Interactions and S-acylation of Sprouty and SPRED proteins

Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2023
Thesis identifier
  • T16812
Person Identifier (Local)
  • 202072536
Qualification Level
Qualification Name
Department, School or Faculty
  • Sprouty (Spry)/SPRED proteins are important regulators of the MAPK/ERK signalling pathway, and dysregulation of this pathway can contribute to development of cancer. The defining feature of Spry/SPRED proteins is a highly conserved cysteine-rich Sprouty domain, which for Spry2 contains 26 cysteine residues. Recent work has shown that the SPR domain is S-acylated by zDHHC17. The aim of this thesis is to explore the mechanisms of interaction and S-acylation of Spry/SPRED proteins by zDHHC17 and identify downstream effects of these interactions. The approaches used included expression of zDHHC17 and Spry/SPRED mutants in HEK-293T and PC12 cells, followed by analysis of protein interactions by immunoprecipitation and analysis of S-acylation using click chemistry methodologies. Protein localisation was examined using confocal microscopy and protein stability measured in cycloheximide-chase experiments. The use of AlphaFold structural predictions and other bioinformatic tools was used to inform these analyses. The results indicate that the interaction of Spry2 and zDHHC17 has a unique dual-stabilisation effect on both the enzyme and substrate. Furthermore, a novel mechanism of recognition/S-acylation of Spry/SPRED proteins, which is distinct from the mechanism of SNAP25 recognition/S-acylation by zDHHC17 was identified. Specifically, the zDABM sequence of Spry2 which is the major interaction site for zDHHC17 was shown to be dispensable for S-acylation. Analysis of SPRED3, which lacks a zDABM sequence, showed that the highly conserved SPR domain contains a novel zDHHC17 binding site that facilitates S-acylation. This thesis also identifies a potential effect of Spry2 on the S-acylation of the SARS-CoV-2 accessory protein, Orf3d, which should be investigated further in future work. Collectively, the findings in this thesis provide important new information on substrate recognition by zDHHC enzymes. This new knowledge can assist in identifying new zDHHC17 substrates and could also be used to develop peptide-based inhibitors that disrupt specific substrate interactions of zDHHC17.
Advisor / supervisor
  • Chamberlain, Luke
Resource Type