Fig 2: Gas and Gai are direct substrates of DHHC5. Palmitoylation of recombinant Gas (A), Gai (B), and GAPDH (C) in vitro with mock immunoprecipitation (-), catalytically inactive form of DHHC5 (DHHS5), and DHHC5 are shown. As a negative control, HA was added to cleave all thioester bonds. DHHC total input as determined by Western blotting is shown below the palmitoylation panels.

Fig 3: DHHC5 is required for signaling downstream of ß-adrenergic receptor (ß-?R) stimulation in cardiomyocytes. (A) Palmitoylation of Gas, Gai, and calnexin in control (Cont) and DHHC5 knockdown (KD) are shown. NRVMs treated with 10 µM ISO for the indicated time points are shown. (B) Fold changes of palmitoylation levels of Gas and Gai normalized to calnexin at the indicated time points compared to 0 min (n = 3) are shown. (C) Quantification of cellular cAMP levels before and after ISO (5 min) in Cont (black) and DHHC5 KD (red) NRVMs (n = 3) are shown. (D) Quantification of beating frequency monitored by Fura-2 before (baseline) and after ISO (+ISO) in Cont (black) and KD (red) cells (n = 5) is shown. *p < 0.05 (paired t-test). N.S. = not significant. To see this figure in color, go online.

Fig 4: Mechanism of Lomitapide’s inhibition of palmitoylation mediated by ZDHHC5.a Binding of Lomitapide and ZDHHC5 substrate domain. b Binding of SSTR5 (307–362 aa) and ZDHHC5 substrate domain. c Binding of Palmitic acid and ZDHHC5 substrate domain. d In the presence of 25 µM Lomitapide, binding of SSTR5 (307–362 aa) and ZDHHC5 substrate domain. e In the presence of 25 µM Lomitapide, binding of Palmitic acid and ZDHHC5 substrate domain. f Co-IP assay showing that SSTR5 interacted with ZDHHC5 in HEK293T cell, and lomitapide inhibits their combination. g As shown in the mechanism diagram, we propose that Lomitapide inhibits pancreatic cancer proliferation by competitively binding the ZDHHC5 substrate to the binding region of SSTR5 and Palmitic acid, preventing palmitoylation.

Fig 5: Single-cell sequencing of normal pancreatic tissue and pancreatic cancer tissue, and TCGA database analysis.a Flow chart of single cell sequencing. b Single cell sequencing of normal pancreas tissue, t-SNE analysis. c Single cell sequencing of tumor sample, t-SNE analysis. d, f ZDHHC5 expressing in normal pancreas tissue (heatmap and t-SNE analysis). e, g ZDHHC5 expressing in tumor sample (heatmap and t-SNE analysis). h TCGA public pancreatic cancer samples, the survival rate of ZDHHC5 (high expression, n = 45) patients and ZDHHC5 (low expression, n = 132) patients (upper) and the expression level of ZDHHC5 in pancreatic cancer patients (n = 178) or normal pancreas samples (n = 4).