S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.-acylation is a reversible posttranslational modification (PTM) involving the attachment of fatty acids onto cysteines (1, 2). This PTM occurs on both soluble and transmembrane (TM) proteins and exerts a number of important effects, including mediating membrane binding (of soluble proteins or soluble loops of TM proteins), regulating protein trafficking and targeting to cholesterol-rich membrane microdomains, and modulating protein stability (3, 4). These actions of S-acylation on a diverse array of cellular proteins affect many important physiological pathways, and defects in this process are linked to a number of major diseases and disorders (2, 5).S-acylation is mediated by the opposing actions of acyltransferases and thioesterases. S-acyltransferase enzymes belong to the zinc finger DHHC domain containing (zDHHC) protein family, which are encoded by 24 distinct genes (6-8). zDHHC enzymes are thought to share the same overall membrane topology, with four to six transmembrane domains and the N and C termini present in the cytosol (9). The catalytic DHHC cysteinerich domain (CRD) of the enzymes lies in a cytosolic loop (9), allowing zDHHC enzymes to modify substrate cysteines present at the cytosol-membrane interface. The S-acylation reaction is thought to proceed through an enzyme-acyl intermediate, where the acyl chain is attached to the cysteine of the DHHC motif via a thioester linkage (often referred to as enzyme "autoacylation") (10, 11). The S-acyl chain is then transferred to a cysteine residue of a substrate protein (10,11). This overall process is referred to as a "ping-pong" reaction mechanism. There has been progress in identifying the zDHHC enzymes that are active against many substrate proteins (2), although we lack a detailed understanding of the protein substrate profiles of individual enzymes and how enzyme-substrate interaction specificity is achieved. Coexpression experiments have suggested that individual zDHHC enzymes might exhibit a level of overlap in their ...

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.

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Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.

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