{"gene":"ZDHHC20","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2016,"finding":"DHHC20 palmitoylates EGFR at cysteine residues within its unstructured C-terminal tail; this palmitoylation 'pins' the C-terminal tail to the plasma membrane, impeding EGFR activation. Mutation of these cysteine residues to alanine is sufficient to activate EGFR signaling, promote cell migration and transformation, and sensitize cells to EGFR tyrosine kinase inhibition.","method":"Mass spectrometry identification of palmitoylated cysteines, site-directed mutagenesis (Cys→Ala), loss-of-function (DHHC20 inhibition), cell migration and transformation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal identification of palmitoylation sites by MS, mutagenesis validation, functional phenotypic readout, replicated across multiple cell lines","pmids":["27153536"],"is_preprint":false},{"year":2017,"finding":"ZDHHC20 enhances IFITM3 palmitoylation and uniquely increases IFITM3 antiviral activity (against influenza) when both are co-overexpressed. ZDHHC20 co-localizes with IFITM3 at lysosomes, unlike ZDHHC3/7/15 which show perinuclear localization, suggesting that the subcellular site of palmitoylation influences IFITM3 activity. Combined knockdown of ZDHHC3 and ZDHHC7 in ZDHHC20-knockout cells was required to reduce endogenous IFITM3 palmitoylation, demonstrating functional redundancy among ZDHHCs.","method":"Overexpression screen (23 mammalian ZDHHCs), ZDHHC knockout cell line library, siRNA knockdown, metabolic palmitoylation labeling, viral infection assay, co-localization by fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — orthogonal overexpression and knockout/knockdown approaches, direct palmitoylation assay, antiviral functional readout, multiple methods in one study","pmids":["29079573"],"is_preprint":false},{"year":2019,"finding":"Human DHHC20 is catalytically active when reconstituted in POPC nanodiscs. Molecular dynamics simulations reveal that DHHC20 induces a drastic deformation/invagination of the cytoplasmic leaflet of the lipid membrane, causing the catalytic Cys (within the DHHC motif) to become hydrated and optimally positioned to undergo autoacylation upon encountering acyl-CoA. This membrane reshaping resolves the paradox of a nucleophilic Cys being exposed to a hydrophobic membrane environment.","method":"Biochemical reconstitution in POPC nanodiscs, microsecond all-atom molecular dynamics simulations","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution plus MD simulation in a single study; computational model not validated by mutagenesis in this paper","pmids":["31858978"],"is_preprint":false},{"year":2021,"finding":"ZDHHC20 mediates S-acylation of the ORAI1 Ca2+ channel at Cys143. This palmitoylation targets ORAI1 to cholesterol-rich lipid raft domains and is required for efficient TCR recruitment and signaling at the immune synapse. Cys143 mutations reduced ORAI1 currents, store-operated Ca2+ entry, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement; the acylation-deficient channel was also recruited less efficiently to the immune synapse along with actin and TCR.","method":"Site-directed mutagenesis (Cys143), patch-clamp electrophysiology, Ca2+ imaging, NFATC1 nuclear translocation assay, IL-2 ELISA, enforced ZDHHC20 overexpression, lipid raft fractionation, live-cell fluorescence microscopy at immune synapse","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis of palmitoylation site, multiple orthogonal functional readouts (currents, Ca2+ entry, cytokine secretion, IS recruitment), mechanistic pathway placement","pmids":["34913437"],"is_preprint":false},{"year":2022,"finding":"Molecular dynamics of membrane-embedded hDHHC20 shows that among acyl-CoA chains of various lengths, only C16 (palmitoyl) adopts a conformation suitable for hDHHC20 autoacylation within the hydrophobic cavity formed by four transmembrane helices. A V185G mutant shifts preference to C18, demonstrating that residues at the cavity ceiling determine acyl-chain length selectivity. An unusual hydrophilic ridge in TM helix 4 of DHHC20 may mediate association with substrate protein TM domains.","method":"Molecular dynamics simulation of membrane-embedded hDHHC20 and mutants with various acyl-CoAs; mutagenesis of cavity residues; experimental validation of spike protein acylation by S→A mutagenesis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — MD-based mechanistic model with partial mutagenesis validation; single study, limited experimental corroboration","pmids":["35563480"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 palmitoylates YTHDF3 at Cys474, preventing its chaperone-mediated autophagic (CMA) degradation and causing accumulation of YTHDF3 protein. Stabilized YTHDF3 in turn leads to abnormal accumulation of MYC mRNA/protein, promoting malignant phenotypes in pancreatic cancer. A YTHDF3-derived peptide competitively inhibiting ZDHHC20-mediated YTHDF3 palmitoylation downregulates MYC and inhibits KRAS-mutant pancreatic cancer progression.","method":"KPC mouse model, acyl-biotin exchange assay, site-directed mutagenesis (Cys474), co-immunoprecipitation, protein half-life assay, competitive peptide inhibitor, in vivo tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct identification of palmitoylation site by ABE plus mutagenesis, mechanistic link to CMA degradation and MYC accumulation, validated in vivo with multiple orthogonal methods","pmids":["38821916"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 palmitoylates fatty acid synthase (FASN) at Cys1471 and Cys1881. This palmitoylation competes with ubiquitination at these sites, blocking FASN degradation via the E3 ubiquitin ligase complex SNX8-TRIM28. ZDHHC20 knockout or pharmacological inhibition, or FASN C1471S/C1881S mutation, accelerates FASN degradation and reduces hepatocarcinogenesis in two chemical carcinogen mouse models.","method":"ZDHHC20 knockout mice (DEN and DEN/CCl4 HCC models), palmitoylation LC-MS, acyl-biotin exchange assay, co-immunoprecipitation, ubiquitination assay, protein half-life assay, site-directed mutagenesis (C1471S/C1881S), immunofluorescence microscopy","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout models, direct palmitoylation site identification by MS+ABE, mutagenesis, ubiquitination competition mechanism defined with multiple orthogonal methods","pmids":["39696259"],"is_preprint":false},{"year":2024,"finding":"zDHHC20 palmitoylates CD80 at Cys261/262/266/271 (transmembrane and cytoplasmic regions). This palmitoylation protects CD80 from ubiquitin-mediated proteasomal degradation, ensures accurate plasma membrane localization, and is required for CD80 costimulatory function in T cell activation. Palmitoylation-deficient CD80 (4CS mutant) loses membrane localization and fails to costimulate T cells.","method":"Metabolic palmitoylation labeling, site-directed mutagenesis (4CS), co-immunoprecipitation, ubiquitination assay, protein stability assay, immunofluorescence, T cell co-stimulation assay","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct palmitoylation site identification, mutagenesis, ubiquitination and stability assays, functional T cell readout; single lab but multiple orthogonal methods","pmids":["38467718"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 was identified in an in vivo shRNA screen as critical for pancreatic cancer metastatic outgrowth. This pro-metastatic function is abrogated in immunocompromised animals and in animals depleted of NK cells, placing ZDHHC20-dependent palmitoylation in the regulation of tumor-innate immune interactions required for distant metastasis. A chemical genetics substrate profiling platform identified multiple novel ZDHHC20 substrates.","method":"In vivo shRNA screen, immunocompromised and NK-cell-depleted mouse models, chemical genetics substrate profiling platform","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic screen with mechanistic follow-up (immune cell depletion), substrate profiling; single study, substrates not fully validated individually","pmids":["38733589"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 overexpression increases phosphorylation of PI3K and AKT in hepatocellular carcinoma cells, while ZDHHC20 knockdown decreases PI3K and AKT phosphorylation. PI3K-AKT pathway inhibitors (LY294002 and MK2206) block the pro-proliferative effect of ZDHHC20 overexpression, placing ZDHHC20 upstream of the PI3K-AKT pathway in HCC.","method":"siRNA knockdown, overexpression, Western blotting for PI3K/AKT phosphorylation, pharmacological inhibition, xenograft mouse model","journal":"Journal of hepatocellular carcinoma","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic epistasis via pharmacological inhibition without direct substrate identification; single lab, no direct palmitoylation mechanism defined","pmids":["39309302"],"is_preprint":false},{"year":2025,"finding":"ZDHHC20 palmitoylates KAP1/TRIM28 at Cys232. This palmitoylation increases chromatin binding of phosphorylated KAP1, enhancing chromatin accessibility and recruitment of DNA damage response components BRCA1 and 53BP1. ATM kinase phosphorylates ZDHHC20 at Ser339 in response to DNA damage, which in turn increases KAP1 palmitoylation, defining an ATM-ZDHHC20-KAP1 axis in the DNA damage response. ZDHHC20 knockout enhances radiosensitivity in mice, tumor cell lines, and xenograft models.","method":"ZDHHC family screen, palmitoylation label-free quantitative proteomics, acyl-biotin exchange (ABE) assay, site-directed mutagenesis (Cys232), chromatin fractionation, BRCA1/53BP1 recruitment assay, ATM phosphorylation site mapping (Ser339), ZDHHC20 knockout mice, xenograft models, radiosensitivity assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct palmitoylation site by ABE+proteomics, upstream kinase (ATM) identified, downstream chromatin mechanism defined, multiple in vivo models; multiple orthogonal methods in one study","pmids":["41109928"],"is_preprint":false},{"year":2026,"finding":"MEF2A transcriptionally activates ZDHHC20 (validated by dual-luciferase reporter and ChIP assays). ZDHHC20 in turn activates the NF-κB signaling pathway, promoting AML cell proliferation and doxorubicin resistance. Overexpression of ZDHHC20 reverses the antitumor effects of MEF2A silencing.","method":"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), siRNA knockdown, overexpression rescue, Western blotting, xenograft mouse model","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct transcriptional regulation validated by ChIP and reporter assay, rescue experiment; NF-κB pathway activation shown but direct palmitoylation substrate not identified","pmids":["41882733"],"is_preprint":false},{"year":2026,"finding":"ZDHHC20 palmitoylates CMPK2 at Cys137 and Cys153, maintaining CMPK2 mitochondrial localization. This palmitoylation is reversed by the thioesterase PPT1. CMPK2 palmitoylation supports ddhCTP production and MAVS stabilization, enabling IFN-I production and antiviral immunity. Palmitic acid activates this pathway; PPT1 deficiency restores CMPK2 palmitoylation and antiviral immunity.","method":"Chemical library screen, palmitoylation assay, site-directed mutagenesis (Cys137/153), subcellular fractionation/mitochondrial localization assay, CMPK2 knockout, PPT1 knockout/inhibition, IFN-I production assay, viral replication assay, in vivo diet and pharmacological intervention","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct palmitoylation site identification with mutagenesis, writer (ZDHHC20) and eraser (PPT1) both identified, mitochondrial localization consequence defined, multiple in vivo validations","pmids":["42011944"],"is_preprint":false},{"year":2026,"finding":"RAB11A acts as a scaffold that recruits ZDHHC20 to promote FGFR3 palmitoylation, inhibiting FGFR3 degradation and retaining it at the plasma membrane in bladder cancer cells. SREBP2 directly activates RAB11A transcription (dependent on its liquid-liquid phase separation capability), placing ZDHHC20 downstream of a SREBP2-RAB11A axis. Knockdown of ZDHHC20 or RAB11A reduced membrane FGFR3 and attenuated tumor growth in xenografts.","method":"Co-immunoprecipitation (RAB11A-ZDHHC20 interaction), promoter-reporter assay (SREBP2→RAB11A), SREBP2 phase-separation mutant (F178A), RAB11A/ZDHHC20 knockdown, immunoblotting for membrane FGFR3, xenograft tumor models","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for scaffold interaction, reporter assay for transcriptional activation, in vivo xenograft; direct FGFR3 palmitoylation site not mapped in this abstract","pmids":["41586981"],"is_preprint":false},{"year":2024,"finding":"A selective, orally bioavailable small-molecule inhibitor SD-066-4 inhibits ZDHHC20 acyltransferase activity. A specific alanine residue in ZDHHC20 accommodates the methyl group of SD-066-4, providing isoform selectivity over other ZDHHCs. SD-066-4 stably reduces EGFR S-acylation in KRAS-mutant cells and blocks growth of KRAS-mutant lung tumors, extending overall survival in a mouse model.","method":"Selective inhibitor identification, mutagenesis identifying selectivity-determining alanine residue, EGFR palmitoylation assay, KRAS-mutant lung tumor mouse model, survival analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with isoform selectivity residue identified by mutagenesis, in vivo tumor model; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.07.18.604152"],"is_preprint":true}],"current_model":"ZDHHC20 is a plasma membrane and lysosome-localized integral membrane palmitoyltransferase that autoacylates via its conserved DHHC catalytic cysteine—facilitated by enzyme-induced membrane deformation that hydrates the catalytic site—and then transfers C16 acyl chains to substrate cysteine residues on diverse proteins including EGFR (C-terminal tail), ORAI1 (Cys143), IFITM3, YTHDF3 (Cys474), FASN (Cys1471/1881), KAP1/TRIM28 (Cys232), CMPK2 (Cys137/153), CD80, and FGFR3, thereby regulating their membrane localization, protein stability (by competing with ubiquitination), and signaling activity in processes spanning oncogenic RTK signaling, Ca2+-dependent immune synapse function, antiviral immunity, and DNA damage repair; ZDHHC20 itself is regulated upstream by ATM-mediated phosphorylation (Ser339) and transcriptional activation by MEF2A, and its activity can be selectively inhibited by acrylamide-based or small-molecule inhibitors that exploit a selectivity-determining alanine residue."},"narrative":{"mechanistic_narrative":"ZDHHC20 is an integral-membrane DHHC palmitoyltransferase that controls the membrane localization, stability, and signaling output of a broad range of substrates by transferring C16 (palmitoyl) acyl chains onto target cysteines [PMID:27153536, PMID:39696259]. Catalysis proceeds through autoacylation at the DHHC-motif cysteine: the enzyme induces a drastic invagination of the cytoplasmic membrane leaflet that hydrates the catalytic cysteine and positions it to react with acyl-CoA [PMID:31858978], and a hydrophobic cavity formed by its transmembrane helices selects specifically for the C16 chain length, with cavity-ceiling residues such as Val185 dictating this preference [PMID:35563480]. A recurring functional logic is that ZDHHC20-mediated palmitoylation competes with ubiquitination to stabilize its substrates—blocking SNX8-TRIM28-dependent degradation of FASN at Cys1471/Cys1881 [PMID:39696259], protecting CD80 from proteasomal turnover [PMID:38467718], and preventing chaperone-mediated autophagic degradation of YTHDF3 at Cys474 to drive MYC accumulation in pancreatic cancer [PMID:38821916]. Through distinct substrates ZDHHC20 governs several processes: it pins the EGFR C-terminal tail to the plasma membrane to restrain receptor activation [PMID:27153536], targets the ORAI1 Ca2+ channel (Cys143) to lipid rafts for immune-synapse signaling [PMID:34913437], promotes IFITM3 antiviral activity at lysosomes [PMID:29079573], sustains CMPK2 mitochondrial localization and MAVS-dependent type I interferon responses [PMID:42011944], and palmitoylates KAP1/TRIM28 (Cys232) to enhance chromatin accessibility and DNA damage repair [PMID:41109928]. ZDHHC20 is itself regulated upstream by ATM-mediated phosphorylation at Ser339 in the DNA damage response [PMID:41109928] and by MEF2A-driven transcription [PMID:41882733], and its activity is selectively druggable via a single isoform-determining alanine residue exploited by small-molecule inhibitors [PMID:bio_10.1101_2024.07.18.604152].","teleology":[{"year":2016,"claim":"Established ZDHHC20 as a functional palmitoyltransferase with a defined substrate, showing that palmitoylation of the EGFR C-terminal tail acts as a brake on receptor activation rather than a passive modification.","evidence":"Mass-spectrometry site identification, Cys→Ala mutagenesis, and migration/transformation assays across cell lines","pmids":["27153536"],"confidence":"High","gaps":["Did not resolve the structural basis of catalysis","Substrate range beyond EGFR unknown at the time"]},{"year":2017,"claim":"Showed that the subcellular site of palmitoylation matters, with ZDHHC20 uniquely enhancing IFITM3 antiviral activity at lysosomes and acting redundantly with other ZDHHCs on endogenous IFITM3.","evidence":"23-enzyme overexpression screen, ZDHHC knockout/knockdown library, metabolic palmitoylation labeling, viral infection and colocalization assays","pmids":["29079573"],"confidence":"High","gaps":["IFITM3 palmitoylation site selectivity by ZDHHC20 not mapped","Mechanism linking localization to antiviral potency not fully defined"]},{"year":2019,"claim":"Resolved how a nucleophilic catalytic cysteine functions within a hydrophobic membrane by showing the enzyme deforms the lipid leaflet to hydrate and position the DHHC cysteine for autoacylation.","evidence":"Reconstitution in POPC nanodiscs and microsecond all-atom molecular dynamics simulations","pmids":["31858978"],"confidence":"Medium","gaps":["MD model not validated by mutagenesis in this study","Substrate hand-off step not addressed"]},{"year":2021,"claim":"Connected ZDHHC20 catalysis to immune-synapse signaling by showing ORAI1 Cys143 palmitoylation directs the channel to lipid rafts required for store-operated Ca2+ entry and TCR-evoked cytokine output.","evidence":"Cys143 mutagenesis, patch-clamp, Ca2+ imaging, NFATC1 translocation, IL-2 ELISA, lipid raft fractionation, live-cell imaging","pmids":["34913437"],"confidence":"High","gaps":["Whether ZDHHC20 is the sole ORAI1 acyltransferase not established","Erasers/turnover of ORAI1 palmitoylation unknown"]},{"year":2022,"claim":"Explained the enzyme's strict C16 acyl-chain preference by identifying a transmembrane-helix cavity whose ceiling residues set chain-length selectivity, with V185G shifting preference to C18.","evidence":"Molecular dynamics of membrane-embedded hDHHC20 and cavity mutants, plus spike-protein acylation S→A validation","pmids":["35563480"],"confidence":"Medium","gaps":["Proposed TM4 hydrophilic ridge role in substrate recognition not experimentally proven","Limited experimental corroboration of the MD model"]},{"year":2024,"claim":"Defined a recurring stabilization mechanism in which ZDHHC20 palmitoylation competes with ubiquitin- or autophagy-mediated degradation, demonstrated on FASN, CD80, and YTHDF3 to drive cancer and immune phenotypes.","evidence":"Knockout mice, LC-MS/ABE site mapping, mutagenesis, ubiquitination and protein half-life assays, T-cell costimulation and in vivo tumor models","pmids":["39696259","38467718","38821916"],"confidence":"High","gaps":["Whether palmitoylation directly occludes ubiquitin sites or acts allosterically not resolved for all substrates","Substrate selection rules among many targets unclear"]},{"year":2024,"claim":"Placed ZDHHC20 in tumor-innate immune crosstalk by identifying it in an in vivo screen as required for pancreatic metastatic outgrowth in an NK-cell-dependent manner, and broadened its substrate repertoire.","evidence":"In vivo shRNA screen, immunocompromised and NK-depleted mouse models, chemical-genetics substrate profiling","pmids":["38733589"],"confidence":"Medium","gaps":["The palmitoylation substrate(s) mediating the NK-dependent effect not individually validated","Direct molecular link to NK recognition unknown"]},{"year":2024,"claim":"Identified a selective, orally bioavailable inhibitor and the structural determinant of its isoform selectivity, showing ZDHHC20 is pharmacologically tractable in KRAS-mutant tumors.","evidence":"Inhibitor SD-066-4 characterization, mutagenesis of selectivity-determining alanine, EGFR palmitoylation assay, KRAS-mutant lung tumor model (preprint)","pmids":["bio_10.1101_2024.07.18.604152"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","On-target selectivity across all ZDHHC isoforms not exhaustively profiled"]},{"year":2025,"claim":"Wired ZDHHC20 into the DNA damage response by showing ATM phosphorylates it at Ser339, which boosts KAP1/TRIM28 Cys232 palmitoylation to enhance chromatin accessibility and DDR factor recruitment.","evidence":"ZDHHC family screen, palmitoylation proteomics + ABE, Cys232 mutagenesis, chromatin fractionation, BRCA1/53BP1 recruitment, ATM site mapping, knockout mice and xenografts","pmids":["41109928"],"confidence":"High","gaps":["How Ser339 phosphorylation alters enzyme activity mechanistically unclear","Whether other DDR substrates exist not addressed"]},{"year":2026,"claim":"Extended ZDHHC20 into antiviral metabolism and identified its eraser, showing CMPK2 Cys137/153 palmitoylation (reversed by PPT1) maintains mitochondrial localization and supports MAVS-dependent type I interferon.","evidence":"Chemical library screen, palmitoylation/mutagenesis, mitochondrial fractionation, CMPK2 and PPT1 knockout, IFN-I and viral replication assays, in vivo diet/pharmacological intervention","pmids":["42011944"],"confidence":"High","gaps":["Dynamics of the ZDHHC20/PPT1 writer-eraser cycle not quantified","How palmitate availability tunes the pathway in vivo not fully defined"]},{"year":2026,"claim":"Identified transcriptional and scaffolding control of ZDHHC20, with MEF2A driving its expression and a SREBP2-RAB11A axis recruiting it to palmitoylate and stabilize membrane FGFR3.","evidence":"Dual-luciferase/ChIP for MEF2A, Co-IP for RAB11A-ZDHHC20, SREBP2 phase-separation mutant, knockdown and xenograft models","pmids":["41882733","41586981"],"confidence":"Medium","gaps":["FGFR3 palmitoylation site not mapped","Direct NF-κB substrate of ZDHHC20 in AML not identified"]},{"year":null,"claim":"How ZDHHC20 selects its diverse substrates and how its catalytic activity is integrated with its many upstream regulators remains unresolved.","evidence":"No single study defines the substrate-recognition code or a unified regulatory model","pmids":[],"confidence":"Low","gaps":["No structural model of substrate engagement","Substrate-selection determinants among dozens of targets unknown","Interplay between ATM phosphorylation, scaffolds, and erasers not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,6,7,5,3,10,12]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,10]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,7,13]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,7,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,3,12]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,6,8,13]}],"complexes":[],"partners":["EGFR","ORAI1","IFITM3","YTHDF3","FASN","TRIM28","CMPK2","RAB11A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5W0Z9","full_name":"Palmitoyltransferase ZDHHC20","aliases":["Acyltransferase ZDHHC20","DHHC domain-containing cysteine-rich protein 20","DHHC20","Zinc finger DHHC domain-containing protein 20"],"length_aa":365,"mass_kda":42.3,"function":"Palmitoyltransferase that could catalyze the addition of palmitate onto various protein substrates (PubMed:27153536, PubMed:29326245, PubMed:33219126). Catalyzes palmitoylation of Cys residues in the cytoplasmic C-terminus of EGFR, and modulates the duration of EGFR signaling by modulating palmitoylation-dependent EGFR internalization and degradation (PubMed:27153536). Has a preference for acyl-CoA with C16 fatty acid chains (PubMed:29326245). Can also utilize acyl-CoA with C14 and C18 fatty acid chains (PubMed:29326245). May palmitoylate CALHM1 subunit of gustatory voltage-gated ion channels and modulate channel gating and kinetics (Microbial infection) Dominant palmitoyltransferase responsible for lipidation of SARS coronavirus-2/SARS-CoV-2 spike protein. Through a sequential action with ZDHHC9, rapidly and efficiently palmitoylates spike protein following its synthesis in the endoplasmic reticulum (ER). In the infected cell, promotes spike biogenesis by protecting it from premature ER degradation, increases half-life and controls the lipid organization of its immediate membrane environment. Once the virus has formed, spike palmitoylation controls fusion with the target cell","subcellular_location":"Golgi apparatus membrane; Cell membrane; Cytoplasm, perinuclear region; Endoplasmic reticulum membrane; Endoplasmic reticulum-Golgi intermediate compartment membrane","url":"https://www.uniprot.org/uniprotkb/Q5W0Z9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZDHHC20","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ZDHHC20","total_profiled":1310},"omim":[{"mim_id":"620907","title":"METALLO-BETA-LACTAMASE DOMAIN-CONTAINING PROTEIN 2; MBLAC2","url":"https://www.omim.org/entry/620907"},{"mim_id":"617972","title":"ZDHHC PALMITOYLTRANSFERASE 20; ZDHHC20","url":"https://www.omim.org/entry/617972"},{"mim_id":"131550","title":"EPIDERMAL GROWTH FACTOR RECEPTOR; EGFR","url":"https://www.omim.org/entry/131550"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZDHHC20"},"hgnc":{"alias_symbol":["FLJ25952","DHHC20"],"prev_symbol":[]},"alphafold":{"accession":"Q5W0Z9","domains":[{"cath_id":"-","chopping":"9-77_160-224","consensus_level":"medium","plddt":96.8327,"start":9,"end":224},{"cath_id":"-","chopping":"81-135","consensus_level":"high","plddt":95.7842,"start":81,"end":135}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5W0Z9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5W0Z9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5W0Z9-F1-predicted_aligned_error_v6.png","plddt_mean":85.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZDHHC20","jax_strain_url":"https://www.jax.org/strain/search?query=ZDHHC20"},"sequence":{"accession":"Q5W0Z9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5W0Z9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5W0Z9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5W0Z9"}},"corpus_meta":[{"pmid":"27153536","id":"PMC_27153536","title":"Inhibition of DHHC20-Mediated EGFR Palmitoylation Creates a Dependence on EGFR Signaling.","date":"2016","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/27153536","citation_count":187,"is_preprint":false},{"pmid":"29079573","id":"PMC_29079573","title":"The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29079573","citation_count":90,"is_preprint":false},{"pmid":"38821916","id":"PMC_38821916","title":"ZDHHC20-mediated S-palmitoylation of YTHDF3 stabilizes MYC mRNA to promote pancreatic cancer progression.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38821916","citation_count":57,"is_preprint":false},{"pmid":"39696259","id":"PMC_39696259","title":"ZDHHC20 mediated S-palmitoylation of fatty acid synthase (FASN) promotes hepatocarcinogenesis.","date":"2024","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39696259","citation_count":52,"is_preprint":false},{"pmid":"34913437","id":"PMC_34913437","title":"S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34913437","citation_count":37,"is_preprint":false},{"pmid":"38733589","id":"PMC_38733589","title":"Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.","date":"2024","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/38733589","citation_count":21,"is_preprint":false},{"pmid":"38467718","id":"PMC_38467718","title":"zDHHC20-driven S-palmitoylation of CD80 is required for its costimulatory function.","date":"2024","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/38467718","citation_count":18,"is_preprint":false},{"pmid":"36262404","id":"PMC_36262404","title":"Charting the Chemical Space of Acrylamide-Based Inhibitors of zDHHC20.","date":"2022","source":"ACS medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/36262404","citation_count":14,"is_preprint":false},{"pmid":"31858978","id":"PMC_31858978","title":"DHHC20 Palmitoyl-Transferase Reshapes the Membrane to Foster Catalysis.","date":"2019","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31858978","citation_count":12,"is_preprint":false},{"pmid":"39309302","id":"PMC_39309302","title":"ZDHHC20 Activates AKT Signaling Pathway to Promote Cell Proliferation in Hepatocellular Carcinoma.","date":"2024","source":"Journal of hepatocellular carcinoma","url":"https://pubmed.ncbi.nlm.nih.gov/39309302","citation_count":8,"is_preprint":false},{"pmid":"35563480","id":"PMC_35563480","title":"Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35563480","citation_count":7,"is_preprint":false},{"pmid":"40842128","id":"PMC_40842128","title":"Multi-Omics Mendelian Randomization Identifies a DNA Methylation-ZDHHC20-Immune Axis Associated With Schizophrenia Risk.","date":"2025","source":"Brain and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/40842128","citation_count":2,"is_preprint":false},{"pmid":"41109928","id":"PMC_41109928","title":"ZDHHC20-mediated S-palmitoylation of KAP1/TRIM28 promotes DNA damage repair.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/41109928","citation_count":1,"is_preprint":false},{"pmid":"41084588","id":"PMC_41084588","title":"DNA Methylation at cg18095732 Modulates ZDHHC20 Expression and Decreases Acne Vulgaris Risk.","date":"2025","source":"Clinical, cosmetic and investigational dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/41084588","citation_count":1,"is_preprint":false},{"pmid":"41882733","id":"PMC_41882733","title":"MEF2A activates the ZDHHC20/NF-κB pathway to inhibit doxorubicin sensitivity and promote the malignant progression of acute myeloid leukemia.","date":"2026","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/41882733","citation_count":0,"is_preprint":false},{"pmid":"42142278","id":"PMC_42142278","title":"Transcriptomic Analysis and Multiple Machine Learning Approaches Identify ZDHHC20 and Its Highly Correlated Gene AK5 as Biomarkers in Multiple System Atrophy.","date":"2026","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/42142278","citation_count":0,"is_preprint":false},{"pmid":"41586981","id":"PMC_41586981","title":"SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression.","date":"2026","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/41586981","citation_count":0,"is_preprint":false},{"pmid":"42011944","id":"PMC_42011944","title":"Palmitic Acid Promotes Antiviral Innate Immunity via ZDHHC20-Mediated CMPK2 Palmitoylation.","date":"2026","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/42011944","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.07.18.604152","title":"A selective S-acyltransferase inhibitor suppresses tumor growth","date":"2024-07-22","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.18.604152","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11550,"output_tokens":4978,"usd":0.05466,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13128,"output_tokens":4303,"usd":0.086608,"stage2_stop_reason":"end_turn"},"total_usd":0.141268,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"DHHC20 palmitoylates EGFR at cysteine residues within its unstructured C-terminal tail; this palmitoylation 'pins' the C-terminal tail to the plasma membrane, impeding EGFR activation. Mutation of these cysteine residues to alanine is sufficient to activate EGFR signaling, promote cell migration and transformation, and sensitize cells to EGFR tyrosine kinase inhibition.\",\n      \"method\": \"Mass spectrometry identification of palmitoylated cysteines, site-directed mutagenesis (Cys→Ala), loss-of-function (DHHC20 inhibition), cell migration and transformation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal identification of palmitoylation sites by MS, mutagenesis validation, functional phenotypic readout, replicated across multiple cell lines\",\n      \"pmids\": [\"27153536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZDHHC20 enhances IFITM3 palmitoylation and uniquely increases IFITM3 antiviral activity (against influenza) when both are co-overexpressed. ZDHHC20 co-localizes with IFITM3 at lysosomes, unlike ZDHHC3/7/15 which show perinuclear localization, suggesting that the subcellular site of palmitoylation influences IFITM3 activity. Combined knockdown of ZDHHC3 and ZDHHC7 in ZDHHC20-knockout cells was required to reduce endogenous IFITM3 palmitoylation, demonstrating functional redundancy among ZDHHCs.\",\n      \"method\": \"Overexpression screen (23 mammalian ZDHHCs), ZDHHC knockout cell line library, siRNA knockdown, metabolic palmitoylation labeling, viral infection assay, co-localization by fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — orthogonal overexpression and knockout/knockdown approaches, direct palmitoylation assay, antiviral functional readout, multiple methods in one study\",\n      \"pmids\": [\"29079573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human DHHC20 is catalytically active when reconstituted in POPC nanodiscs. Molecular dynamics simulations reveal that DHHC20 induces a drastic deformation/invagination of the cytoplasmic leaflet of the lipid membrane, causing the catalytic Cys (within the DHHC motif) to become hydrated and optimally positioned to undergo autoacylation upon encountering acyl-CoA. This membrane reshaping resolves the paradox of a nucleophilic Cys being exposed to a hydrophobic membrane environment.\",\n      \"method\": \"Biochemical reconstitution in POPC nanodiscs, microsecond all-atom molecular dynamics simulations\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution plus MD simulation in a single study; computational model not validated by mutagenesis in this paper\",\n      \"pmids\": [\"31858978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZDHHC20 mediates S-acylation of the ORAI1 Ca2+ channel at Cys143. This palmitoylation targets ORAI1 to cholesterol-rich lipid raft domains and is required for efficient TCR recruitment and signaling at the immune synapse. Cys143 mutations reduced ORAI1 currents, store-operated Ca2+ entry, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement; the acylation-deficient channel was also recruited less efficiently to the immune synapse along with actin and TCR.\",\n      \"method\": \"Site-directed mutagenesis (Cys143), patch-clamp electrophysiology, Ca2+ imaging, NFATC1 nuclear translocation assay, IL-2 ELISA, enforced ZDHHC20 overexpression, lipid raft fractionation, live-cell fluorescence microscopy at immune synapse\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis of palmitoylation site, multiple orthogonal functional readouts (currents, Ca2+ entry, cytokine secretion, IS recruitment), mechanistic pathway placement\",\n      \"pmids\": [\"34913437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Molecular dynamics of membrane-embedded hDHHC20 shows that among acyl-CoA chains of various lengths, only C16 (palmitoyl) adopts a conformation suitable for hDHHC20 autoacylation within the hydrophobic cavity formed by four transmembrane helices. A V185G mutant shifts preference to C18, demonstrating that residues at the cavity ceiling determine acyl-chain length selectivity. An unusual hydrophilic ridge in TM helix 4 of DHHC20 may mediate association with substrate protein TM domains.\",\n      \"method\": \"Molecular dynamics simulation of membrane-embedded hDHHC20 and mutants with various acyl-CoAs; mutagenesis of cavity residues; experimental validation of spike protein acylation by S→A mutagenesis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — MD-based mechanistic model with partial mutagenesis validation; single study, limited experimental corroboration\",\n      \"pmids\": [\"35563480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 palmitoylates YTHDF3 at Cys474, preventing its chaperone-mediated autophagic (CMA) degradation and causing accumulation of YTHDF3 protein. Stabilized YTHDF3 in turn leads to abnormal accumulation of MYC mRNA/protein, promoting malignant phenotypes in pancreatic cancer. A YTHDF3-derived peptide competitively inhibiting ZDHHC20-mediated YTHDF3 palmitoylation downregulates MYC and inhibits KRAS-mutant pancreatic cancer progression.\",\n      \"method\": \"KPC mouse model, acyl-biotin exchange assay, site-directed mutagenesis (Cys474), co-immunoprecipitation, protein half-life assay, competitive peptide inhibitor, in vivo tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct identification of palmitoylation site by ABE plus mutagenesis, mechanistic link to CMA degradation and MYC accumulation, validated in vivo with multiple orthogonal methods\",\n      \"pmids\": [\"38821916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 palmitoylates fatty acid synthase (FASN) at Cys1471 and Cys1881. This palmitoylation competes with ubiquitination at these sites, blocking FASN degradation via the E3 ubiquitin ligase complex SNX8-TRIM28. ZDHHC20 knockout or pharmacological inhibition, or FASN C1471S/C1881S mutation, accelerates FASN degradation and reduces hepatocarcinogenesis in two chemical carcinogen mouse models.\",\n      \"method\": \"ZDHHC20 knockout mice (DEN and DEN/CCl4 HCC models), palmitoylation LC-MS, acyl-biotin exchange assay, co-immunoprecipitation, ubiquitination assay, protein half-life assay, site-directed mutagenesis (C1471S/C1881S), immunofluorescence microscopy\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout models, direct palmitoylation site identification by MS+ABE, mutagenesis, ubiquitination competition mechanism defined with multiple orthogonal methods\",\n      \"pmids\": [\"39696259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"zDHHC20 palmitoylates CD80 at Cys261/262/266/271 (transmembrane and cytoplasmic regions). This palmitoylation protects CD80 from ubiquitin-mediated proteasomal degradation, ensures accurate plasma membrane localization, and is required for CD80 costimulatory function in T cell activation. Palmitoylation-deficient CD80 (4CS mutant) loses membrane localization and fails to costimulate T cells.\",\n      \"method\": \"Metabolic palmitoylation labeling, site-directed mutagenesis (4CS), co-immunoprecipitation, ubiquitination assay, protein stability assay, immunofluorescence, T cell co-stimulation assay\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct palmitoylation site identification, mutagenesis, ubiquitination and stability assays, functional T cell readout; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38467718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 was identified in an in vivo shRNA screen as critical for pancreatic cancer metastatic outgrowth. This pro-metastatic function is abrogated in immunocompromised animals and in animals depleted of NK cells, placing ZDHHC20-dependent palmitoylation in the regulation of tumor-innate immune interactions required for distant metastasis. A chemical genetics substrate profiling platform identified multiple novel ZDHHC20 substrates.\",\n      \"method\": \"In vivo shRNA screen, immunocompromised and NK-cell-depleted mouse models, chemical genetics substrate profiling platform\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic screen with mechanistic follow-up (immune cell depletion), substrate profiling; single study, substrates not fully validated individually\",\n      \"pmids\": [\"38733589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 overexpression increases phosphorylation of PI3K and AKT in hepatocellular carcinoma cells, while ZDHHC20 knockdown decreases PI3K and AKT phosphorylation. PI3K-AKT pathway inhibitors (LY294002 and MK2206) block the pro-proliferative effect of ZDHHC20 overexpression, placing ZDHHC20 upstream of the PI3K-AKT pathway in HCC.\",\n      \"method\": \"siRNA knockdown, overexpression, Western blotting for PI3K/AKT phosphorylation, pharmacological inhibition, xenograft mouse model\",\n      \"journal\": \"Journal of hepatocellular carcinoma\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic epistasis via pharmacological inhibition without direct substrate identification; single lab, no direct palmitoylation mechanism defined\",\n      \"pmids\": [\"39309302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZDHHC20 palmitoylates KAP1/TRIM28 at Cys232. This palmitoylation increases chromatin binding of phosphorylated KAP1, enhancing chromatin accessibility and recruitment of DNA damage response components BRCA1 and 53BP1. ATM kinase phosphorylates ZDHHC20 at Ser339 in response to DNA damage, which in turn increases KAP1 palmitoylation, defining an ATM-ZDHHC20-KAP1 axis in the DNA damage response. ZDHHC20 knockout enhances radiosensitivity in mice, tumor cell lines, and xenograft models.\",\n      \"method\": \"ZDHHC family screen, palmitoylation label-free quantitative proteomics, acyl-biotin exchange (ABE) assay, site-directed mutagenesis (Cys232), chromatin fractionation, BRCA1/53BP1 recruitment assay, ATM phosphorylation site mapping (Ser339), ZDHHC20 knockout mice, xenograft models, radiosensitivity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct palmitoylation site by ABE+proteomics, upstream kinase (ATM) identified, downstream chromatin mechanism defined, multiple in vivo models; multiple orthogonal methods in one study\",\n      \"pmids\": [\"41109928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MEF2A transcriptionally activates ZDHHC20 (validated by dual-luciferase reporter and ChIP assays). ZDHHC20 in turn activates the NF-κB signaling pathway, promoting AML cell proliferation and doxorubicin resistance. Overexpression of ZDHHC20 reverses the antitumor effects of MEF2A silencing.\",\n      \"method\": \"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), siRNA knockdown, overexpression rescue, Western blotting, xenograft mouse model\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct transcriptional regulation validated by ChIP and reporter assay, rescue experiment; NF-κB pathway activation shown but direct palmitoylation substrate not identified\",\n      \"pmids\": [\"41882733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZDHHC20 palmitoylates CMPK2 at Cys137 and Cys153, maintaining CMPK2 mitochondrial localization. This palmitoylation is reversed by the thioesterase PPT1. CMPK2 palmitoylation supports ddhCTP production and MAVS stabilization, enabling IFN-I production and antiviral immunity. Palmitic acid activates this pathway; PPT1 deficiency restores CMPK2 palmitoylation and antiviral immunity.\",\n      \"method\": \"Chemical library screen, palmitoylation assay, site-directed mutagenesis (Cys137/153), subcellular fractionation/mitochondrial localization assay, CMPK2 knockout, PPT1 knockout/inhibition, IFN-I production assay, viral replication assay, in vivo diet and pharmacological intervention\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct palmitoylation site identification with mutagenesis, writer (ZDHHC20) and eraser (PPT1) both identified, mitochondrial localization consequence defined, multiple in vivo validations\",\n      \"pmids\": [\"42011944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RAB11A acts as a scaffold that recruits ZDHHC20 to promote FGFR3 palmitoylation, inhibiting FGFR3 degradation and retaining it at the plasma membrane in bladder cancer cells. SREBP2 directly activates RAB11A transcription (dependent on its liquid-liquid phase separation capability), placing ZDHHC20 downstream of a SREBP2-RAB11A axis. Knockdown of ZDHHC20 or RAB11A reduced membrane FGFR3 and attenuated tumor growth in xenografts.\",\n      \"method\": \"Co-immunoprecipitation (RAB11A-ZDHHC20 interaction), promoter-reporter assay (SREBP2→RAB11A), SREBP2 phase-separation mutant (F178A), RAB11A/ZDHHC20 knockdown, immunoblotting for membrane FGFR3, xenograft tumor models\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for scaffold interaction, reporter assay for transcriptional activation, in vivo xenograft; direct FGFR3 palmitoylation site not mapped in this abstract\",\n      \"pmids\": [\"41586981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A selective, orally bioavailable small-molecule inhibitor SD-066-4 inhibits ZDHHC20 acyltransferase activity. A specific alanine residue in ZDHHC20 accommodates the methyl group of SD-066-4, providing isoform selectivity over other ZDHHCs. SD-066-4 stably reduces EGFR S-acylation in KRAS-mutant cells and blocks growth of KRAS-mutant lung tumors, extending overall survival in a mouse model.\",\n      \"method\": \"Selective inhibitor identification, mutagenesis identifying selectivity-determining alanine residue, EGFR palmitoylation assay, KRAS-mutant lung tumor mouse model, survival analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with isoform selectivity residue identified by mutagenesis, in vivo tumor model; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.07.18.604152\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ZDHHC20 is a plasma membrane and lysosome-localized integral membrane palmitoyltransferase that autoacylates via its conserved DHHC catalytic cysteine—facilitated by enzyme-induced membrane deformation that hydrates the catalytic site—and then transfers C16 acyl chains to substrate cysteine residues on diverse proteins including EGFR (C-terminal tail), ORAI1 (Cys143), IFITM3, YTHDF3 (Cys474), FASN (Cys1471/1881), KAP1/TRIM28 (Cys232), CMPK2 (Cys137/153), CD80, and FGFR3, thereby regulating their membrane localization, protein stability (by competing with ubiquitination), and signaling activity in processes spanning oncogenic RTK signaling, Ca2+-dependent immune synapse function, antiviral immunity, and DNA damage repair; ZDHHC20 itself is regulated upstream by ATM-mediated phosphorylation (Ser339) and transcriptional activation by MEF2A, and its activity can be selectively inhibited by acrylamide-based or small-molecule inhibitors that exploit a selectivity-determining alanine residue.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZDHHC20 is an integral-membrane DHHC palmitoyltransferase that controls the membrane localization, stability, and signaling output of a broad range of substrates by transferring C16 (palmitoyl) acyl chains onto target cysteines [#0, #6]. Catalysis proceeds through autoacylation at the DHHC-motif cysteine: the enzyme induces a drastic invagination of the cytoplasmic membrane leaflet that hydrates the catalytic cysteine and positions it to react with acyl-CoA [#2], and a hydrophobic cavity formed by its transmembrane helices selects specifically for the C16 chain length, with cavity-ceiling residues such as Val185 dictating this preference [#4]. A recurring functional logic is that ZDHHC20-mediated palmitoylation competes with ubiquitination to stabilize its substrates—blocking SNX8-TRIM28-dependent degradation of FASN at Cys1471/Cys1881 [#6], protecting CD80 from proteasomal turnover [#7], and preventing chaperone-mediated autophagic degradation of YTHDF3 at Cys474 to drive MYC accumulation in pancreatic cancer [#5]. Through distinct substrates ZDHHC20 governs several processes: it pins the EGFR C-terminal tail to the plasma membrane to restrain receptor activation [#0], targets the ORAI1 Ca2+ channel (Cys143) to lipid rafts for immune-synapse signaling [#3], promotes IFITM3 antiviral activity at lysosomes [#1], sustains CMPK2 mitochondrial localization and MAVS-dependent type I interferon responses [#12], and palmitoylates KAP1/TRIM28 (Cys232) to enhance chromatin accessibility and DNA damage repair [#10]. ZDHHC20 is itself regulated upstream by ATM-mediated phosphorylation at Ser339 in the DNA damage response [#10] and by MEF2A-driven transcription [#11], and its activity is selectively druggable via a single isoform-determining alanine residue exploited by small-molecule inhibitors [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established ZDHHC20 as a functional palmitoyltransferase with a defined substrate, showing that palmitoylation of the EGFR C-terminal tail acts as a brake on receptor activation rather than a passive modification.\",\n      \"evidence\": \"Mass-spectrometry site identification, Cys\\u2192Ala mutagenesis, and migration/transformation assays across cell lines\",\n      \"pmids\": [\"27153536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of catalysis\", \"Substrate range beyond EGFR unknown at the time\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed that the subcellular site of palmitoylation matters, with ZDHHC20 uniquely enhancing IFITM3 antiviral activity at lysosomes and acting redundantly with other ZDHHCs on endogenous IFITM3.\",\n      \"evidence\": \"23-enzyme overexpression screen, ZDHHC knockout/knockdown library, metabolic palmitoylation labeling, viral infection and colocalization assays\",\n      \"pmids\": [\"29079573\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IFITM3 palmitoylation site selectivity by ZDHHC20 not mapped\", \"Mechanism linking localization to antiviral potency not fully defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved how a nucleophilic catalytic cysteine functions within a hydrophobic membrane by showing the enzyme deforms the lipid leaflet to hydrate and position the DHHC cysteine for autoacylation.\",\n      \"evidence\": \"Reconstitution in POPC nanodiscs and microsecond all-atom molecular dynamics simulations\",\n      \"pmids\": [\"31858978\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MD model not validated by mutagenesis in this study\", \"Substrate hand-off step not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected ZDHHC20 catalysis to immune-synapse signaling by showing ORAI1 Cys143 palmitoylation directs the channel to lipid rafts required for store-operated Ca2+ entry and TCR-evoked cytokine output.\",\n      \"evidence\": \"Cys143 mutagenesis, patch-clamp, Ca2+ imaging, NFATC1 translocation, IL-2 ELISA, lipid raft fractionation, live-cell imaging\",\n      \"pmids\": [\"34913437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZDHHC20 is the sole ORAI1 acyltransferase not established\", \"Erasers/turnover of ORAI1 palmitoylation unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Explained the enzyme's strict C16 acyl-chain preference by identifying a transmembrane-helix cavity whose ceiling residues set chain-length selectivity, with V185G shifting preference to C18.\",\n      \"evidence\": \"Molecular dynamics of membrane-embedded hDHHC20 and cavity mutants, plus spike-protein acylation S\\u2192A validation\",\n      \"pmids\": [\"35563480\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Proposed TM4 hydrophilic ridge role in substrate recognition not experimentally proven\", \"Limited experimental corroboration of the MD model\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a recurring stabilization mechanism in which ZDHHC20 palmitoylation competes with ubiquitin- or autophagy-mediated degradation, demonstrated on FASN, CD80, and YTHDF3 to drive cancer and immune phenotypes.\",\n      \"evidence\": \"Knockout mice, LC-MS/ABE site mapping, mutagenesis, ubiquitination and protein half-life assays, T-cell costimulation and in vivo tumor models\",\n      \"pmids\": [\"39696259\", \"38467718\", \"38821916\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether palmitoylation directly occludes ubiquitin sites or acts allosterically not resolved for all substrates\", \"Substrate selection rules among many targets unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed ZDHHC20 in tumor-innate immune crosstalk by identifying it in an in vivo screen as required for pancreatic metastatic outgrowth in an NK-cell-dependent manner, and broadened its substrate repertoire.\",\n      \"evidence\": \"In vivo shRNA screen, immunocompromised and NK-depleted mouse models, chemical-genetics substrate profiling\",\n      \"pmids\": [\"38733589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The palmitoylation substrate(s) mediating the NK-dependent effect not individually validated\", \"Direct molecular link to NK recognition unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a selective, orally bioavailable inhibitor and the structural determinant of its isoform selectivity, showing ZDHHC20 is pharmacologically tractable in KRAS-mutant tumors.\",\n      \"evidence\": \"Inhibitor SD-066-4 characterization, mutagenesis of selectivity-determining alanine, EGFR palmitoylation assay, KRAS-mutant lung tumor model (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.07.18.604152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"On-target selectivity across all ZDHHC isoforms not exhaustively profiled\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Wired ZDHHC20 into the DNA damage response by showing ATM phosphorylates it at Ser339, which boosts KAP1/TRIM28 Cys232 palmitoylation to enhance chromatin accessibility and DDR factor recruitment.\",\n      \"evidence\": \"ZDHHC family screen, palmitoylation proteomics + ABE, Cys232 mutagenesis, chromatin fractionation, BRCA1/53BP1 recruitment, ATM site mapping, knockout mice and xenografts\",\n      \"pmids\": [\"41109928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Ser339 phosphorylation alters enzyme activity mechanistically unclear\", \"Whether other DDR substrates exist not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Extended ZDHHC20 into antiviral metabolism and identified its eraser, showing CMPK2 Cys137/153 palmitoylation (reversed by PPT1) maintains mitochondrial localization and supports MAVS-dependent type I interferon.\",\n      \"evidence\": \"Chemical library screen, palmitoylation/mutagenesis, mitochondrial fractionation, CMPK2 and PPT1 knockout, IFN-I and viral replication assays, in vivo diet/pharmacological intervention\",\n      \"pmids\": [\"42011944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the ZDHHC20/PPT1 writer-eraser cycle not quantified\", \"How palmitate availability tunes the pathway in vivo not fully defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified transcriptional and scaffolding control of ZDHHC20, with MEF2A driving its expression and a SREBP2-RAB11A axis recruiting it to palmitoylate and stabilize membrane FGFR3.\",\n      \"evidence\": \"Dual-luciferase/ChIP for MEF2A, Co-IP for RAB11A-ZDHHC20, SREBP2 phase-separation mutant, knockdown and xenograft models\",\n      \"pmids\": [\"41882733\", \"41586981\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FGFR3 palmitoylation site not mapped\", \"Direct NF-\\u03baB substrate of ZDHHC20 in AML not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ZDHHC20 selects its diverse substrates and how its catalytic activity is integrated with its many upstream regulators remains unresolved.\",\n      \"evidence\": \"No single study defines the substrate-recognition code or a unified regulatory model\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of substrate engagement\", \"Substrate-selection determinants among dozens of targets unknown\", \"Interplay between ATM phosphorylation, scaffolds, and erasers not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 6, 7, 5, 3, 10, 12]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 10]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 7, 13]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 7, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 3, 12]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 6, 8, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EGFR\", \"ORAI1\", \"IFITM3\", \"YTHDF3\", \"FASN\", \"TRIM28\", \"CMPK2\", \"RAB11A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}