Please Note: The main page lists projects via BBSRC Research Theme(s) quoted and then relevant Topic(s).
Dynamic S-acylation of the β2-Adrenergic Receptor
Secondary Supervisor(s): Dr Giuseppe Deganutti
University of Registration: Coventry University
BBSRC Research Themes:
Project Outline
Post-translational modifications such as S-acylation convey tight control on the functions of proteins such as G protein-coupled receptors (GPCRs)1; our recent characterisation of several key deacylases and our GPCR expertise will enable us to investigate these control mechanisms in the prototypical GPCR, the b2-adrenergic receptor (b2AR, involved in cardiac perfusion and vasodilation, striated muscles contraction, and smooth muscle relaxation).
S-acylation involves the reversible attachment of fatty acids (predominantly palmitate) onto proteins; because it regulates almost all aspects of human physiology, a deeper understanding may lead to novel therapeutic opportunities. In humans, S-acylation is mediated by a family of 23 ZDHHC S-acyltransferases and reversed in a subset of proteins by deacylases belonging to the metabolic serine hydrolase (mSH) superfamily. Dynamic S-acylation regulates many fundamental physiological processes, including signalling, cell migration and division, communication, metabolism, and immune function. It’s been estimated that up to 20% of the human proteome is S-acylated, regulating the localisation and function of thousands of soluble and transmembrane proteins including receptors, transporters, ion channels, signalling molecules, membrane fusion proteins and scaffolding proteins2.
A family of S-acylated receptor proteins, the G protein–coupled receptor (GPCR) superfamily, is the largest family of membrane receptors (comprising >800 members) having diverse physiological functions. GPCRs are multiply modified by S-acylation and various other PTMs, which regulate their structure, stability, activity and function1. The prototypical S-acylated ligand-activated GPCR, the β2AR, is stably S-acylated at its C-terminal cytoplasmic tail and dynamically S-acylated on a cysteine residue (Cys-265) within its third intracellular loop3. Dynamic S-acylation of β2AR at Cys-265 is catalysed by the S-acyltransferases ZDHHC9/14/18 and reversed by the deacylase APT1, however, other mSH deacylases may also be involved3. Understanding how the S-acylation machinery interacts with β2AR and how the deacylases catalyse the release of the attached fatty acid is unknown and can open a new branch of β2AR pharmacology.
Objectives
The objective of this research is:
- To define the full complement of APTs involved in β2AR deacylation.
- To understand the molecular mechanisms of β2AR regulation by the S-acylation regulatory machinery; and,
- To explore the cardiac functional consequences and therapeutic opportunities of tilting the β2AR S-acylation equilibrium.
Methods
This project will employ a multidisciplinary approach combining molecular modelling and cell, molecular, biochemical and chemical biology methods.
Biorthogonal labelling of cells with palmitic acid analogues coupled with click chemistry and western blotting will be used to assess changes in S-acylation quantitatively. Co-immunoprecipitation approaches will be used to measure protein-protein interactions. Mutagenesis by PCR will be used to inform and verify computational analysis. Confocal microscopy including live-cell imaging will be used to assess changes in protein localisation.
The PGR will learn in silico techniques covering molecular modelling, molecular docking, and advanced molecular dynamics simulations (e.g. multiple walker supervised molecular dynamics) to study the interactions between β2AR and S-acyltransferases ZDHHC9/14/18 or the deacylase APT1, and rationalise experimental results.
Key References
- Patwardhan et al (2021) Pharmacol Rev. 73, 120–151. DOI: 10.1124/pharmrev.120.000082
- Mesquita et al (2024) Mechanisms and functions of protein S-acylation. Nat Rev Mol Cell Biol 25, 488-509. DOI:10.1038/s41580-024-00700-8.
- Adachi et al (2016). S-Palmitoylation of a Novel Site in the β2-Adrenergic Receptor Associated with a Novel Intracellular Itinerary. J Biol Chem 291, 20232-20246. DOI:10.1074/jbc.M116.725762.
