Background: The homeostasis of palmitoylation and depalmitoylation is involved in various cellular processes, the disruption of which induces severe physiological consequences. Acyl-protein thioesterase (APT) and palmitoyl-protein thioesterases (PPT) catalyze the depalmitoylation process. The natural mutation in human PPT1 caused neurodegenerative disease, yet the understanding of APT1 remains to be elucidated. While the deletion of APT1 in mice turned out to be potentially embryonically lethal, the decoding of its function strictly relied on the identification of its substrates. Objective: To determine the potential substrates of APT1 by using the generated human APT1 knockout cell line. Methods : The combined techniques of palmitoyl-protein enrichment and massspectrometry were used to analyze the different proteins. Palmitoyl-proteins both in HEK293T and APT1-KO cells were extracted by resin-assisted capture (RAC) and data independent acquisition (DIA) quantitative method of proteomics for data collection. Results: In total, 382 proteins were identified. The gene ontology classification segregated these proteins into diverse biological pathways e.g. endoplasmic reticulum process and ubiquitin-mediated proteolysis. A few potential substrates were selected for verification; indeed, major proteins were palmitoylated. Importantly, their levels of palmitoylation were clearly changed in APT1-KO cells. Interestingly, the proliferation of APT1-KO cells escalated dramatically as compared to that of the WT cells, which could be rescued by APT1 overexpression. Conclusion: Our study provides a large scale of potential substrates of APT1, thus facilitating the understanding of its intervened molecular functions.

The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.