S-acylation is a post-translational modification that can regulate protein localization, function, and turnover by reversible attachment of fatty acid to specific cysteine residues. It affects over 20% of proteins, with membrane proteins making up the largest group, followed by cytosolic, ciliary, and nuclear proteins.
This process is mediated by a family of 23 ZDHHC enzymes, which exhibit varying expression levels and substrate specificities. The studies on ZDHHCs conducted so far have mostly been performed in cell cultures, and no comprehensive study has examined all ZDHHCs at the organismal level. Addressing this gap was the first objective of our study.
We chose to study zdhhcs expression during early zebrafish development. Initially, we analyzed zdhhc spatial expression by in situ mRNA hybridization and reanalysed scRNA-seq data to classify zdhhcs by cell type. We then focused on a subset of zdhhcs, about one-third of which were expressed in the inner ear, an organ enriched in cilia. Downregulation of some of these genes resulted in phenotypes suggestive of cilia defects, leading us to hypothesize that ZDHHCs might play a role in cilia function or biogenesis.
To test our hypothesis, we chose hair cells in the zebrafish inner ear, which are an established model for studying primary cilia. Among cilia-related zdhhcs we concentrated on zdhhc4, which showed the most significant cilia-related phenotypes upon knockdown. Using light-sheet microscopy on transgenic zebrafish with fluorescent hair cells, we observed altered hair cell numbers without major morphological defects upon zdhhc4 knockdown, indicating zdhhc4's potential role in ciliated cell biogenesis. The inability to obtain a zdhhc4 knockout zebrafish and cell line, suggesting its lethality, further highlighted its essential role in cellular function.
We then explored signaling pathways potentially regulated by zdhhc4 by examining cilia-related gene expression patterns upon zdhhc4 knockdown. Misexpression of atoh1a and spaw, similar to that seen upon knockdown of arl13b, which encodes an S-acylated protein crucial to ciliogenesis, led us to hypothesize that ARL13B might be a ZDHHC4 target. Interestingly, functional experiments in zebrafish suggested that there might be more targets for ZDHHC4.
To rigorously identify ZDHHC4 targets, we developed an approach combining zebrafish and human omics data. This approach identified eight proteins, including RFX3 and ARL13B, as potential ZDHHC4 targets. We confirmed RFX3 and ARL13B S-acylation in human RPE-1 cells and identified ZDHHC2 and ZDHHC4 as enzymes responsible for their S-acylation.
In summary, zdhhcs exhibit tissue-specific expression, with many concentrated in the nervous system and some in the inner ear, where cilia are abundant. We identified ZDHHC4 as an enzyme that may play an important role in primary cilia biogenesis. Finally, our approach revealed key cilia biogenesis regulators, RFX3 and ARL13B, as direct targets of ZDHHC4. The experiments in human cell lines confirmed their S-acylation by ZDHHC4 as well as by another cilia-specific enzyme, ZDHHC2.