation, that has been identified in histone proteins, indicating that lysine fatty acylation might be involved in very diverse cell pathways and biological functions in mammalian cells. However, the understanding of this class of PTMs is very poor due to the limited techniques available for detecting the acylated lysine side sites. This thesis focuses on lysine long-chain fatty acylation (LCFA) and aims at developing methodologies and a workflow for detecting and characterising lysine-acylated proteins in mammalian cells. An array of tools including metabolic labelling, proteomics, inhibition of background PTMs are employed in this study, which provides the first attempted global profiling of long chain lysine fatty acylated proteins in mammalian cells. The major challenge of this work is the presence of two most common types of fatty acylation in mammalian cells - N-terminal myristoylation and S-palmitoylation - which hinder the detection of lysine LCFA. Further to this, observation of lysine acylated proteins is also hindered by potential long-chain deacylation on lysine residues by proteins such as the sirtuin family. Strategies are developed and optimised in the thesis to 1) decrease background noise caused by N‑terminal myristoylation and S‑palmitoylation and 2) increase lysine LCFA level through sirtuin inhibition and N-terminal myristoylation knock-down, along with chemical tagging and LC-MS/MS to unpick this intriguing and understudied class of PTMs.