Fig. 1: Design of functional validation of a chemogenetic depalmitoylase.
From: Engineered depalmitoylases enable selective manipulation of protein localization and function
a Schematic shows the molecular architecture of Abhd17 depalmitoylases. b To engineer a chemogenetically-activated depalmitoylase, Abhd17c is split with FRB attached to the N-terminus, and FKBP to C-terminus. c AlphaFold models of FacePalm-17C with FKBP/FRB dimerization. d Confocal images show changes in CaVβ2A localization: (1) baseline (top), (2) Abhd17C (middle), and (3) Abhd17c S/A mutant. Scale bar, 10 µm e Confocal images show time-dependent changes in colocalization of FacePalm-17C NT (top) and CT (middle) and subsequent changes in CaVβ2A localization (bottom). Scale bar, 5 µm f Diary plot shows changes in Pearson’s correlation showing colocalization of FacePalm-17C NT and CT. Each dot, mean ± s.e.m. n = 12 cells from two independent transfections. g Diary plot quantifies normalized cytosolic fluorescence of CaVβ2A with FacePalm-17C (black) or with FacePalm-17C S/A mutant (red). Each dot, mean ± s.e.m. n = 35 (FacePalm-17C) and 11 (S/A mutant) from two independent transfections. h Acyl-RAC assay confirms FacePalm-17C induced a reduction in palmitoylation of CaVβ2A-YFP. Top, palmitoylated fraction. Bottom, total input. HA, hydroxylamine. Palmitoylated fraction and input are derived from the same experiment and processed in parallel. i Bar graph shows the relative density of palmitoylated CaVβ2A captured and eluted from resin compared to total input. Each bar, mean ± s.e.m from three independent trials. ****p < 0.0001 by unpaired two-tailed t-test. j Ba2+ current recordings of CaV2.2/CaVβ2A show minimal inactivation with (blue) and without (black) rapamycin. With FacePalm, rapamycin boosts inactivation of CaV2.2/CaVβ2A. k Bar graph summarizes changes in inactivation quantified as r800, the fraction of current remaining following 800 ms of depolarization. Each bar, mean ± s.e.m. ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison’s test. CaV2.2/CaVβ2A without FacePalm-17C (baseline, n = 6; +rapa, n = 5). CaV2.2/CaVβ2A with FacePalm-17C (baseline, n = 10; +rapa, n = 12). Source data are provided as a Source Data file.
