A CHEMICAL BIOLOGY APPROACH TO IDENTIFY NOVEL SUBSTRATES OF THE CELL SURFACE PROTEIN ACYL TRANSFERASE ZDHHC5

Supervisors: 

Prof Nicholas Tomkinson, Pure and Applied Chemistry, (University of Strathclyde)

Prof William Fuller, School of Cardiovascular & Metabolic Health (University of Glasgow)

Summary: 

Dynamic S-acylation of integral membrane and membrane associated proteins regulates numerous aspects of their function, including localisation, protein interactions, and activity. Understanding physiological control of S-acylation is therefore central to understanding how it controls organism physiology. S-Acylation is catalysed by integral membrane zDHHC-PAT enzymes and reversed by thioesterases, but to date very little information exists about how these enzymes recognise their substrates, or how their activities are regulated.1  

In the heart, the cell surface enzyme zDHHC5 S-acylates ion channels and their regulators that directly control action potential dynamics and calcium handling, and hence cardiac output. In the brain, zDHHC5 S-acylation of substrates is central to learning and memory. However, a systematic evaluation of zDHHC5 substrates has not been conducted, which means the breadth of cellular processes regulated by zDHHC5 remains to be determined. This project will employ a novel chemical biology approach to identify zDHHC5 substrates. 

S-Acylation occurs by autoacylation of the catalytic cysteine residue of the zDHHC enzyme by an acyl coenzyme A followed by transfer of the acyl group to the cysteine of a substrate protein, regenerating the free zDHHC enzyme. To transfer the acyl group from the zDHHC enzyme to the substrate the reactive cysteines of both the zDHHC enzyme and the substrate protein must be attached to the same carbon atom simultaneously. This juxtaposition of the two sulfur atoms is essential for the addition-elimination mechanism involved in acyl group transfer.2 Drawing inspiration from this natural ping-pong mechanism for acyl group transfer we will prepare reactive probe molecules that are able to form a covalent bond with both the zDHHC enzyme and its substrate partner, joining the two proteins and allowing the partners to be identified using mass spectrometry. 

Several warheads have been designed to react irreversibly with cysteine residues including acrylamides and chloroacetamides which have led to the identification of covalent inhibitors of the zDHHC family of enzymes. We will use one of these, CMA,3 a known inhibitor of zDHHC5, as the starting point to this project. We will add a second electrophilic group to the CMA scaffold to also capture the nucleophilic cysteine residue of protein substrates. Variation of the linker between these two reactive functionalities will allow the distance and trajectory between each electrophilic centre to be optimised. Initially these tool compounds will be assayed in vitro against known peptide substrates of zDHHC5 with the adducts being verified through mass spectrometry.4 On identification of compounds that form a ternary complex with both the zDHHC5 enzyme and a substrate peptide we will then examine known protein substrates of the enzyme. On optimisation of this in vitro protocol, we will proceed to a discovery phase in cells. This will involve over expression of a HaloTag-zDHHC5 construct and addition of the optimised bis-electrophilic species to capture HaloTag-zDHHC5-substrate complexes. The HaloTag will allow development of a simple workflow for the rapid isolation and purification of zDHHC5 adducts and therefore enable the identification of zDHHC5 cellular substrate networks. Parallel, funded projects in the NCOT and WF labs will identify signalling pathways that regulate zDHHC5 activity, enabling us to characterise the impact of these pathways on substrate recruitment by zDHHC5. 

The project draws upon expertise and techniques embedded within the NCOT and WF laboratories and will provide critical insight into the fundamental biology of this important class of enzyme.