Dynamic S-acylation, the post-translational reversible attachment of fatty acids ontocysteine amino acids of proteins, allows for the dynamic control of protein membraneaffinity and function. In humans, the addition of fatty acids onto substrate proteins iscatalysed by 23 ‘DHHC’ S-acyltransferases and reversed by acyl-protein thioesterases(APTs). Whilst twenty-one enzymes from the metabolic serine hydrolase superfamily havebeen identified as putative APTs, only seven have been confirmed as deacylases. SNAP25(synaptosomal-associated protein of 25 kDa) are a family of SNARE (solubleN-ethylmaleimide sensitive factor attachment protein receptor) proteins essential forintracellular membrane fusion and exocytosis. Dynamic S-acylation of SNAP25 within itscentral cysteine rich domain (CRD) regulates SNAP25 membrane attachment, subcellularlocalisation and SNARE complex association; however, how SNAP25 deacylation isregulated is unknown. Using bioorthogonal labelling and click chemistry to analyseS-acylation dynamics, and fractionation approaches to measure membrane association, thiswork identifies α/β-hydrolase domain (ABHD)-containing metabolic serine hydrolasesresponsible for deacylating SNAP25 family isoforms. While ABHD16A is capable ofpromoting the deacylation and solubilisation of all SNAP25 family isoforms, ABHD13 isonly active against SNAP25b, the most abundant isoform in adult brains. Furthermore,using a mutagenesis approach, the molecular basis for ABHD13 selectivity against SNAP25isoforms was identified. ABHD13 was shown to be selective for SNAP25a/SNAP23mutants that mirror the phenylalanine-cysteine configuration of the SNAP25b CRD.Conversely, introducing a cysteine-cysteine motif in SNAP25b to mirror the CRD ofSNAP25a/SNAP23 prevented ABHD13-mediated deacylation. Further characterisationdetermined that ABHD16A activity was higher than ABHD13 under similar conditions.Moreover, subsequent experiments demonstrated that modulating ABHD16A expression,but not ABHD13, regulated SNAP25b S-acylation dynamics in PC12 cells, suggesting thatABHD13 could play a specialised role in SNAP25 deacylation. Collectively, these resultsxviiirepresent the first description of APT selectivity at the amino acid level in full lengthproteins and this new knowledge may assist in the identification of further ABHD13/16Asubstrates in the future
Identification and characterisation of ABHD13 and ABHD16A as novel deacylases of the SNAP25 family of proteins
Mejuto Miranda, L. (Author). Aug 2024
Student thesis: Doctoral Thesis › Doctor of Philosophy