Temporal Profiling Establishes a Dynamic S-Palmitoylation Cycle
| Autor: | Brent R. Martin, Sang Joon Won |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Proteomics
0301 basic medicine Proteome G protein Lipoylation GTPase 01 natural sciences Biochemistry Mass Spectrometry Organofluorophosphonates Article 03 medical and health sciences Palmitoylation Humans Enzyme Inhibitors chemistry.chemical_classification 010405 organic chemistry Peripheral membrane protein HEK 293 cells technology industry and agriculture General Medicine 0104 chemical sciences Cell biology Kinetics HEK293 Cells 030104 developmental biology Enzyme chemistry Fatty Acids Unsaturated Molecular Medicine Indicators and Reagents lipids (amino acids peptides and proteins) Thiolester Hydrolases Bioorthogonal chemistry Protein Processing Post-Translational |
| Zdroj: | ACS Chemical Biology. 13:1560-1568 |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/acschembio.8b00157 |
| Popis: | S-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling S-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic S-palmitoylation largely remains a mystery. Here we present a pulsed bioorthogonal S-palmitoylation assay for temporal analysis of S-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies S-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state S-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. S-palmitoylation profiling of APT1(−/−)/APT2(−/−) mouse brains similarly show no change in S-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic S-palmitoylated proteins, raising the possibility that S-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state S-palmitoylation across the proteome. |
| Databáze: | OpenAIRE |
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