Cysteine-string Protein (CSPα) has been linked to neurodegeneration and mutations in CSPα are known to cause Adult-onset Ceroid Lipofuscinosis (ANCL). CSPα's normal function relies on its targeting to synaptic vesicles by palmitoylation - the attachment offatty acids to cysteine residues. Palmitoylation is mediated by several Palmitoyl-Transferase (PAT) enzymes, such as zDHHC3, zDHHC7 and zDHHC17. zDHHC17 bindsto all its known substrates such as, CSPα through its ankyrin repeat domain by recognising CSPα's zDHHC ankyrin-repeat binding motif (zDABM), which contains the consensus sequence ΨβXXQP, where proline-175 is the central residue for CSPα's association. Despite the known requirement of the zDABM for CSPα binding to zDHHC17,the importance of the zDABM for palmitoylation has not yet been demonstrated.Therefore, this investigation determined how disruption of the zDABM affected palmitoylation of CSPα. Furthermore, PAT co-expression with CSPα has been shown to increase aggregation in ANCL mutants in vitro; therefore, we also aimed to determine the importance of the zDABM in the aggregation of ANCL mutant CSPα. in-silico methods were also used to predict changes in post-translational modifications (PTMs), and changes in the monomeric and homodimeric structure of CSPα caused by ANCL mutations. It was found that disruption of the zDABM sequence led to a zDHHC17-dependent increase in the levels of both palmitoylated and non-palmitoylated CSPα.Moreover, disruption of the zDABM sequence had no influence on the aggregation of ANCL mutants. A method for solubilisation of the CSPα aggregates using Triton-X100lysis and Sodium Dodecyl Sulfate (SDS) incubation was identified; however, further investigation of the solubilised aggregates was not possible due to restrictions on laboratory work. Structural predictions of CSPα identified that proline-175 in the zDABM sequence directly interacts with L115. Furthermore, this interaction, in addition to the structure and localisation of the zDABM sequence is considerably compromised in theANCL mutants. Structural homodimer predictions also found altered dimer formation fo rthe L115R mutant that could provide a mechanism of aggregation that is independent of palmitoylation. Collectively, these analyses have uncovered potential novel functions of proline-175 in the zDABM of CSPα, which may be relevant to both the expression of the protein and its aggregation in ANCL.
|Date of Award||23 Apr 2021|
- University Of Strathclyde
|Supervisor||Luke Chamberlain (Supervisor)|