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Small-molecule inhibition of APT1 affects Ras localization and signaling


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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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.).

Author information




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.

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Correspondence to Philippe I H Bastiaens or Herbert Waldmann.

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The authors declare no competing financial interests.

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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).

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