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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Chamberlain LH, Shipston MJ (2015) The physiology of protein S-acylation. Physiol Rev 95:341–376
Fukata Y, Fukata M (2010) Protein palmitoylation in neuronal development and synaptic plasticity. Nat Rev Neurosci 11:161–175
Linder ME, Deschenes RJ (2007) Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8:74–84
El-Husseini AE, Schnell E, Chetkovich DM et al (2000) PSD-95 involvement in maturation of excitatory synapses. Science 290:1364–1368
Topinka JR, Bredt DS (1998) N-terminal palmitoylation of PSD-95 regulates association with cell membranes and interaction with K+ channel, Kv1.4. Neuron 20:125–134
Craven SE, El-Husseini AE, Bredt DS (1999) Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22:497–509
Webb Y, Hermida-Matsumoto L, Resh MD (2000) Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275:261–270
El-Husseini AE, Schnell E, Dakoji S et al (2002) Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108:849–863
Fukata Y, Dimitrov A, Boncompain G et al (2013) Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J Cell Biol 202:145–161
Noritake J, Fukata Y, Iwanaga T et al (2009) Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95. J Cell Biol 186:147–160
Fukata M, Fukata Y, Adesnik H et al (2004) Identification of PSD-95 palmitoylating enzymes. Neuron 44:987–996
Fukata Y, Iwanaga T, Fukata M (2006) Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells. Methods 40:177–182
Camp LA, Hofmann SL (1993) Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J Biol Chem 268:22566–22574
Duncan JA, Gilman AG (1998) A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J Biol Chem 273:15830–15837
Yeh DC, Duncan JA, Yamashita S et al (1999) Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin. J Biol Chem 274:33148–33154
Martin BR, Wang C, Adibekian A et al (2012) Global profiling of dynamic protein palmitoylation. Nat Methods 9:84–89
Yokoi N, Fukata Y, Sekiya A et al (2016) Identification of PSD-95 depalmitoylating enzymes. J Neurosci 36:6431–6444
Howie J, Reilly L, Fraser NJ et al (2014) Substrate recognition by the cell surface palmitoyl transferase DHHC5. Proc Natl Acad Sci U S A 111:17534–17539
Percher A, Ramakrishnan S, Thinon E et al (2016) Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation. Proc Natl Acad Sci U S A 113:4302–4307
Percher A, Thinon E, Hang H (2017) Mass-tag labeling using acyl-PEG exchange for the determination of endogenous protein S-fatty acylation. Curr Protoc Protein Sci 89:14.17.11–14.17.11
Hurst CH, Turnbull D, Plain F et al (2017) Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods. BioTechniques 62:69–75
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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