AbstractS-Palmitoylation is the reversible addition of 16-carbon fatty acids onto cysteine residues of proteins. The functional consequences of palmitoylation are diverse and it has been demonstrated to play a critical role in several physiological and disease pathways. DHHC palmitoyl acyl transferase (DHHC-PAT) enzymes are responsible for catalysing the addition of acyl groups onto cysteine residues and there are 23 human DHHC-PAT isoforms. All isoforms share a conserved DHHC catalytic motif and they display heterogeneity in their N- and C- termini. This coupled with differing sub-cellular localisations is thought to be the basis of substrate specificity.
DHHC5 is a cell surface localised DHHC-PAT which has a characteristic, long cytoplasmic C-tail which is responsible for substrate recruitment. DHHC5 has an established role in hippocampal learning and memory formation. It is also known to palmitoylate phospholemman (PLM), thereby regulating cardiac sodium pump activity. However, the regulatory mechanisms governing substrate recruitment by DHHC5 are not understood and it is thought that there are more, currently unidentified DHHC5 substrates. The aims of this project were to i) identify the PLM binding site on the DHHC5 C-tail and investigate how PLM recruitment to DHHC5 is regulated, and ii) develop and use a peptide-based array to identify novel DHHC5 substrates.
A small peptide-based array identified a PLM binding site on the DHHC5 C-tail, positioned just after the final transmembrane domain. Further interrogation of this region revealed that DHHC5 is palmitoylated on its C-tail at a di-cysteine motif situated close to the PLM binding site at residues 236/237. Over-expression of DHHC20 increased palmitoylation of DHHC5 in HEK293 cells, but not when the di-cysteine motif was mutated. DHHC20 overexpression in HEK293 cells also increased coimmunoprecipitation of PLM with DHHC5 and again the di-cysteine motif was required for this. Therefore, it was concluded that DHHC20 palmitoylates DHHC5 at a di-cysteine motif on its C-tail, and regulates recruitment of PLM to DHHC5.
A peptide-based array covering the entire DHHC5 C-tail was developed and used to purify interacting proteins from rat heart and mouse cerebellum tissue lysates. Mass spectrometry (LC-MS/MS) was used to identify DHHC5 interacting proteins and a stringent filtering process identified high-confidence putative DHHC5 substrates. Several candidate proteins identified by LC-MS/MS were tested using a validation protocol, resulting in the identification of two novel DHHC5 substrates; FAT/CD36 and Gai2. Palmitoylation of GFP-tagged FAT/CD36 and Gai2 was reduced in DHHC5 KO cells compared to HEK293 cells. This was rescued by overexpression of DHHC5 but not DHHC17 in DHHC5 KO cells, confirming these as DHHC5 substrates.
FAT/CD36 is responsible for the majority of long chain fatty acid uptake in several tissues and has particular importance in the heart. Further investigation of FAT/CD36 as a DHHC5 substrate revealed that palmitoylation by DHHC5 does not contribute to cell surface localisation of FAT/CD36. FAT/CD36 activity was measured indirectly by using Oil Red-O staining to determine the intracellular lipid content. There was significantly less Oil Red-O staining in DHHC5 KO cells overexpressing FAT/CD36 compared to HEK 293 cells and this was rescued by overexpression of DHHC5 but not DHHC17 in the KO cells. Therefore, it was concluded that palmitoylation of FAT/CD36 by DHHC5 increases FAT/CD36 mediated fatty acid uptake.
Furthermore, insulin has been shown to increase DHHC5 C-tail palmitoylation in adult rat ventricular myocytes. This suggests that insulin may regulate palmitoylation of DHHC5 by DHHC20 and therefore recruitment of FAT/CD36 to DHHC5. However, the mechanism for this remains unclear. FAT/CD36 palmitoylation was elevated in a rat model of type 2 diabetes, which may contribute to increased fatty acid uptake and lipotoxicity in the diabetic heart.
Overall, this project has identified novel regulatory mechanisms governing substrate recruitment by DHHC5. Furthermore, two novel DHHC5 substrates have been identified by a peptide-based array of the DHHC5 C-tail. A novel insulin-DHHC signalling axis has been identified which may be responsible for regulating FAT/CD36 palmitoylation and therefore fatty acid uptake.
|Date of Award||2018|
|Sponsors||British Heart Foundation|
|Supervisor||Niall Fraser (Supervisor), Tim Hales (Supervisor) & Will Fuller (Supervisor)|