Investigating the mechanisms and outcomes of Sprouty-2 S-acylation

  • Carolina Locatelli

Student thesis: Doctoral Thesis

Abstract

S-acylation is a reversible post-translational modification (PTM) that regulates proteins in several ways, including by modulating trafficking, stability and protein interactions. This PTM is mediated by twenty-three zinc finger DHHC (zDHHC) enzymes that display distinct activity and substrate selectivity profiles. Sprouty (SPRY) proteins are regulators of Receptor Tyrosine Kinase (RTK) signalling and play a major role in maintaining cellular homeostasis. Indeed, their deregulation is linked to tumorigenesis, developmental defects and neurological diseases.;The defining feature of SPRY proteins is a large cysteine-rich domain (CRD), which is modified by S-acylation. In this thesis, cell-based and cell-free assays, extensive mutagenesis analysis and confocal imaging techniques were employed to examine the mechanisms and outcomes of SPRY2 S-acylation. The results presented show that SPRY2 is differentially modified by zDHHC enzymes: zDHHC17 displayed apparent selectivity for Cys-265/268, whereas zDHHC7 and zDHHC3 mediated more expansive S-acylation of the CRD. S-acylation mediated by both zDHHC17 and zDHHC7 required the presence of a novel NDK (Asn-Asp-Lys) motif in the CRD.;Regardless of the modifying enzyme, S-acylation correlated with increased levels of SPRY2 protein, which is linked to an increase in SPRY2 stability. Furthermore, confocal microscopy revealed that S-acylation is also required for efficient plasma membrane targeting. The interaction between SPRY2 and zDHHC17 was further explored and although SPRY2 interacted with the ankyrin repeat (ANK) domain of zDHHC17 through a canonical mechanism, this mode of binding was dispensable for S-acylation. Binding assays suggested the presence of an additional lower affinity interaction site, which might involve amino acid regions 155-290 and/or 106-120 of SPRY2.;Finally, evidence is presented that zDHHC17 forms dimers or higher-order oligomers, which involve ANK-ANK interactions. It is speculated that the formation of these zDHHC17 oligomers might be relevant to the function of the enzyme. Overall, this thesis highlights that S-acylation regulates multiple aspects of SPRY2, including protein stability and plasma membrane targeting. As expression levels and localisation are two fundamental properties of a protein and because alterations in these properties can cause disease, we propose that targeting SPRY2 S-acylation might represent a novel therapeutic strategy in cancer and other diseases.
Date of Award29 Apr 2021
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorLuke Chamberlain (Supervisor) & Christian Wozny (Supervisor)

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