Palmitoylation in neurodegeneration, analysis of cysteine-string protein mutants linked with neuronal ceroid lipofuscinosis

Neuronal Ceroid Lipofuscinoses (NCLs) are neurodegenerative lysosomal-storage disorders characterized by intracellular accumulation of autofluorescent material. Mutations in the DNAJC5 gene encoding Cysteine-String Protein alpha (CSPα) cause autosomal-dominant adult-onset NCL (ANCL). The disease-causing mutations occur within the cysteine-stringdomain (CSD), a region of the protein that is extensively palmitoylated. It has been shown that the ANCL CSPα mutants form aggregates and that this is dependent on palmitoylation. As aggregation is a common feature of neurodegenerative disorders, aggregates formed in ANCL are likely to contribute, together with loss-of-function effects, to disease pathology. The aims of this project were to: (i) investigate features of CSPα that are important for aggregate formation; (ii) identify molecular changes that occur in ANCL; and (iii) identify mechanisms regulating CSPα turnover to provide insight into pathways that might be perturbed in ANCL. A cluster of palmitoylated cysteines in the CSD were identified as essential for aggregation of the ANCL CSPα mutants, further supporting an association between palmitoylation and aggregation. Analysis of the expression levels of different proteins in post-mortem brain revealed a massive increase in expression of the palmitoyl thioesterase enzyme PPT1 in ANCL samples, which is intriguing as PPT1 functions to depalmitoylate proteins during their degradation and mutations in the PPT1 gene cause infantile NCL. Further analysis identified several other proteins that had altered expression levels including α-synuclein. Degradation of both wild-type and mutant CSPα proteins was mediated by the proteasome but was independent of lysine ubiquitination. This pathway mediated rapid degradation of non-palmitoylated protein, and blocking proteasome activity enhanced the formation of mutant aggregates. It will be important in future work to determine how mutations in CSPα affect protein degradation pathways to cause the characteristic NCL lysosomal morphology, and whether there is a role for the lysosome in degradation of palmitoylated or aggregated CSPα.Neuronal Ceroid Lipofuscinoses (NCLs) are neurodegenerative lysosomal-storage disorders characterized by intracellular accumulation of autofluorescent material. Mutations in the DNAJC5 gene encoding Cysteine-String Protein alpha (CSPα) cause autosomal-dominant adult-onset NCL (ANCL). The disease-causing mutations occur within the cysteine-stringdomain (CSD), a region of the protein that is extensively palmitoylated. It has been shown that the ANCL CSPα mutants form aggregates and that this is dependent on palmitoylation. As aggregation is a common feature of neurodegenerative disorders, aggregates formed in ANCL are likely to contribute, together with loss-of-function effects, to disease pathology. The aims of this project were to: (i) investigate features of CSPα that are important for aggregate formation; (ii) identify molecular changes that occur in ANCL; and (iii) identify mechanisms regulating CSPα turnover to provide insight into pathways that might be perturbed in ANCL. A cluster of palmitoylated cysteines in the CSD were identified as essential for aggregation of the ANCL CSPα mutants, further supporting an association between palmitoylation and aggregation. Analysis of the expression levels of different proteins in post-mortem brain revealed a massive increase in expression of the palmitoyl thioesterase enzyme PPT1 in ANCL samples, which is intriguing as PPT1 functions to depalmitoylate proteins during their degradation and mutations in the PPT1 gene cause infantile NCL. Further analysis identified several other proteins that had altered expression levels including α-synuclein. Degradation of both wild-type and mutant CSPα proteins was mediated by the proteasome but was independent of lysine ubiquitination. This pathway mediated rapid degradation of non-palmitoylated protein, and blocking proteasome activity enhanced the formation of mutant aggregates. It will be important in future work to determine how mutations in CSPα affect protein degradation pathways to cause the characteristic NCL lysosomal morphology, and whether there is a role for the lysosome in degradation of palmitoylated or aggregated CSPα

Substrate selectivity in the zDHHC family of S-acyltransferases

S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results in anchoring of soluble proteins to membrane compartments or in the trafficking of membrane proteins to different compartments. Recent work has shown that although S-acylation of some proteins may involve very weak interactions with zDHHC enzymes, a pool of zDHHC enzymes exhibit strong and specific interactions with substrates, thereby recruiting them for S-acylation. For example, the ankyrin-repeat domains of zDHHC17 and zDHHC13 interact specifically with unstructured consensus sequences present in some proteins, thus contributing to substrate specificity of these enzymes. In addition to this new information on zDHHC enzyme protein substrate specificity, recent work has also identified marked differences in selectivity of zDHHC enzymes for acyl-CoA substrates and has started to unravel the underlying molecular basis for this lipid selectivity. This review will focus on the protein and acyl-CoA selectivity of zDHHC enzymes

A cluster of palmitoylated cysteines are essential for aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis

Autosomal-dominant adult-onset neuronal cero id lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSP α ). The disease- causing mutations, which result in substituti on of leucine-115 with an arginine (L115R) or deletion of the neig hbouring leucine-116 ( Δ L116) in the cysteine-string domain cause CSP α 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-str ing domain, however the regions of the mutant proteins that drive aggregatio n have not been determined. The importance of specific residues in the cysteine-string domain was in vestigated, revealing that a central core of palmitoylated cysteines is essential for aggregation of ANCL CSP α mutants. Interestingly, palmitoylated monomers of ANCL CSP α mutants were shown to be short-lived compared with wild-type CSP α, suggesting that the mutants eith er 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 CSP α that promote aggregation in the presence of L115R/ Δ L116 mutations and reveal a change in the lifetime of palmitoyla ted monomers of the mutant proteins

The zDHHC family of S-acyltransferases

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology