J Biol Chem. 2013 Sep 20;288(38):27002-18.
Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii
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 and Dominique Soldati-Favre
Department of Microbiology and Molecular Medicine, University Medical Center (CMU), University of Geneva, Rue Michel-Servet 1, CH-1211 Geneva, Switzerland.
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 modelling 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.
Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and animals and is able to establish a life–long persistent chronic infection. Prevalence varies from region to region but the WHO estimates that 30-50 % of the world’s population have been exposed to T. gondii and are chronically infected.
Congenital toxoplasmosis occurs during pregnancy while acquired toxoplasmosis occurs to through ingestion of raw meat (intermediate hosts) or contact with oocysts that are shed by the cats (definitive host). In humans, T. gondii is responsible for severe medical complications in immuno-compromised individuals. When transmitted congenitally, it can cause abortion or lead to mental retardation in infected babies. In recent years chronic infection has been increasingly connected with mental health disorders.
Figure 1: This scheme represents the dynamic S-palmitoylation cycle. Both soluble and membrane proteins can be S-palmitoylated.
Post-translational modifications (PTMs) are essential for cellular function and have already been considered targets for intervention in parasitic infections, as the players and pathways required for modification can be distinguished between infectious agent and host (Jomaa et al. 1999; Eastman et al. 2006). Palmitoylation involves the post-translational addition of the 16-carbon fatty acid palmitate to cysteine residues. Palmitate can be attached in two ways, firstly, via an amide bond (N-palmitoylation), a reaction that is irreversible. In contrast S-palmitoylation, in which the palmitate is attached to a cysteine residue via a thioester bond, is a reversible reaction (Fig. 1). The reversibility and fast reaction kinetics of the palmitoylation/depalmitoylation cycle can serve as key and versatile regulator of protein function within the cell. Palmitoylation has been shown to play a crucial role in several proteins implicated in parasite motility and invasion. The current knowledge on protein palmitoylation and palmitoylation machinery in T. gondii was recently reviewed (Frénal et al. 2014).
Figure 2: A – Replication assay, treatment of intracellular parasites during 24 hours to monitor growth. Compounds administered at 10mM B – Invasion assay, extracellular parasites treated with compounds prior to invasion DMSO control shown in each category for comparison only.
We focussed our attention on the depalmitoylation step of the cycle. Depalmitoylation is performed by Acyl Protein Thioesterases (APTs) and only two enzymes were reported to be capable of depalmitoylation in the context of a dynamic cycle. We therefore reasoned that depalmitoylation was a more appropriate target for therapeutic intervention than the palmitoylation step, which is fulfilled by large family of protein S-acyl transferases (PATs) that tend to exhibit functional redundancy.
T. gondii tachyzoites go through an asexual replication cycle in the tissues of the intermediate host. During this lytic cycle the parasites invade host cells in an active manner, go through several rounds of replication, then egress ready to rapidly invade new host cells. In tissue culture, it is possible to dissect each step of the lytic cycle individually. We determined the impact of a series of b-lactone and triazole urea small molecule inhibitors designed against the human APT1 (hAPT1) on tachyzoite propagation (b-lactones – RM448, RM449 and inactive compound FD242, triazole urea – AA401). We reported severe defects in the replication step of the lytic cycle upon treatment with 10mM concentrations of both b-lactone and triazole urea compounds (Fig. 2A). A severe invasion defect was observed upon treatment of extracellular parasites with b-lactone compounds including FD242 whereas AA401 did not affect invasion (Fig. 2B). In the related parasite Plasmodium falciparum, which causes malaria, treatment with b-lactone compounds inhibited egress (Fig. 3), again suggesting these inhibitors were disrupting an essential process.
Figure 3: Test of P. falciparum lytic cycle in absence (A) or presence (B) of 10mM b-lactone compound RM449. In absence of inhibitor parasites egress and reinvade red blood cells, and ring stage parasites can be seen. In presence of inhibitor, development is blocked at schizont stage and parasites fail to egress.
To search for the existence of putative acyl protein thioesterases in T. gondii, we took two parallel approaches:
1) We performed a BLAST search of the T. gondii genome using hAPT1 as a template and detected a close homologue that shared 37% identity, hereafter called Acyl Serine Hydrolase 1 (TgASH1). All critical residues are aligned (Fig. 4) and the T. gondii protein is predicted to fold to create a functional catalytic pocket. Furthermore, activity assays confirmed that TgASH1 has activity against fatty acid chains similar to hAPT1. Other genes coding for less conserved, putative acyl protein thioesterases were also identified and characterized.
2) Using a serine hydrolase probe (FP-Rhodamine) we determined the extent of active serine hydrolases in the parasites. A competition experiment with the probe and inhibitor identified the serine hydrolase targeted by the inhibitors (Fig. 5). This experiment revealed that the b-lactone inhibitors targeted not only ASH1 but also other targets. The triazole urea AA401 was highly specific and highly effective at inhibiting TgASH1 at low concentrations that did not compromise parasite viability.
We therefore hypothesised that the effects observed with b-lactone inhibitors had off target deleterious effects. As further evidence for this, the genetic ablation of TgASH1 gene was not detrimental to parasite survival.
Figure 4: Alignment of a selection of eukaryotic APT1 homologues. P70470 – Rattus norvegicus; O75608 – Homo sapiens; Q54T49 – Dictyostelium discoideum; Q12354 – Saccharomyces cerevisiae; TGME49_228290 – Toxoplasma gondii; NCLIV_045170 – Neospora caninum. Produced with Multalin
Post-translational modifications play central roles in fine-tuning the localisation, stability and function of proteins. Palmitoylation-depalmitoylation cycle has recently emerged as an important component of the PTM toolbox. The depalmitoylating enzyme APT1 is a highly conserved in eukaryotes suggestive of an essential role. We characterized TgASH1 as the closest homologue of hAPT1. Unexpectedly, TgASH1 is dispensable for parasite survival. We identified three potential candidates that might play redundant functions with TgASH1. The individual function of these enzymes is yet to be investigated and it may be that multiple gene knockouts are required to confidently assess the importance of the palmitoyation cycle in parasite infection. Recent preliminary studies in Trypanosoma brucei, another protozoan parasite, have suggested that APT1 may have a role in bloodstream form and during differentiation of the parasites (Alsford et al. 2011).
Figure 5: Identification of inhibitor targets. The FP-Rhodamine serine hydrolase probe and inhibitors both bind in the catalytic site of active serine hydrolases. A – A competition experiment performed by pre-treating cell lysates with the inhibitors can identify which enzymes the inhibitors are targeting by monitoring subsequent loss of binding of the FP-Rh probe. B – b-lactone inhibitors appear to target multiple enzymes at concentrations as low as 1mM while only at higher concentrations does the triazole urea compound AA401 have unspecific targets.
Alsford, S. et al., 2011. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Research, 21(6), pp.915–924.
Eastman, R.T. et al., 2006. Thematic review series: lipid posttranslational modifications. Fighting parasitic disease by blocking protein farnesylation. Journal of lipid research, 47(2), pp.233–240. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16339110.
Frénal, K., Kemp, L.E. & Soldati-Favre, D., 2014. Emerging roles for protein S-palmitoylation in Toxoplasma biology. International Journal for Parasitology, 44, pp.121–131.
Jomaa, H. et al., 1999. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science, 285(5433), pp.1573–1576. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10477522.