J Biol Chem. 2013 Aug 2. [Epub ahead of print]

Characterisation of a serine hydrolase targeted by acyl protein thioesterase inhibitors in Toxoplasma gondii

Kemp LE, Rusch M, Adibekian A, Bullen HE, Graindorge A, Freymond C, Rottmann M, Braun-Breton C, Baumeister S, Porfetye AT, Vetter IR,Hedberg C, Soldati-Favre D.

Source

University of Geneva, Switzerland;

Abstract

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification impacting on 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 Palmitoyl Acyl Transferases (PATs). Dynamic palmitoylation cycles require the action of an Acyl Protein Thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this post-translational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of beta-lactone or triazole urea-based inhibitors that target human APT1 show 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, was identified as the closest homologue to human APT1 and APT2 and characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a similar specificity to APTs and homology modelling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the beta-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 resistant to gene disruption due either to an experimental failure or to the essential nature of the protein.

KEYWORDS:

Acyl protein thioesterase, Enzyme inhibitors, Hydrolases, Parasitology, Plasmodium falcuparum, Post translational modification, Protein palmitoylation, Serine hydrolase, Toxoplasma gondii, inhibitor

PMID:

 

23913689

 

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