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PLoS One. 2018 Jan 24;13(1):e0190255. doi: 10.1371/journal.pone.0190255. eCollection 2018.

Development of an activity-based probe for acyl-protein thioesterases.

Author information

1
Cancer Biology Program, Stanford University School of Medicine, Stanford, California, United States of America.
2
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
3
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, United States of America.
4
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Abstract

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.

PMID:
29364904
PMCID:
PMC5783350
DOI:
10.1371/journal.pone.0190255
[Indexed for MEDLINE]
Free PMC Article

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