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

Area of Science:

• Biochemistry
• Cell Biology
• Parasitology

Background:

• Protein palmitoylation is a vital post-translational modification (PTM) regulating protein stability, trafficking, and interactions.
• S-palmitoylation, catalyzed by palmitoyl acyl transferases (PATs), involves adding palmitate to cysteine residues.
• Acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs) remove palmitate, making them key regulators of this PTM.

Purpose of the Study:

• To develop and apply a cell-permeable activity-based probe (ABP) targeting APTs in mammalian cells and *Toxoplasma gondii*.
• To investigate the activity of human APTs (HsAPTs) and identify a potential APT in *T. gondii*.
• To assess the relationship between APT activity and total protein concentration in different cell lines.

Main Methods:

• Synthesis of a focused library of substituted chloroisocoumarins to identify APT-targeting scaffolds.
• Development of a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT), with nanomolar affinity for HsAPT1 and HsAPT2.
• In situ profiling of HsAPT activity in mammalian cells and detection of an APT in *T. gondii* (TgPPT1) using JCP174-BT.

Main Results:

• Identification of a chloroisocoumarin scaffold with high affinity for human APTs.
• Successful synthesis and application of the fluorescent ABP, JCP174-BT.
• Demonstration of JCP174-BT's utility in profiling APT activity in mammalian cells and detecting an APT in *T. gondii*.
• Observation of discordance between HsAPT activity and total protein levels in certain cell lines.

Conclusions:

• The developed ABP, JCP174-BT, is a valuable tool for studying APTs in various biological systems.
• APT activity levels may not be accurately reflected by total protein concentration, emphasizing the need for activity-based probes.
• This research provides insights into protein palmitoylation regulation and its potential targets in parasites.