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Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion

Nature Chemical Biology volume 9, pages 651–656 (2013)Cite this article

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Abstract

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

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Figure 1: Substituted chloroisocoumarins enhance Toxoplasma host-cell invasion, induce microneme secretion and enhance motility.
Figure 2: TgPPT1 is a target of JCP174.
Figure 3: Conditional knockdown and direct knockout genetically validates TgPPT1 as the target of the enhancers.
Figure 4: The enhancers inhibit TgPPT1 activity.
Figure 5: TgPPT1 is a bona fide thioesterase, and TgPPT1 inhibition increases membrane partitioning of palmitoylated substrates.

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References

  1. Sibley, L.D. Intracellular parasite invasion strategies. Science 304, 248–253 (2004).

    Article  CAS  Google Scholar 

  2. Hall, C.I. et al. Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion. Proc. Natl. Acad. Sci. USA 108, 10568–10573 (2011).

    Article  CAS  Google Scholar 

  3. Arrizabalaga, G. & Boothroyd, J.C. Role of calcium during Toxoplasma gondii invasion and egress. Int. J. Parasitol. 34, 361–368 (2004).

    Article  CAS  Google Scholar 

  4. Billker, O., Lourido, S. & Sibley, L.D. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5, 612–622 (2009).

    Article  CAS  Google Scholar 

  5. Roos, D.S., Donald, R.G., Morrissette, N.S. & Moulton, A.L. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 45, 27–63 (1994).

    Article  CAS  Google Scholar 

  6. Keeley, A. & Soldati, D. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa. Trends Cell Biol. 14, 528–532 (2004).

    Article  CAS  Google Scholar 

  7. Carruthers, V.B., Giddings, O.K. & Sibley, L.D. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell. Microbiol. 1, 225–235 (1999).

    Article  CAS  Google Scholar 

  8. Soldati, D., Dubremetz, J.F. & Lebrun, M. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int. J. Parasitol. 31, 1293–1302 (2001).

    Article  CAS  Google Scholar 

  9. Carruthers, V.B. & Sibley, L.D. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol. Microbiol. 31, 421–428 (1999).

    Article  CAS  Google Scholar 

  10. Lourido, S. et al. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359–362 (2010).

    Article  CAS  Google Scholar 

  11. Carruthers, V.B., Sherman, G.D. & Sibley, L.D. The Toxoplasma adhesive protein MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases. J. Biol. Chem. 275, 14346–14353 (2000).

    Article  CAS  Google Scholar 

  12. Huynh, M.H. & Carruthers, V.B. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathog. 2, e84 (2006).

    Article  Google Scholar 

  13. Black, M.W., Arrizabalaga, G. & Boothroyd, J.C. Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress. Mol. Cell. Biol. 20, 9399–9408 (2000).

    Article  CAS  Google Scholar 

  14. Heynekamp, J.J. et al. Isocoumarin-based inhibitors of pancreatic cholesterol esterase. Bioorg. Med. Chem. 16, 5285–5294 (2008).

    Article  CAS  Google Scholar 

  15. Powers, J.C. et al. Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design. J. Cell. Biochem. 39, 33–46 (1989).

    Article  CAS  Google Scholar 

  16. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  Google Scholar 

  17. Haedke, U., Gotz, M., Baer, P. & Verhelst, S.H. Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases. Bioorg. Med. Chem. 20, 633–640 (2012).

    Article  CAS  Google Scholar 

  18. Weerapana, E., Speers, A.E. & Cravatt, B.F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414–1425 (2007).

    Article  CAS  Google Scholar 

  19. Duncan, J.A. & Gilman, A.G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein α subunits and p21(RAS). J. Biol. Chem. 273, 15830–15837 (1998).

    Article  CAS  Google Scholar 

  20. Santos, J.M., Hedberg, C. & Soldati-Favre, D. Protein acylation: new potential targets for intervention against the apicomplexa in Apicomplexan Parasites: Molecular Approaches Toward Targeted Drug Development Vol. 2 (eds. Becker, K. & Selzer, P.M.) 335–357 (Wiley-Blackwell, 2011).

  21. Herm-Götz, A. et al. Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat. Methods 4, 1003–1005 (2007).

    Article  Google Scholar 

  22. Dekker, F.J. et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol. 6, 449–456 (2010).

    Article  CAS  Google Scholar 

  23. Fox, B.A., Ristuccia, J.G., Gigley, J.P. & Bzik, D.J. Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining. Eukaryot. Cell 8, 520–529 (2009).

    Article  CAS  Google Scholar 

  24. Duncan, J.A. & Gilman, A.G. Characterization of Saccharomyces cerevisiae acyl-protein thioesterase 1, the enzyme responsible for G protein α subunit deacylation in vivo. J. Biol. Chem. 277, 31740–31752 (2002).

    Article  CAS  Google Scholar 

  25. Lourido, S., Tang, K. & Sibley, L.D. Distinct signalling pathways control Toxoplasma egress and host-cell invasion. EMBO J. 31, 4524–4534 (2012).

    Article  CAS  Google Scholar 

  26. Rees-Channer, R.R. et al. Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 149, 113–116 (2006).

    Article  CAS  Google Scholar 

  27. Frénal, K. et al. Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8, 343–357 (2010).

    Article  Google Scholar 

  28. Martin, B.R., Wang, C., Adibekian, A., Tully, S.E. & Cravatt, B.F. Global profiling of dynamic protein palmitoylation. Nat. Methods 9, 84–89 (2012).

    Article  CAS  Google Scholar 

  29. Garrison, E. et al. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma. PLoS Pathog. 8, e1003049 (2012).

    Article  CAS  Google Scholar 

  30. McCoy, J.M., Whitehead, L., van Dooren, G.G. & Tonkin, C.J. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells. PLoS Pathog. 8, e1003066 (2012).

    Article  CAS  Google Scholar 

  31. Beck, J.R. et al. A Toxoplasma palmitoyl acyl transferase and the palmitoylated Armadillo Repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress. PLoS Pathog. 9, e1003162 (2013).

    Article  CAS  Google Scholar 

  32. Mueller, C. et al. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host Microbe 13, 289–301 (2013).

    Article  CAS  Google Scholar 

  33. Prescott, G.R., Gorleku, O.A., Greaves, J. & Chamberlain, L.H. Palmitoylation of the synaptic vesicle fusion machinery. J. Neurochem. 110, 1135–1149 (2009).

    Article  CAS  Google Scholar 

  34. Del Carmen, M.G., Mondragon, M., Gonzalez, S. & Mondragon, R. Induction and regulation of conoid extrusion in Toxoplasma gondii. Cell. Microbiol. 11, 967–982 (2009).

    Article  CAS  Google Scholar 

  35. Carey, K.L., Westwood, N.J., Mitchison, T.J. & Ward, G.E. A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. Proc. Natl. Acad. Sci. USA 101, 7433–7438 (2004).

    Article  CAS  Google Scholar 

  36. Kremer, K. et al. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes. PLoS Pathog. 9, e1003213 (2013).

    Article  CAS  Google Scholar 

  37. Jones, M.L., Collins, M.O., Goulding, D., Choudhary, J.S. & Rayner, J.C. Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis. Cell Host Microbe 12, 246–258 (2012).

    Article  CAS  Google Scholar 

  38. Alonso, A.M. et al. Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii. Mol. Biochem. Parasitol. 184, 39–43 (2012).

    Article  CAS  Google Scholar 

  39. Hirakawa, S., Saito, R., Ohara, H., Okuyama, R. & Aiba, S. Dual oxidase 1 induced by TH2 cytokines promotes STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B in human epidermal keratinocytes. J. Immunol. 186, 4762–4770 (2011).

    Article  CAS  Google Scholar 

  40. Speers, A.E. & Cravatt, B.F. Activity-based protein profiling (ABPP) and click chemistry (CC)-ABPP by MudPIT mass spectrometry. Curr. Protoc. Chem. Biol. 1, 29–41 (2009).

    PubMed  PubMed Central  Google Scholar 

  41. Huynh, M.H. & Carruthers, V.B. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot. Cell 8, 530–539 (2009).

    Article  CAS  Google Scholar 

  42. Lodoen, M.B., Gerke, C. & Boothroyd, J.C. A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell. Microbiol. 12, 55–66 (2010).

    Article  CAS  Google Scholar 

  43. Donald, R.G. & Roos, D.S. Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis. Mol. Biochem. Parasitol. 91, 295–305 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the Bogyo and Boothroyd labs for discussions that shaped the direction of the project; G. Ward (University of Vermont) for discussions and the GAP45 antibody; B. Martin for discussions regarding the 17-ODA metabolic labeling experiments; G. Arrizabalaga (Indiana University) and M. Treeck (Stanford University) for the TgCDPK3-HA parasite line; B. Cravatt (Scripps Research Institute) for FP-rho; T. Wandless (Stanford University) for Shield-1; and the Stanford high-throughput imaging facility for assistance with imaging the attachment and invasion assays. This work was funded by a Burroughs Wellcome Trust New Investigators in Pathogenesis Award (to M.B.), by the US National Institutes of Health grants R01-AI078947 and EB005011 (to M.B.) and RO1 AI21423 (to J.C.B.).

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Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Matthew A Child, Leslie O Ofori, Victoria E Albrow, Megan Garland, Paul W Bowyer & Matthew Bogyo

  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Carolyn I Hall, John C Boothroyd & Matthew Bogyo

  3. Department of Microbiology, Immunology and Molecular Genetics, University of California–Los Angeles, Los Angeles, California, USA

    Josh R Beck & Peter J Bradley

  4. Department of Chemistry, Georgia Institute of Technology, Atlanta, Georgia, USA

    James C Powers

  5. Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA

    Eranthie Weerapana

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Contributions

M.A.C. designed and performed the majority of the experiments, analyzed the data, generated the figures and wrote the manuscript. C.I.H. performed the original high-throughput screen and various biochemical and cell biological studies. C.I.H., V.E.A. and P.W.B. characterized the enhancer phenotype. V.E.A. synthesized JCP174-IA and JCP174-alk. J.C.P. and L.O.O. synthesized JCP174, JCP222 and JCP362. J.R.B. generated the TgPPT1-HAdd parasite line under the supervision of P.J.B. M.G. contributed to the CDPK3 experiments. E.W. performed MS experiments. E.W. and J.C.B. intellectually contributed to the decision to pursue TgPPT1 as the functionally relevant target of JCP174. M.B. supervised the project, designed and analyzed experiments and wrote parts of the manuscript.

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Correspondence to Matthew Bogyo.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 3 and 4. (PDF 10179 kb)

Supplementary Tables 1 and 2

Suplementary Table 1: Mass spectrometry data of identified peptides from FP-Biotin labeling and TOP-ABPP using the JCP174-alk probe. Supplementary Table 2: List of the top 6 hits from the total list of identified targets of FP-Rho and JCP174-alk. (XLS 66 kb)

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Child, M., Hall, C., Beck, J. et al. Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nat Chem Biol 9, 651–656 (2013). https://doi.org/10.1038/nchembio.1315

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