Small-molecule inhibition of APT1 affects Ras localization and signaling

Journal name:
Nature Chemical Biology
Volume:
6,
Pages:
449–456
Year published:
DOI:
doi:10.1038/nchembio.362
Received
Accepted
Published online

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.

At a glance

Figures

  1. Development of an APT1 inhibitor based on protein structure similarity clustering (PSSC).
    Figure 1: Development of an APT1 inhibitor based on protein structure similarity clustering (PSSC).

    (a) The ligand-sensing core of APT1 was chosen as starting point for a Dali/FSSP search, which yielded gastric lipase as a structurally similar hit. It is known that gastric lipase is inhibited by tetrahydrolipstatin, so the PSSC postulates that structurally similar inhibitors should inhibit APT1. Thus, the β-lactone motif present in tetrahydrolipstatin was chosen as the core motif of a new compound collection. Subsequent biochemical screening then identified inhibitors of APT1 in the β-lactone compound collection. (b) The ligand-sensing core, extracted from the crystal structure of APT1 (PDB 1FJ2). The catalytic triad amino acids—Ser114, His203 and Asp169—are shown as purple sticks. The two lower α-helix fragments belong to chain B and result from the dimeric crystal structure found in the PDB file. (c) Structural overlay of the ligand-sensing cores of APT1 and gastric lipase. APT1 is shown in blue and red. Gastric lipase is shown in green and purple. (d) Overlay of the ligand-sensing cores of gastric lipase (orange) and APT1 (green). The catalytic triad (Asp, His and Ser) of each enzyme is shown as sticks (also shown in the enlarged image). The structural similarity between both ligand-sensing cores validates the PSSC concept.

  2. Palmostatin B specifically inhibits depalmitoylation.
    Figure 2: Palmostatin B specifically inhibits depalmitoylation.

    (a) Structure of the CysFar semisynthetic protein, which serves as a substrate for palmitoylation upon injection. Confocal time-lapse images of MDCK cells expressing the Golgi marker GalT-CFP before and after microinjecton of CysFar. Cells were incubated for 80 min with 1 μM palmostatin B (Palm B), or equivalent DMSO as control, before the experiment. Scale bar, 10 μm. (b) Structure of the PalFar semisynthetic protein, which serves as a substrate for depalmitoylation upon injection. Confocal time-lapse images of MDCK cells expressing GalT-CFP preincubated with 1 μM palmostatin B, or equivalent DMSO as a control, for 55 min before and after microinjecton of PalFar. Scale bar, 10 μm. (c) Top, quantitative ratiometric analysis of CysFar accumulation at the Golgi in the presence of palmostatin B using GalT-CFP fluorescence as reference. Plateau values of Cy3 fluorescence at the Golgi were normalized to 1 (n = 5). Bottom, contrast of PalFar fluorescence at the Golgi over adjacent areas (membranes/cytosol, n = 5) compared to CysFar fluorescence contrast (n = 5) in the presence of 1 μM palmostatin B. (d) Thioesterase inhibition by palmostatin B increases the fraction of palmitoylated NRas in live cells, as measured by the ABE method23. An inhibitor of cellular palmitoylation (2-BP) is shown as a negative control. Quantification of western blots (arbitrary fluorescence units (A.U.) using infrared dye–labeled antibodies on the same gel) represents data from four independent experiments with eight independent runs per treatment (Supplementary Fig. 12a). (e) FLIM of live cells showing specific binding of APT1-GFP (donor) to TAMRA-labeled palmostatin B (acceptor). Lifetime and intensity maps of cells after incubation with acceptor showing a reduction in GFP fluorescence lifetime due to FRET. Graph shows the reduction in APT1-GFP lifetime as compared to the GFP-GFP (control) lifetime upon incubation with the acceptor. The lower limit for the APT1/palmostatin B–bound fraction is estimated to be 40%.

  3. Palmostatin B causes redistribution of palmitoylated Ras isoforms.
    Figure 3: Palmostatin B causes redistribution of palmitoylated Ras isoforms.

    (a) Representative example of changes in distribution of Citrine-NRas with respect to Cherry-HRasC181S,C184S in MDCK cells at various time points after treatment with 1 μM palmostatin B (see also Supplementary Fig. 8), observed under confocal microscopy. Columns show red-green channel overlays and intensity scatter plots for Citrine-NRas (green) and Cherry-HRasC181S,C184S (red), demonstrating their increase in colocalization over time. (b) Manders' coefficients (R)40 for colocalization of Citrine-NRas and Cherry-HRasC181S,C184S over time after palmostatin B treatment, as compared to the changes caused by treatment with 2-BP. Solid lines show a logistic fit for comparison. (c) Representative example of changes in distribution of Citrine-HRas with respect to Cherry-HRasC181S,C184S in MDCK cells at various time points after treatment with 10 μM palmostatin B. (d) Representative example of stable distribution of Citrine-KRas with respect to Cherry-HRasC181S,C184S in MDCK cells at various time points after treatment with 10 μM palmostatin B. Scale bar, 10 μm.

  4. Downregulation of APT1 increases the steady-state palmitoylation level of Ras.
    Figure 4: Downregulation of APT1 increases the steady-state palmitoylation level of Ras.

    (a) Efficacy of APT1 RNA interference (RNAi). The bar graph shows the decrease in relative APT1 mRNA content in NRas-transfected cells for two siRNA concentrations. (RT-qPCR quantification of mRNA APT1 knockdown in MDCK cells treated with 100 nM anti-canine APT1 siRNA (Δ(ΔCt)) = 1.8 cycles w.r.t. GAPDH control; see also Supplementary Methods). (b) Downregulation of APT1 by RNAi increases the levels of NRas palmitoylation, as measured by the acyl biotin–exchange assay (Supplementary Fig. 12b) in arbitrary fluorescence units (A.U.) on the same gel. NT, non-targeting. The effect of canine APT1 knockdown can be rescued by the expression of human APT1 in MDCK cells (Supplementary Fig. 12c). (c) Ratiometric image analysis of the redistribution of palmitoylatable Ras isoforms to endomembranes after downregulation of APT1. A significant decrease of the plasma membrane (PM)/Golgi intensity ratio of both Citrine-HRas and Citrine-NRas occurs in cells treated with anti-APT1 siRNA. For Citrine-NRas, cells treated with palmostatin B showed a similar decrease in PM/Golgi intensity ratio in MDCK and MDCK-F3 cells (Supplementary Fig. 12d). PM/Golgi ratios from control cells treated with either NT siRNA or DMSO as vehicle control (indicated by horizontal line) were used to normalize the data.

  5. Palmostatin B causes compartment-specific inhibition of Ras activity.
    Figure 5: Palmostatin B causes compartment-specific inhibition of Ras activity.

    (a) Altered HRas activity profile in palmostatin B–treated cells. Confocal time lapse images of EGF-induced membrane translocation of Cherry-RafRBD to Citrine-HRas in MDCK cells coexpressing GalT-CFP in the presence of 1 μM palmostatin B for 45 min. Scale bar, 10 μm. Arrows indicate absence of activated Citrine-HRas on the Golgi as compared to the control case. The graph shows ratiometric quantification of Cherry-RafRBD translocation to the plasma membrane (straight lines) and to the Golgi (dotted lines) in the presence of palmostatin B (n = 4) using local Citrine-HRas intensities as reference. Maximal values for plasma membrane translocation were normalized to 1 and the Golgi response in the same cell were scaled with this normalization factor. (b) The representative control case, showing an unperturbed spatial activity profile for HRas after EGF induction. Arrows indicate accumulation of Raf-RBD-mCherry on the Golgi. Scale bar, 10 μm.

  6. Palmostatin B–induced phenotypic reversion of HRasG12V-transformed MDCK-F3 cells.
    Figure 6: Palmostatin B–induced phenotypic reversion of HRasG12V-transformed MDCK-F3 cells.

    (a) Above, HRasG12V-transformed MDCK-F3 cells show changes in overall cell shape comparable to nontransformed MDCK cells, after overnight treatment with 50 μM palmostatin B. Below, confocal images of E-cadherin immunostaining of HRasG12V-transformed MDCK-F3 cells show restoration of E-cadherin expression at the cell-cell interfaces after treatment with 50 μM palmostatin B. Transformed and untransformed MDCK cells treated with DMSO (vehicle) are shown as controls (see also Supplementary Fig. 13b). E-cadherin staining at the cell junctions is correlated with contact inhibition35. Scale bars, 20 μm. (b) Cell circularity distribution (n > 400 cells for each case) of HRasG12V-transformed MDCK-F3 cells approaches that of untransformed MDCK cells upon treatment with palmostatin B. (c) KRasG12V-mediated rescue of the transformed palmostatin B–treated cells. Circularity distributions (n > 400 cells for each case) of MDCK-F3 cells transfected with KRasG12V did not change significantly upon treatment with palmostatin B.

  7. Synthesis and biochemical characterization of palmostatins A–D.
    Scheme 1: Synthesis and biochemical characterization of palmostatins A–D.

    (a) Palmostatins A and B were synthesized by Cy2BOTf anti-selective aldol condensation according to a known procedure20, followed by chiral auxiliary cleavage and subsequent β-lactonization (Supplementary Methods and Supplementary Scheme 1). (b) Palmostatins C and D were synthezised by syn-selective aldol condensations according to a known procedure21, followed by chiral auxiliary cleavage and subsequent β-lactonization. (c) By analogy to the mechanism of gastric lipase inhibition by β-lactones palmostatin B inhibits APT1 by fast formation of a covalent enzyme-inhibitor complex (kI, IC50 = 670 nM) by acylation of the nucleophilic Ser114 residue in the catalytic triad. The resulting APT1–palmostatin B complex hydrolyzes slowly (kII) into active APT1 and the corresponding β-hydroxy acid. Nonenzymatic hydrolysis of the β-lactone determined by a rate constant kIII also occurs, characterizing its stability in aqueous solution. PM, plasma membrane.

Compounds

4 compounds View all compounds
  1. Palmostatin A
    Compound 1 Palmostatin A
  2. Palmostatin B
    Compound 2 Palmostatin B
  3. Palmostatin C
    Compound 3 Palmostatin C
  4. Palmostatin D
    Compound 4 Palmostatin D

Accession codes

Referenced accessions

Protein Data Bank

References

  1. Bijlmakers, M.J. & Marsh, M. The on-off story of protein palmitoylation. Trends Cell Biol. 13, 3242 (2003).
  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, 233242 (2009).
  3. Greaves, J. & Chamberlain, L.H. Palmitoylation-dependent protein sorting. J. Cell Biol. 176, 249254 (2007).
  4. Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 17461752 (2005).
  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.
  6. Resh, M.D. Use of analogs and inhibitors to study the functional significance of protein palmitoylation. Methods 40, 191197 (2006).
  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, 1583015837 (1998).
  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, 3314833154 (1999).
  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, 1672116726 (2004).
  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, 232239 (2005).
  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, 11371146 (2000).
  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, 36003609 (1994).
  14. Holm, L. & Park, J. DaliLite workbench for protein structure comparison. Bioinformatics 16, 566567 (2000).
  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, 22662274 (2002).
  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, 10811085 (1987).
  17. Hadváry, P. et al. The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase. J. Biol. Chem. 266, 20212027 (1991).
  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, 46004603 (2008).
  19. Wang, Z. et al. β-lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana . Nat. Chem. Biol. 4, 557563 (2008).
  20. Inoue, T. et al. Boron-mediated aldol reaction of carboxylic esters: complementary anti- and syn-selective asymmetric aldol reactions. J. Org. Chem. 67, 52505256 (2002).
  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, 894902 (2001).
  22. Bader, B. et al. Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403, 223226 (2000).
  23. Kuhn, K. et al. Synthesis of functional Ras lipoproteins and fluorescent derivatives. J. Am. Chem. Soc. 123, 10231035 (2001).
  24. Coleman, R.A. et al. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes. Biochim. Biophys. Acta 1125, 203209 (1992).
  25. Drisdel, R.C. & Green, W.N. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36, 276285 (2004).
  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, 261270 (2000).
  29. Chiu, V.K. et al. Ras signalling on the endoplasmic reticulum and the Golgi. Nat. Cell Biol. 4, 343350 (2002).
  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, 351357 (2006).
  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, 100116 (2006).
  32. Karaguni, I.M. et al. The new sulindac derivative IND 12 reverses Ras-induced cell transformation. Cancer Res. 62, 17181723 (2002).
  33. Mueller, O. et al. Identification of potent Ras signaling inhibitors by pathway-selective phenotype-based screening. Angew. Chem. Int. Ed. 43, 450454 (2004).
  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, 24352447 (1989).
  35. Onder, T.T. et al. Loss of E-cadherin promotes metastasis via multipledownstream transcriptional pathways. Cancer Res. 68, 36453654 (2008).
  36. Deck, P. et al. Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. 44, 49754980 (2005).
  37. Biel, M. et al. Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chem. Eur. J. 12, 41214143 (2006).
  38. Takeichi, M. Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell Biol. 5, 806811 (1993).
  39. Downward, J. Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 1122 (2003).
  40. Manders, E., Verbeek, F. & Alten, J. Measurement of co-localisation of objects in dual-colour confocal images. J. Microsc. 169, 375382 (1993).

Download references

Author information

  1. These authors contributed equally to this work.

    • Frank J Dekker,
    • Oliver Rocks,
    • Nachiket Vartak &
    • Sascha Menninger

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, Ithaca, New York, USA.

    • John W Kramer &
    • Geoffrey W Coates
  5. Chemical Genomics Centre of the Max Planck Gesellschaft, Dortmund, Germany.

    • Daniel Rauh
  6. 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.).

    • Frank J Dekker &
    • Oliver Rocks

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (516K)

    Supplementary Methods, Supplementary Results, Supplementary Figures 1–13, Supplementary Schemes 1–2 and Supplementary Tables 1–5

Additional data