Abstract
Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSPα). The disease-causing mutations, which result in substitution of leucine-115 with an arginine (L115R) or deletion of the neighbouring leucine-116 (∆L116) in the cysteine-string domain cause CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the cysteine-string domain, however the regions of the mutant proteins that drive aggregation have not been determined. The importance of specific residues in the cysteine-string domain was investigated, revealing that a central core of palmitoylated cysteines is essential for aggregation of ANCL CSPα mutants. Interestingly, palmitoylated monomers of ANCL CSPα mutants were shown to be short-lived compared with wild-type CSPα, suggesting that the mutants either have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates. These findings provide new insight into the features of CSPα that promote aggregation in the presence of L115R/∆L116 mutations and reveal a change in the lifetime of palmitoylated monomers of the mutant proteins.
Highlights
An increased aggregation of the adult-onset neuronal ceroid lipofuscinosis (ANCL) CSPα mutants compared with wild-type protein was seen with bacterially-produced recombinant proteins, which lack palmitoyl modifications[13], it is unclear if these aggregates/oligomers are the same as those formed from palmitoylated proteins in cells
We previously proposed that palmitoylated ANCL CSPα mutants present within aggregates may be inaccessible to PPT1 and that the resulting deficit in degradative protein depalmitoylation could be the trigger for this lysosomal-storage disorder[6]
To explore the aggregation process further, we have examined how specific palmitoylated cysteines contribute to this process by generating and analysing a panel of ANCL CSPα mutants bearing specific cysteine-to-alanine substitutions
Summary
An increased aggregation of the ANCL CSPα mutants compared with wild-type protein was seen with bacterially-produced recombinant proteins, which lack palmitoyl modifications[13], it is unclear if these aggregates/oligomers are the same as those formed from palmitoylated proteins in cells. Levels of the lysosomal thioesterase enzyme PPT1 (which removes acyl chains from palmitoylated proteins during their degradation) were recently shown to be markedly increased in brain samples from ANCL patients[15], further supporting a link between palmitoylation and ANCL. We previously proposed that palmitoylated ANCL CSPα mutants present within aggregates may be inaccessible to PPT1 and that the resulting deficit in degradative protein depalmitoylation could be the trigger for this lysosomal-storage disorder[6]. Given the previous identified links between palmitoylation and aggregation[6], this study has focused on the importance of specific cysteines in the CSD for aggregation
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