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Abstract

Protein S-acylation is a reversible lipid modification that regulates the trafficking, stability, and signaling of neuronal proteins. This process is mediated by a family of 23 protein acyltransferases (PATs), also known as ZDHHC enzymes. Among these, ZDHHC21 is highly expressed in the brain and is unique in that it contains a calmodulin (CaM)-binding site. Previous work has shown that ZDHHC21 binds calmodulin in a calcium-dependent manner and that this interaction enhances its enzymatic activity, raising the possibility that ZDHHC21 may play a role in activity-dependent synaptic plasticity. However, when we examined ZDHHC21-CaM interactions in primary rat cortical neurons, we did not observe increased binding following bicuculline-induced network activity, thapsigargin-mediated ER calcium release, or direct calcium elevation. This contrasts with findings in non-neuronal systems, such as T cells, where calcium release robustly enhances ZDHHC21-CaM binding. Consistent with this, a ΔF233 mutant previously reported to disrupt CaM association in non-neuronal cells retained CaM binding in neurons, suggesting cell-type-specific regulation of this interaction. Given the lack of activity-dependent modulation, we next investigated the basal function of ZDHHC21. Using shRNA-mediated knockdown, we found that loss of ZDHHC21 increased dendritic length and branching, indicating that ZDHHC21 acts as a negative regulator of dendritic growth. Knockdown also induced marked increase in spontaneous calcium bursting. In contrast, overexpression of ZDHHC21 had no effect on dendritic morphology or spontaneous activity. Finally, proximity-dependent biotinylation (TurboID) identified candidate ZDHHC21 interactors and substrates enriched for synaptic scaffolding proteins, trafficking regulators, and signaling molecules. Further work is needed to determine which of these substrates is regulating dendrite outgrowth and neuronal excitability.