Thesis

Analysis and functional characterisation of the zDHHC9/GCP16 protein complex

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17548
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Abstract
  • S-Acylation is a reversible post-translational modification that affects many proteins, influencing their localisation, interactions, stability and activity. S-Acylation is mediated by 23 zinc finger DHHC enzymes (zDHHCs), which are predominantly catalytically autonomous. However, zDHHC9 requires an accessory protein, GCP16, for S-acylation activity. We hypothesise that interfering with the zDHHC9/GCP16 interaction offers a novel approach for selective inhibition of this enzyme. However, before inhibitors of the zDHHC9/GCP16 complex can be rationally designed, it is important to understand the mechanisms and regulatory consequences of this interaction. This thesis characterises the bidirectional effects of zDHHC9/GCP16 complex formation on the S-acylation and stability of both proteins in mammalian cells, also highlighting critical residues in the binding interfaces at the N- and C-terminal regions of GCP16. The stabilisation of GCP16 required S-acylation by zDHHC9 and, indeed, non-acylated GCP16 mutants were more rapidly degraded by the proteasome; interestingly, the presence of non-acylated cysteines appeared to be linked to GCP16 degradation. Furthermore, comparison of non-acylated GCP16 mutants with either intact cysteines or cysteine-to-alanine substitutions suggested that the cysteine residues in GCP16 are also important for membrane association before S-acylation. This suggests a model where cysteines and surrounding hydrophobic residues initially target GCP16 to the membrane and subsequent S-acylation (perhaps by driving deeper membrane insertion) protects the protein from degradation – this may provide a mechanism to ensure that GCP16 is always complexed with partner zDHHC enzymes by ensuring the rapid degradation of non-complexed protein. Finally, analysis of mutant forms of GCP16 with disrupted interaction with zDHHC9, demonstrated that the formation of an intact zDHHC9/GCP16 complex is critical for dendritic growth in hippocampal neurons. Overall, this study provides a detailed characterisation of the bidirectional regulation of the zDHHC9/GCP16 interaction, providing new insights that can underpin development of selective inhibitors of zDHHC9.
Advisor / supervisor
  • Chamberlain, Luke H

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