Fig 2: Attenuated 5-HT1AR palmitoylation in the prefrontal cortex (PFC) of individuals with major depressive disorder (MDD) who died by suicide correlates with reduced expression of ZDHHC21. a 5-HT1AR palmitoylation in the PFC and cerebellum from the control and individuals with MDD who died by suicide (PFC, n = 5; cerebellum, n = 4). b Normalized level of 5-HT1AR palmitoylation in the PFC and cerebellum of individuals with MDD who died by suicide in comparison with control subjects (two-tailed t test; see also Supplementary Fig. 8). c Relative expression levels of ZDHHC5, -9, and -21 in the PFC (left) and cerebellum (right) of control (n = 14) and individuals with MDD who died by suicide (n = 12). Data points represent the means ± SEM (*P < 0.05; ***P < 0.001; two-tailed t test). d N1E cells were transfected with microRNAs as indicated and the expression of ZDHHC21 was determined by reverse transcriptase-PCR (see also Supplementary Table 1). Data points represent mean ± SEM from at least three independent experiments (*P < 0.05; ***P < 0.001; two-tailed t test). e N1E cells were transfected with 5-HT1AR along with miR-30e, miR30-a, or shRNA against ZDHHC21. Palmitoylation of 5-HT1AR was analyzed by acyl-biotinyl exchange. Western blots shown are representative of at least three independent experiments. f Analysis of miR-30a, -30e, and -200a expression in the PFC of control and individuals with MDD who died by suicide. (*P < 0.05, **P < 0.01; two-tailed t test). Source data are available as a Source Data file

Fig 3: DHHC5 is a putative palmitoyl acyltransferase for Cdc42-palm.A, representative Western blots of ABE experiments to validate palmitoylation of Cdc42 after overexpression of either WT or dominant-negative (DN) mutant of HA-tagged DHHC5. The upper panel represents the palmitoylation of Cdc42-palm; middle panel shows the expression of GFP-tagged Cdc42. In both cases, anti-GFP antibody was used for detection. The lowest panel shows expression of HA-tagged DHHC5 protein (detected with anti-HA antibody). B, quantification of the relative palmitoylation level of GFP-tagged Cdc42 in the presence of DHHC5 WT or its DN mutant. N = 4, one-way ANOVA with Holm–Sidak's multiple comparisons tests. *p < 0.05, **p < 0.01, ***p < 0.0001. C, analysis of the global palmitoylation levels assessed after quantification of the silver staining gels (D) after ABE. ABE, acyl-biotinyl exchange; HA, hemagglutinin.

Fig 4: Functional impact of DHHC5-mediated Cdc42 palmitoylation of gene transcription.A, schematic representation of dual-luciferase reporter assay. B, neuroblastoma N1E-115 cells were transiently transfected with constructs indicated along with the SRE-driven luciferase reporter construct as well as CMV-driven reporter construct. Luciferase activity was measured 16 h post-transfection. The results are shown as mean ± SD from nine experiments. *p < 0.01; ****p < 0.0001; two-way ANOVA with Bonferroni’s multiple comparison test. CMV, cytomegalovirus promotor; Fire, Firefly luciferase; Ren, Renilla luciferase, SRE, serum response element; SRF, serum response factor.

Fig 5: Interaction between DHHC5 and Cdc42 in neuroblastoma cells.A, N1E-115 cells were transfected with GFP-tagged DHHC5 WT or its dominant negative mutant (DN) together with mCherry-tagged Cdc42 WT or its cysteine mutants, as indicated. Subcellular distribution of recombinant proteins was analyzed in living cell by confocal microscopy. Representative images are shown. Corresponding fluorescence intensity profiles display localization of GFP-DHHC5 (green) and mCherry-Cdc42 (red) within individual cells. The scale bar represents 20 µm. B, quantification of the fluorescence intensity ratio for the coexpressed proteins. The plasma membrane region (assessed by DHHC5 channel) and the whole cell were selected for analyses as two different regions of interest (ROIs). The integrated density (area × mean intensity) was quantified in each ROI for mCherry-Cdc42 channel. After that, the fraction Cdc42-palm resided at the plasma membrane in comparison to whole cell Cdc42-palm intensity was calculated; *p < 0.05,< ****p < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparison test. C, N1E-115 cells were cotransfected with HA-tagged DHHC5 and indicated GFP-tagged Cdc42, followed by immunoprecipitation (IP) with an anti-GFP antibody and Western blot with anti-HA antibody. As a control, mixed lysates from the single transfected cells (mix) were applied to IP. Left: input (expression) for the immunoprecipitation, top: DHHC5 detected with anti-HA antibody. Second panel: expression of Cdc42 variants and corresponding mutants detected with anti-GFP antibody. Similar order in the single transfections below on the left. Right: proteins detected after IP(co-IP) with anti-GFP antibody. Top panel: immunoprecipitated HA-tagged DHHC5 visualized with anti-HA antibody. Second panel: Cdc42 variants detected with anti-GFP antibody. Similar order in the single transfections below on the right. Representative Western blots are shown (N = 3). co-IP, coimmunoprecipitation; HA, hemagglutinin.