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.
Highlights
Post-translational modification (PTM) of proteins enables diversification of function beyond the raw coding capacity of the genome
Using a similar small-molecule approach, we demonstrated that the Toxoplasma gondii orthologue of HsAPT1 (TgPPT1) is the target of a substituted chloroisocoumarin [12] and its inhibition in extracellular T. gondii tachyzoites enhanced their ability to invade host cells, highlighting a critical role for this PTM in establishing the initial point of contact between the parasite and its host cell
We screened a focused library of substituted chloroisocoumarins to identify compounds that inhibited HsAPT1 and HsAPT2 activity
Summary
Post-translational modification (PTM) of proteins enables diversification of function beyond the raw coding capacity of the genome. PTMs alter the functional state of proteins, often serving as switch-like modifications that regulate transduction of intracellular signals [1]. PTMs play integral roles in a range of processes including the maintenance of homeostasis [2], the cellular response to stress [3], and even host-pathogen interactions [4]. Proteins responsible for the addition and removal of PTMs, so-called writers and erasers, regulate the specificity of these modifications.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
