Fig. 5: Engineering a depalmitoylation feedback loop for NRas.

a Schematic shows palmitoylation cycle of NRas. Increased NRas activity and downstream signaling results in activation of Erk. We engineer a depalmitoylase that is activated by Erk as a negative feedback loop to inhibit NRas in an activity-dependent manner. b Design strategy for Erk-activated depalmitoylase (dePalm-er). We engineer the N-terminus of Abhd17C with a WW motif. The C-terminus of Abhd17C is equipped with an Erk substrate from cdc24C along with an FQFP ERK-docking site. Increased Erk-activity results in phosphorylation of Erk-substrate that then promotes dimerization of the N- and C-terminal regions of Abhd17C, resulting in activation of the depalmitoylase. c Confocal images show either wildtype (top) or Q[61]K mutant NRas (bottom) tagged with YFP in the presence of Golgi-targeted mCherry (golgin-mcherry). Left, at baseline both wild-type and mutant NRas exhibit strong membrane localization. Middle, co-expression of Abhd17c increases golgi-localization of both wild-type and mutant NRas. Right, co-expression of dePalm-er results in increased golgi-localization of Q[61]K mutant NRas but not wild-type. Scale bar, 10 µm. d Bar graph summarizes Pearson’s correlation between NRas (yellow fluorescence) and golgi marker (red fluorescence). Each Bar, mean ± s.e.m. ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test. For wild-type NRas, at baseline, n = 47 cells; with Abhd17c, n = 51 cells; with dePalm-er, n = 65 cells. For Q[61]K mutant NRas, at baseline, n = 48 cells; with Abhd17c, n = 52 cells; with dePalm-er, n = 72 cells from 3 independent transfections. Source data are provided as a Source Data file.