Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay
| Autor: | Yuko Fukata, Norihiko Yokoi, Masaki Fukata, Takashi Kanadome |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Scaffold protein Chemistry technology industry and agriculture Serine hydrolase 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Enzyme Thioesterase Biochemistry Palmitoylation Hydrolase lipids (amino acids peptides and proteins) Lipid modification Gene 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493995318 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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