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parallel, we developed the cell-permeable small molecule APT1-inhibitor denotedPalmostatin B (Figure 2b) through the knowledge-driven PSSC approach [5]. Bycombining the semi-synthetic protein tools with the conditional chemical knockdown ofAPT1, we could observe the inhibition of H- and N-Ras depalmitoylation events by liveconfocal microscopy. Short-term Palmostatin B knockdown of APT1 prevents Rasactivation on the Golgi, which enables studies on compartment specific signalling.Alternatively, long term Palmostatin B treatment disturbs H- and N-Ras precise steady statelocalization, causing unspecific entropy driven redistribution to endomembranes, thusdown regulating global Ras signalling. This is reflected by the phenotypic backtransformation of oncogenic Ras transformed MDCKf3 cells, as well as reduction in Erkphosphorylation. We verified the long-term effect by siRNA mediated inhibition of APT1expression in MDCKf3 cells and observed similar Ras-delocalization and phenotypicreversal. In summary, we have demonstrated that Palmostatin B is an excellent tool todissect Ras-protein dynamics in a time-dependent manner in vivo.abCy3Cy3N-RasN-Ras; 110 min 1µMFig. 2. (a) Semi-synthetic lipidatedN-Ras protein probes. The scissorsindicate site of APT1 depalmitoylation.(b) Palmostatin B, a potentsmall molecule inhibitor of APT1.The development of APT1 inhibitors hasgained little attention in pharmaceutical industry, asit has intuitively been expected that inhibition ofdepalmitoylation would increase the portion of Rasin active palmitoylated form, which would facilitateoncogenic signalling. However, the discovery thatdynamic palmitoylation / depalmitoylation eventsare required to maintain proper Ras localizationindicates that inhibition of Ras depalmitoylationwill attenuate Ras-mediated signalling, thus makingthe depalmitoylation event more attractive to target,compared to the palmitoylation step. In a recentstudy, we have found the palmitoylation-reaction ofproteins at the Golgi to be a rather unspecificgeneric sorting machinery for palmitoylatedproteins, thus being unwanted to target by smallmoleculeinhibitors [6]. Furthermore, it opens anew and unexpected entry towards furtherdevelopment of conceptually new approaches inoncology that target depalmitoylation specifically.However, the selectivity of both Palmostatin B andthe enzyme APT1 remain to be investigated further.It can be expected that many biological functionsare regulated by reversible palmitoylation eventscontrolling protein localization and that additionalthioesterases will be identified in the near future.Such a development will further increase theimportance of thioesterase inhibitors such aspalmostatin B, as well as of semi-synthetic lipidatedproteins to probe biological function in vivo.AcknowledgmentsWe thank all our collaboration partners, listed as co-authors in references 1-2 and 4-6, as well as thefunding agencies supporting the Ras-project over the years, in particular DFG through SFB 642,Alexander von Humboldt Foundation and the Max-Planck Gesellschaft.References1. Waldmann, H., Wittinghofer, A. Angew. Chem. Int. Ed. 39, 4192-4214 (2000).2. Rocks, O., et al. Science 307, 1746 (2005).3. a). Sugimoto, H., Hayashi, H., Yamashita, S. J. Biol. Chem. 271, 7705 (1996); b). Duncan, J.A.,Gilman, A.G. J. Biol. Chem. 273, 15830 (1998).4. Dekker, F.J., et al. Nat. Chem. Biol. 6, 449 (2010).5. Dekker, F.J., Koch, M.A., Waldmann, H. Curr. Op. Chem. Biol. 9, 232 (2005).6. Rocks, O., et al. Cell 141, 458. (2010).43