Post-translational modifications play a crucial role in regulating the function of many biological molecules. Lipid modifications, in particular, via prenylation, acylation, myristoylation and palmitoylation help to direct membrane localization, protein-protein interactions, cell signaling, subcellular trafficking and vesicle transport. Among them, palmitoylation is the only reversible process. This modification consists in the addition of a palmitic acid forming a thioester bond with cysteine residues of both soluble and membrane proteins. The thioester linkage is hydrolyzed by acyl protein thioesterase enzymes (APTs), which belong to the α/β hydrolase family of serine hydrolases. The first soluble thioesterase to be characterized was the human APT1, which catalyzes depalmitoylation of signaling regulators like Gα and H/N-Ras. This structure revealed a non-symmetric homodimeric organization where the catalytic pocket is occluded by the dimer interface. The human APT1 is itself palmitatoylated in position 2 and acts as substrate for itself. Recently, we have solved new X-ray structures for the wild-type and the catalytic inactive form (S119A) of human APT1, which reveal (i) a dimeric interface suggesting a novel mode of interaction with the biological membrane, and (ii) a previously unseen electron density adjacent to the catalytic pocket that allowed to study the enzyme-substrate adduct via molecular simulation. Moreover, this new evidence suggested the existence of a druggable pocket that can favorably accommodate a variety of small molecule compounds as observed from a molecular docking campaign on several libraries. We are currently testing in vitro the most promising compounds from this in silico screening to identify high affinity hits able to inhibit human APT1.

New insight into the catalytic and inhibition mechanism of the human acyl protein thioesterase

251st National Meeting (2016)

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