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Small-molecule inhibition of APT1 affects Ras localization and signaling

Nature Chemical Biology volume 6, pages 449–456 (2010)Cite this article

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Abstract

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization—and thereby unregulated signaling—caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

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Figure 1: Development of an APT1 inhibitor based on protein structure similarity clustering (PSSC).
Figure 2: Palmostatin B specifically inhibits depalmitoylation.
Figure 3: Palmostatin B causes redistribution of palmitoylated Ras isoforms.
Figure 4: Downregulation of APT1 increases the steady-state palmitoylation level of Ras.
Figure 5: Palmostatin B causes compartment-specific inhibition of Ras activity.
Figure 6: Palmostatin B–induced phenotypic reversion of HRasG12V-transformed MDCK-F3 cells.

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References

  1. Bijlmakers, M.J. & Marsh, M. The on-off story of protein palmitoylation. Trends Cell Biol. 13, 32–42 (2003).

    Article  CAS  Google Scholar 

  2. Jennings, B.C. et al. 2-Bromopalmitate and 2-(2-hydroxy-5-nitro- benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro. J. Lipid Res. 50, 233–242 (2009).

    Article  CAS  Google Scholar 

  3. Greaves, J. & Chamberlain, L.H. Palmitoylation-dependent protein sorting. J. Cell Biol. 176, 249–254 (2007).

    Article  CAS  Google Scholar 

  4. Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).

    Article  CAS  Google Scholar 

  5. Rocks, O. et al. The palmitoylation machinery is a generic sorting system for peripheral membrane proteins. Cell Published online 30 April 2010; doi:10.1016/j.cell.2010.04.007.

    Article  CAS  Google Scholar 

  6. Resh, M.D. Use of analogs and inhibitors to study the functional significance of protein palmitoylation. Methods 40, 191–197 (2006).

    Article  CAS  Google Scholar 

  7. Duncan, J.A. & Gilman, A.G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein α subunits and p21RAS. J. Biol. Chem. 273, 15830–15837 (1998).

    Article  CAS  Google Scholar 

  8. Yeh, D.C. et al. Depalmitoylation of endothelial nitric-oxide synthase by acyl- protein thioesterase 1 is potentiated by Ca2+-calmodulin. J. Biol. Chem. 274, 33148–33154 (1999).

    Article  CAS  Google Scholar 

  9. Koch, M.A. et al. Compound library development guided by protein structure similarity clustering and natural product structure. Proc. Natl. Acad. Sci. USA 101, 16721–16726 (2004).

    Article  CAS  Google Scholar 

  10. Dekker, F.J., Koch, M.A. & Waldmann, H. Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics. Curr. Opin. Chem. Biol. 9, 232–239 (2005).

    Article  CAS  Google Scholar 

  11. Devedjiev, Y. et al. Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 Å. Structure 8, 1137–1146 (2000).

    Article  CAS  Google Scholar 

  12. Wetzel, S. Similarity in Chemical and Protein Space: Finding Novel Starting Points for Library Design (Thesis, Dortmund University, 2009). MPG E-Doc System (http://edoc.mpg.de/), ID:442195.0.

  13. Holm, L. & Sander, C. The FSSP database of structurally aligned protein fold families. Nucleic Acids Res. 17, 3600–3609 (1994).

    Google Scholar 

  14. Holm, L. & Park, J. DaliLite workbench for protein structure comparison. Bioinformatics 16, 566–567 (2000).

    Article  CAS  Google Scholar 

  15. Roussel, A. et al. Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor. J. Biol. Chem. 277, 2266–2274 (2002).

    Article  CAS  Google Scholar 

  16. Weibel, E.K. et al. Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. I. Producing organism, fermentation, isolation and biological activity. J. Antibiot. 40, 1081–1085 (1987).

    Article  CAS  Google Scholar 

  17. Hadváry, P. et al. The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase. J. Biol. Chem. 266, 2021–2027 (1991).

    PubMed  Google Scholar 

  18. Böttcher, T. & Sieber, S.A. β-Lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes. Angew. Chem. Int. Ed. 47, 4600–4603 (2008).

    Article  Google Scholar 

  19. Wang, Z. et al. β-lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana. Nat. Chem. Biol. 4, 557–563 (2008).

    Article  CAS  Google Scholar 

  20. Inoue, T. et al. Boron-mediated aldol reaction of carboxylic esters: complementary anti- and syn-selective asymmetric aldol reactions. J. Org. Chem. 67, 5250–5256 (2002).

    Article  CAS  Google Scholar 

  21. Crimmins, M.T. et al. Asymmetric aldol additions: use of titanium tetrachloride and (–)-sparteine for the soft enolization of N-acyl oxazolidinones, oxazolidinethiones, and thiazolidinethiones. J. Org. Chem. 66, 894–902 (2001).

    Article  CAS  Google Scholar 

  22. Bader, B. et al. Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403, 223–226 (2000).

    Article  CAS  Google Scholar 

  23. Kuhn, K. et al. Synthesis of functional Ras lipoproteins and fluorescent derivatives. J. Am. Chem. Soc. 123, 1023–1035 (2001).

    Article  CAS  Google Scholar 

  24. Coleman, R.A. et al. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes. Biochim. Biophys. Acta 1125, 203–209 (1992).

    Article  CAS  Google Scholar 

  25. Drisdel, R.C. & Green, W.N. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36, 276–285 (2004).

    Article  CAS  Google Scholar 

  26. Grecco, H.E. et al. In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays. Nat. Methods (in the press).

  27. Silvius, J.R. Lipidated peptides as tools for understanding the membrane interactions of lipid-modified proteins. in Peptide-Lipid Interactions, Current Topics in Membranes vol. 52 (ed. Simon, S.A. & McIntosh, T.J.) 372-397 (Academic, 2002).

  28. Webb, Y., Hermida-Matsumoto, L. & Resh, M.D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J. Biol. Chem. 275, 261–270 (2000).

    Article  CAS  Google Scholar 

  29. Chiu, V.K. et al. Ras signalling on the endoplasmic reticulum and the Golgi. Nat. Cell Biol. 4, 343–350 (2002).

    Article  CAS  Google Scholar 

  30. Rocks, O., Peyker, A. & Bastiaens, P.I. Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors. Curr. Opin. Cell Biol. 18, 351–357 (2006).

    Article  CAS  Google Scholar 

  31. Matallanas, D. et al. Distinct utilization of effectors and biological outcomes resulting from site-specific Ras activation: Ras functions in lipid rafts and Golgi complex are dispensable for proliferation and transformation. Mol. Cell. Biol. 26, 100–116 (2006).

    Article  CAS  Google Scholar 

  32. Karaguni, I.M. et al. The new sulindac derivative IND 12 reverses Ras-induced cell transformation. Cancer Res. 62, 1718–1723 (2002).

    CAS  Google Scholar 

  33. Mueller, O. et al. Identification of potent Ras signaling inhibitors by pathway-selective phenotype-based screening. Angew. Chem. Int. Ed. 43, 450–454 (2004).

    Article  CAS  Google Scholar 

  34. Behrens, J. et al. Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol. 108, 2435–2447 (1989).

    Article  CAS  Google Scholar 

  35. Onder, T.T. et al. Loss of E-cadherin promotes metastasis via multipledownstream transcriptional pathways. Cancer Res. 68, 3645–3654 (2008).

    Article  CAS  Google Scholar 

  36. Deck, P. et al. Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. 44, 4975–4980 (2005).

    Article  CAS  Google Scholar 

  37. Biel, M. et al. Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chem. Eur. J. 12, 4121–4143 (2006).

    Article  CAS  Google Scholar 

  38. Takeichi, M. Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell Biol. 5, 806–811 (1993).

    Article  CAS  Google Scholar 

  39. Downward, J. Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22 (2003).

    Article  CAS  Google Scholar 

  40. Manders, E., Verbeek, F. & Alten, J. Measurement of co-localisation of objects in dual-colour confocal images. J. Microsc. 169, 375–382 (1993).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to H. Schütz for technical assistance with the western blots and immunofluorescence experiments. This work was financially supported by the Max-Planck Gesellschaft, the Deutsche Forschungsgemeinschaft, the Netherlands Organization for Scientific Research (NWO) (TALENT-stipendium for F.J.D.) and the European Union (Marie Curie fellowship for F.J.D.). N.V. is grateful to the International Max Planck Research School-Chemical Biology for providing a doctoral fellowship. C.H. and R.B. are grateful to the Alexander von Humboldt Foundation for postdoctoral fellowships. We thank the US National Institutes of Health for a Chemical/Biology Interface (CBI) Training Grant (to J.W.K.).

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Author notes

  1. Frank J Dekker & Oliver Rocks

    Present address: Present addresses: Pharmaceutical Gene Modulation, University Centre for Pharmacy, Groningen, The Netherlands (F.J.D.). Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (O.R.).,

  2. Frank J Dekker, Oliver Rocks, Nachiket Vartak and Sascha Menninger: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany

    Frank J Dekker, Sascha Menninger, Christian Hedberg, Rengarajan Balamurugan, Stefan Wetzel, Steffen Renner, Marc Gerauer, Beate Schölermann, Marion Rusch, Luc Brunsveld & Herbert Waldmann

  2. Fachbereich Chemie, Universität Dortmund, Dortmund, Germany

    Frank J Dekker, Sascha Menninger, Christian Hedberg, Rengarajan Balamurugan, Stefan Wetzel, Steffen Renner, Marc Gerauer, Marion Rusch, Luc Brunsveld, Philippe I H Bastiaens & Herbert Waldmann

  3. Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany

    Oliver Rocks, Nachiket Vartak & Philippe I H Bastiaens

  4. Department of Chemistry and Chemical Biology, Baker Laboratory, New York, Ithaca, USA

    John W Kramer & Geoffrey W Coates

  5. Chemical Genomics Centre of the Max Planck Gesellschaft, Dortmund, Germany

    Daniel Rauh

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Contributions

P.I.H.B. and H.W. conceived the project and designed experiments; J.W.K. and G.J.C. provided a diverse compound collection; S.W. and S.R. performed the cheminformatics and bioinformatics work; M.G. supplied semisynthetic RAS proteins; S.M. performed the initial screening for hits; S.M. and N.V. performed in vitro palmitoylation assays; F.J.D., R.B., C.H. and M.R. synthesized the palmostatins and their fluorescent derivatives and performed in vitro biochemical assays; B.S. performed ABE assays; N.V. and O.R. performed cell biological and bioimaging experiments; N.V. performed data analysis for imaging and western blots; C.H., L.B. and D.R. supervised scientific work; N.V., C.H., O.R., P.I.H.B. and H.W. wrote the paper.

Corresponding authors

Correspondence to Philippe I H Bastiaens or Herbert Waldmann.

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Supplementary Methods, Supplementary Results, Supplementary Figures 1–13, Supplementary Schemes 1–2 and Supplementary Tables 1–5 (PDF 3897 kb)

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Dekker, F., Rocks, O., Vartak, N. et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat Chem Biol 6, 449–456 (2010). https://doi.org/10.1038/nchembio.362

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