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Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii
Authors:Louise E. Kemp  Marion Rusch  Alexander Adibekian  Hayley E. Bullen  Arnault Graindorge  Céline Freymond  Matthias Rottmann  Catherine Braun-Breton  Stefan Baumeister  Arthur T. Porfetye  Ingrid R. Vetter  Christian Hedberg  Dominique Soldati-Favre
Abstract:
In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.
Keywords:Enzyme Inhibitors   Hydrolases   Parasitology   Posttranslational Modification   Protein Palmitoylation   Acyl-protein Thioesterase   Plasmodium falciparum   Serine Hydrolase   Toxoplasma gondii   Inhibitor
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