Fig 2: PPT1 depalmitoylates the IgG domain of synaptic adhesion molecules.4. (A) Domain organization of IgG domain-containing synaptic adhesion molecules identified as high-confidence PPT1 substrates with palmitoylation sites represented as lipid chains. IG-L–Ig-like; FN3 –fibronectin 3; TM–transmembrane. (B) IgG domain-containing synaptic adhesion molecules show high homology surrounding the palmitoylated cysteines. Red indicates high homology; green indicates low homology. The 2 conserved cysteines at the N-terminal (light blue) and carboxyl terminus (dark blue) of this domain are both identified as carbamidomethylated in different experiments. (C) Representative IgG domain structure (CADM2; PDB, 3M45) with position of palmitoylated cysteines highlighted in blue. (D) Activity of PPT1 enzyme measured from WT or PPT1 KO primary neuronal culture medium. Data presented as mean relative fluorescence normalized to total protein concentration ± SD (n = 6 cultures per genotype; ** p > 0.01; S1 Data). (E) Endogenous syncam2 (CADM2) and synaptophysin 1 (SYPH) colocalize on MAP2+ neurites in WT and PPT1 KO mouse primary neuronal cultures (Scale bars: 10 µm; enlarged ROIs). (F) Quantifications of syncam2 and synaptophysin 1 puncta number (per 10-µm neurite segment), average puncta size (µm2), and colocalization (Pearson correlation coefficient). Data presented as mean of 5 ROIs per culture ± SD (n = 4 cultures; * p > 0.5; S1 Data). Ig, Immunoglobulin; KO, knockout; PPT1, palmitoyl protein thioesterase 1; ROS, region of interest; WT, wild-type.

Fig 3: Generation of the proteome and palmitome from WT and PPT1 KO brains.(A) Proteins were isolated from WT and PPT1 KO whole brains or synaptosomes for proteomic analysis by LFQ-MS to identify overall protein expression (proteome; left) or following Acyl RAC to identify palmitoylated proteins (palmitome; right). (B) Schematic of Acyl RAC. (i) Disulfide bonds were reduced with TCEP and residual thiol groups were blocked with NEM. (ii) Palmitate groups were hydrolyzed with HA, leaving behind an unblocked free thiol. (iii) Free thiols were bound to thiopropyl sepharose beads, allowing for isolation of palmitoylated proteins. These proteins were then (iv) eluted from beads with DTT, (v) digested, and (vi) alkylated with iodoacetamide, followed by (vii) LFQ-MS/MS analysis. (C) Pull-down and immunoblotting of palmitoylated proteins. Addition of HA allowed CSPa, dynamin-1, and SNAP-25, all of which are palmitoylated proteins, to be pulled down on thiopropyl sepharose beads, but not endophilin or actin, which are not palmitoylated. Note that addition of HA removes palmitate and causes a molecular weight shift of palmitoylated CSPa to depalmitoylated CSPa. (D) Quantification of western blot +HA band intensity (A.U), plotted as a ratio of KO/WT (S1 Data). CSPa is a known PPT1 substrate and shows elevated palmitoylation in KO/WT, while SNAP-25 does not (n = 3 to 9 experiments). Acyl RAC, Acyl Resin-Assisted Capture; HA, hydroxylamine; KO, knockout; LFQ-MS, Label-Free Quantification Mass Spectrometry; NEM, N-ethylmaleimide; PPT1, palmitoyl protein thioesterase 1; TCEP, tris(2-carboxyethyl)phosphine; WT, wild-type.

Fig 4: PPT1-mediated depalmitoylation facilitates disulfide bond formation.(A) Schematic of modified Acyl RAC. (i) Free thiols were blocked with NEM prior to reduction of disulfide bonds with TCEP. (ii) Free thiols generated by TCEP treatment were modified with heavy-labeled NEM (dNEM). iii. Acyl RAC was then conducted as previously described (Fig 1Bii–iv). (B) Pipeline for PTM analysis of Acyl RAC peptide data. Peptides were aggregated from 3 independent proteomic screens with 3 biological replicates. All peptides were filtered by individual PTMs or by coincidence signatures where 2 distinct PTMs were found to modify the same cysteine residue. (C) Global distribution of modified peptides. Bars represent percent (%) average normalized abundance of the different peptide classes. The value of each replicate is calculated as the summed abundance of peptides with the given PTM normalized to the total abundance for all detected peptides (S1 Data). Cysteine residues with PTMs are interpreted as follows: carbamidomethyl (carba.) = palmitate, NEM = free thiol, dNEM = disulfide bond, other or no moieties (Other/None). (D–F) Ratios of PTM normalized abundance for peptides where 2 PTMs are found to modify the same cysteine residue. The value of each replicate is calculated as the summed abundance of peptides with the first PTM divided by the summed normalized abundance of peptides with the second PTM (mean ± SD; * p < 0.05, unpaired, 2-tailed t test; S1 Data). (G) Peptide analysis of a high-confidence PPT1 substrate, Sodium/Potassium transporting ATPase subunit ß1 (AT1B2). Normalized abundance fold change (PPT1 KO/WT) of carbamidomethyl AT1B2 peptides (1.5-fold change indicated by blue dashed line). Palmitoylated cysteine sites marked with an asterisk (*) were confirmed to be PPT1 substrates in the validation screen. There is a persistence of palmitates on cysteines that normally participate in disulfide bonds in PPT1 KO (C129, C160, C177, and C261; S5 Table), while C10, which is not predicted to form a disulfide linkage, appears unchanged (S1 Data). (H) Detected AT1B2 peptides are indicated. For those sequences with cysteines predicted to form disulfide bonds, carba. and dNEM moieties were found to modify that cysteine residue. (I) Ribbon structure of AT1B2 (SWISS-MODEL; P14231) with disulfide bonds highlighted. Bar graph and peptide sequences are color coded according to location in the ribbon structure. Acyl RAC, Acyl Resin-Assisted Capture; dNEM, d5-N-ethylmaleimide; KO, knockout; NEM, N-ethylmaleimide; PPT1, palmitoyl protein thioesterase 1; PTM, posttranslational modification; TCEP, tris(2-carboxyethyl)phosphine; WT, wild-type.

Fig 5: Putative PPT1 substrates are enriched at the synapse and display persistent palmitoylation.(A) Venn diagram of 3 palmitome experiments exhibits 1,379 common proteins. (B) Volcano plot indicates 242 proteins are significantly differentially palmitoylated (4 decrease; 238 increase; 1.5-fold, p < 0.05; blue lines; S2 Table, S1 Data). Inset shows significantly increased proteins. The p-value was calculated using a 2-tailed t test on 3 biological and 3 technical replicates. While the technical replicates do not meet the t test condition for independence, we chose to proceed in this manner to generate more putative substrates that could later be validated with higher stringency. (C) Venn diagram of 3 independent proteome experiments exhibits 1,826 common proteins. (D) Volcano plot of fold change between genotypes (PPT1 KO/WT littermates) for proteins identified in all 3 biological replicates. A total of 11 proteins are significantly differentially expressed (1 decrease (PPT1); 10 increase; 1.5-fold, p < 0.05; blue lines; S1 Data). Other depalmitoylating enzymes (green points) do not display significant compensatory up-regulation of protein expression. (E) Palmitoylated protein expression is somewhat correlated between genotypes (R2 = 0.899; m = 1.134 ± 0.0197), with increased palmitoylation in PPT1 KO (S1 Data). (F) Protein expression is almost perfectly correlated between genotypes (R2 = 0.9966; m = 0.969 ± 0.0016; S1 Data). Red lines indicate 1:1 WT to PPT1 KO protein expression ratio. (G) Venn diagram of 1,209 common proteins between whole brain proteome and palmitome (n = 3 each). (H) Protein expression levels compared to palmitoylation levels for significantly changed proteins in palmitome. Proteins in orange region are increased (1.5-fold, p < 0.05) in both proteome and palmitome. Proteins in blue region display decreased or unchanged protein expression and increased palmitoylation. Red line indicates equal expression and palmitoylation levels (x = y). While NNTM has a high fold change, it does not meet the p-value criterion for significantly increased protein expression (p = 0.0522) and is therefore excluded from this category (S1 Data). ASAH1, Acid Ceramidase; CATD, Cathepsin D; KO, knockout; PPT1, palmitoyl protein thioesterase 1; TPP1, Tripeptidyl Peptidase 1; WT, wild-type.