Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Abstract

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively re-distribute S-palmitoylated proteins towards different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The de-palmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

APT2 Inhibition Restores Scribble Localization and S -Palmitoylation in Snail-Transformed Cells

“…This inhibitor concentration is ~4-times higher than the K i , suggesting ML349 is ready cell permeable and achieves equivalent inhibition in vitro and in living cells. ML348 had no significant effect on Scrib membrane localization, similar to an inactive ML349 derivative where the sulfone is reduced (Won et al, 2016). Importantly, Palmostatin B does not enhance Scrib perimeter localization to a higher level than ML349.…”

“…Both APT1 and APT2 are annotated as protein de-palmitoylases (Tom and Martin, 2013), and are widely expressed and active across nearly all tissues (Bachovchin et al, 2010). Both enzymes are targets of several classes of covalent serine hydrolase inhibitors, including β-lactones (Dekker et al, 2010), triazole ureas (Adibekian et al, 2010c), and N -hydroxyhydantoin carbamates (Cognetta et al, 2015), as well as the reversible isoform selective piperazine amide inhibitors ML348 and ML349 (Adibekian et al, 2012; Davda and Martin, 2014; Won et al, 2016). The β-lactone inhibitor Palmostatin B is the most widely used APT inhibitor, and is primarily selective for both APT1 and APT2 at low micromolar concentrations.…”

The role of S‐acylation in protein trafficking

“…[36][37][38][39] The crystal structures of APT1 and APT2 have been determined, revealing an architecture containing a central structure made up of parallel β sheets, connected by loops and surrounded by α-helices. 38,40 Experimental evidence obtained in various laboratories (including ours) has indicated that both enzymes are mainly cytosolic with a highly hydrophilic character. 25,26,41,42 Recently, APT1 and APT2 were found to undergo palmitoylation on cysteine 2, [43][44][45] which was suggested to facilitate the steady-state membrane localization and function of these thioesterases.…”

“…APT1 and APT2 contain a catalytic triad made up of serine, histidine and aspartic acid, and also a glycine‐X‐serine‐X‐glycine motif, which is characteristic of the large family of α/β hydrolases and serine hydrolases . The crystal structures of APT1 and APT2 have been determined, revealing an architecture containing a central structure made up of parallel β sheets, connected by loops and surrounded by α‐helices . Experimental evidence obtained in various laboratories (including ours) has indicated that both enzymes are mainly cytosolic with a highly hydrophilic character .…”

“…Particularly, it will allow exploring if the manipulation of protein palmitoylation represents an opportunity to fight diseases in which acylation or its misregulation is involved. In this sense, greater advances have been obtained for APTs than for PATs, with the recent discovery of the human APT1 and APT2 crystal structures, as well as isoform‐selective inhibitors, which have provided new ways to probe the function of each enzyme . More importantly, these pharmacological approaches represent a substantial advance for the development of rational therapies to control the oncogenic pathways driven by acylated proteins, as recently described for H‐ and N‐Ras, and also for the Scrib tumor suppressor in Snail‐overexpressing epithelial cells, in which a common feature of the inhibitors was to modulate the membrane binding and intracellular distribution of the target protein.…”