Abstract
Palmitoylation is a reversible posttranslational lipid modification of proteins involved in a wide range of cellular functions. More than a thousand proteins are estimated to be palmitoylated. In neurons, PSD-95, a major postsynaptic scaffold protein, requires palmitoylation for its specific accumulation at the synapse and dynamically cycles between palmitoylated and depalmitoylated states. Although palmitoylating enzymes of PSD-95 have been well characterized, little is known about the depalmitoylating enzymes (e.g., thioesterases for palmitoylated PSD-95). An elegant pharmacological analysis has suggested that subsets of α/β hydrolase domain (ABHD)-containing proteins of the metabolic serine hydrolase superfamily involve thioesterases for palmitoylated proteins. Here, we describe a systematic method to screen the ABHD serine hydrolase genes, which unveiled ABHD17 as the depalmitoylating enzyme for PSD-95. Furthermore, we introduce the acyl-PEGyl exchange gel-shift (APEGS) method that enables quantification of palmitoylation levels/stoichiometries on proteins in various biological samples and can be used to monitor the dynamic depalmitoylation process of proteins.
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Acknowledgments
We thank previous lab members Atsushi Sekiya and Tatsuro Murakami for their scientific contribution. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (Grant numbers 17K14969 to N.Y.; 15H04279 to Y.F; 16H01371, 16K14560, 17H03678, 17H05709, 18H04873 to M.F.); the Ministry of Health, Labour and Welfare (Intramural Research Grant [H27-7] for Neurological and Psychiatric Disorders to Y.F.); Takeda Science Foundation to Y.F and M.F.; The Japan Epilepsy Research Foundation and The Kato Memorial Trust for Nambyo Research to Y.F.; and The Naito Foundation and The Hori Sciences and Arts Foundation to M.F.
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Kanadome, T., Yokoi, N., Fukata, Y., Fukata, M. (2019). Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay. In: Linder, M. (eds) Protein Lipidation. Methods in Molecular Biology, vol 2009. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9532-5_7
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DOI: https://doi.org/10.1007/978-1-4939-9532-5_7
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