{"gene":"ZDHHC20","run_date":"2026-04-28T23:00:24","timeline":{"discoveries":[{"year":2016,"finding":"DHHC20 palmitoylates EGFR at cysteine residues within its C-terminal tail; this palmitoylation 'pins' the unstructured C-terminal tail to the plasma membrane, impeding EGFR activation. Loss of palmitoylation (via DHHC20 inhibition or Cys-to-Ala mutation) increases sustained EGFR signaling and sensitizes cancer cells to EGFR tyrosine kinase inhibition.","method":"Mass spectrometry identification of palmitoylated cysteines; cysteine-to-alanine mutagenesis; DHHC20 knockdown; cell migration and transformation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (MS, mutagenesis, KD, functional readouts) in a single study; highly cited foundational paper","pmids":["27153536"],"is_preprint":false},{"year":2017,"finding":"ZDHHC20 palmitoylates IFITM3 and uniquely colocalizes with it at lysosomes, unlike ZDHHC3, 7, and 15, which show perinuclear localization. ZDHHC20 overexpression specifically enhances IFITM3 antiviral activity against influenza, and the lysosomal site of palmitoylation may influence IFITM3 function.","method":"ZDHHC library overexpression screen; ZDHHC knockout cell lines; colocalization imaging; influenza infection assay; siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — systematic screen across 23 ZDHHCs with functional readout, replicated in KO lines and siRNA knockdown","pmids":["29079573"],"is_preprint":false},{"year":2019,"finding":"Human DHHC20 is catalytically active when reconstituted in POPC nanodiscs and induces a drastic deformation of the cytoplasmic leaflet of the lipid membrane, hydrating the catalytic cysteine of the conserved DHHC motif to enable autoacylation by acyl-CoA.","method":"Biochemical reconstitution in nanodiscs; microsecond all-atom molecular dynamics simulations","journal":"Biophysical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution combined with extensive computational modeling providing structural mechanism","pmids":["31858978"],"is_preprint":false},{"year":2021,"finding":"ZDHHC20 S-acylates ORAI1 at Cys143, targeting it to lipid rafts/cholesterol-rich domains; this is required for TCR recruitment and signaling at the immune synapse, long-lasting Ca2+ elevation, NFATC1 translocation, and IL-2 secretion in Jurkat T cells.","method":"Cys143 mutagenesis; patch-clamp (ORAI1 currents); store-operated Ca2+ entry measurements; NFATC1 translocation assay; IL-2 secretion assay; enforced ZDHHC20 expression; immune synapse imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of palmitoylation site combined with multiple functional readouts (electrophysiology, Ca2+ imaging, cytokine secretion)","pmids":["34913437"],"is_preprint":false},{"year":2022,"finding":"Molecular dynamics of hDHHC20 reveals that only C16 acyl-CoA adopts a conformation suitable for autoacylation within the hydrophobic cavity formed by four transmembrane helices; V185G mutation at the cavity ceiling shifts selectivity toward C18, and an unusual hydrophilic ridge in TM helix 4 is implicated in protein substrate association.","method":"Molecular dynamics simulations of membrane-embedded hDHHC20 with mutants; acyl-CoA binding modeling; mutagenesis of spike protein residues reducing S-acylation","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 computationally, Tier 3 experimentally — primarily MD simulations with limited direct biochemical validation","pmids":["35563480"],"is_preprint":false},{"year":2022,"finding":"Structure-activity relationship study of acrylamide-based inhibitors identified a specific alanine residue in zDHHC20 that accommodates inhibitor selectivity; cyanomyracrylamide and derivatives inhibit zDHHC20 acyltransferase activity with isoform selectivity over the homologous zDHHC2.","method":"SAR medicinal chemistry; enzymatic inhibition assays against zDHHC20 and zDHHC2","journal":"ACS medicinal chemistry letters","confidence":"Medium","confidence_rationale":"Tier 2 — systematic SAR with functional inhibition assays identifying selectivity determinant","pmids":["36262404"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 palmitoylates YTHDF3 at Cys474, protecting it from chaperone-mediated autophagic degradation, leading to accumulation of YTHDF3 protein, increased MYC mRNA stability, and promotion of pancreatic cancer progression. A YTHDF3-derived peptide competing for this palmitoylation site reduces MYC expression and tumor growth.","method":"KPC mouse model; acyl-biotin exchange assay; site-directed mutagenesis (Cys474); protein stability/half-life assays; lysosomal pathway inhibitors; peptide competition assay; xenograft models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of palmitoylation site with mechanistic follow-up in multiple models including in vivo","pmids":["38821916"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 palmitoylates fatty acid synthase (FASN) at Cys1471 and Cys1881, competing with ubiquitin-proteasome degradation via E3 ligase complex SNX8-TRIM28; loss of palmitoylation (ZDHHC20 KO or C1471S/C1881S mutation) accelerates FASN degradation and reduces hepatocarcinogenesis.","method":"ZDHHC20 knockout mice; chemical carcinogen HCC models; palmitoylation LC-MS; acyl-biotin exchange assay; co-immunoprecipitation; ubiquitination assays; protein half-life assays; mutagenesis (C1471S/C1881S)","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of palmitoylation sites, in vivo KO models, multiple biochemical assays identifying E3 ligase competition mechanism","pmids":["39696259"],"is_preprint":false},{"year":2024,"finding":"zDHHC20 palmitoylates CD80 at Cys261/262/266/271 in the transmembrane and cytoplasmic regions, protecting CD80 from ubiquitination and degradation, ensuring plasma membrane localization, and enabling its T cell costimulatory function; palmitoylation-deficient (4CS) CD80 loses membrane targeting and costimulatory activity.","method":"Metabolic labeling; acyl-biotin exchange assay; mutagenesis (4CS); co-immunoprecipitation; ubiquitination assay; immunofluorescence; T cell activation assay","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of all palmitoylation sites with multiple orthogonal readouts (ubiquitination, localization, function)","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 effect is abrogated in immunocompromised animals and NK-cell-depleted animals, indicating ZDHHC20 modulates tumor-innate immune interactions to enable metastasis. Chemical genetics substrate profiling identified multiple ZDHHC20 substrates mediating this effect.","method":"In vivo shRNA screen; NK-cell depletion; chemical genetics substrate profiling; immunocompromised animal models","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic screen with mechanistic follow-up linking ZDHHC20 to innate immune evasion, but substrates not fully validated","pmids":["38733589"],"is_preprint":false},{"year":2024,"finding":"ZDHHC20 activates the PI3K-AKT signaling pathway in hepatocellular carcinoma; knockdown decreases phosphorylation of PI3K and AKT and reduces proliferation, while overexpression increases PI3K/AKT phosphorylation and promotes proliferation; PI3K/AKT inhibitors block ZDHHC20-driven proliferation.","method":"ZDHHC20 knockdown/overexpression; Western blot for p-PI3K and p-AKT; CCK-8, colony formation, apoptosis assays; xenograft model; pharmacological inhibitors (LY294002, MK2206)","journal":"Journal of hepatocellular carcinoma","confidence":"Medium","confidence_rationale":"Tier 2 — KD/OE with defined pathway readout and inhibitor confirmation, but specific palmitoylation substrate linking ZDHHC20 to PI3K/AKT not identified","pmids":["39309302"],"is_preprint":false},{"year":2024,"finding":"A selective, orally bioavailable acyltransferase inhibitor SD-066-4 inhibits ZDHHC20 by interacting with a specific alanine residue providing isoform selectivity; SD-066-4 stably reduces EGFR S-acylation in KRAS-mutant cells and blocks growth of KRAS-mutant lung tumors in vivo.","method":"Small molecule inhibitor development; mutagenesis identifying selectivity-determining alanine; EGFR palmitoylation assay; KRAS-mutant cell/tumor growth assays; in vivo tumor model","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — selective inhibitor with defined binding residue and in vivo validation; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.07.18.604152"],"is_preprint":true},{"year":2025,"finding":"ZDHHC20 palmitoylates KAP1/TRIM28 at Cys232; this palmitoylation is induced by ATM-dependent phosphorylation of ZDHHC20 at Ser339 after DNA damage. Palmitoylated KAP1 shows increased chromatin binding and promotes recruitment of DDR factors BRCA1 and 53BP1, enhancing DNA damage repair.","method":"ZDHHC family screen; acyl-biotin exchange assay; palmitoylation label-free quantitative proteomics; ZDHHC20 KO mice and xenografts; phospho-site mutagenesis; chromatin fractionation; BRCA1/53BP1 recruitment assays; radiosensitivity assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — site-specific mutagenesis, proteomics, in vivo KO, and multiple mechanistic readouts linking ATM phosphorylation of ZDHHC20 to KAP1 palmitoylation and DDR","pmids":["41109928"],"is_preprint":false},{"year":2026,"finding":"ZDHHC20 catalyzes palmitoylation of CMPK2 at Cys137 and Cys153, maintaining its mitochondrial localization; this promotes ddhCTP production and MAVS stabilization for antiviral IFN-I responses. Depalmitoylation by PPT1 reverses this. Palmitic acid activates this pathway, and PPT1 inhibition restores CMPK2 palmitoylation and antiviral immunity.","method":"Metabolic chemical library screen; palmitoylation assays; CMPK2 KO; PPT1 inhibitor (DC661); IFN-I measurement; viral replication assay; mutagenesis of Cys137/153; in vivo diet and inhibitor administration","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1–2 — site-specific mutagenesis with writer (ZDHHC20) and eraser (PPT1) identified, functional readouts in cells and in vivo","pmids":["42011944"],"is_preprint":false},{"year":2026,"finding":"RAB11A acts as a scaffold that recruits ZDHHC20 to palmitoylate FGFR3, promoting its plasma membrane retention and preventing degradation in bladder cancer. SREBP2 transcriptionally activates RAB11A (dependent on its liquid-liquid phase separation). Knockdown of RAB11A or ZDHHC20 reduces membrane FGFR3 and attenuates tumor growth in xenografts.","method":"Co-immunoprecipitation; promoter-reporter assay; SREBP2 F178A phase-separation mutant; RAB11A/ZDHHC20 knockdown; xenograft models; immunoblotting for membrane FGFR3; functional proliferation/apoptosis assays","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, reporter assay, and in vivo KD evidence but palmitoylation site on FGFR3 not directly validated by mutagenesis","pmids":["41586981"],"is_preprint":false},{"year":2026,"finding":"MEF2A transcriptionally activates ZDHHC20 (validated by dual-luciferase reporter and ChIP assays), and ZDHHC20 in turn activates the NF-κB pathway to promote AML cell proliferation, glycolysis, and doxorubicin resistance; ZDHHC20 overexpression rescues antitumor effects of MEF2A silencing.","method":"Dual-luciferase reporter assay; ChIP assay; MEF2A knockdown/ZDHHC20 overexpression rescue; NF-κB pathway western blot; xenograft model; functional proliferation/apoptosis/glycolysis assays","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2–3 — ChIP and reporter assay establish transcriptional relationship; epistasis rescue places ZDHHC20 downstream of MEF2A and upstream of NF-κB, but palmitoylation substrate linking ZDHHC20 to NF-κB not identified","pmids":["41882733"],"is_preprint":false}],"current_model":"ZDHHC20 is an integral membrane palmitoyltransferase that autoacylates at its conserved DHHC catalytic cysteine—facilitated by membrane deformation it induces—and then transfers C16 palmitoyl chains to substrate cysteine residues on diverse proteins including EGFR, IFITM3, ORAI1, YTHDF3, FASN, CD80, KAP1/TRIM28, CMPK2, and FGFR3, thereby regulating their membrane localization, stability (competing with ubiquitin-proteasome degradation), and signaling activity in contexts ranging from antiviral immunity and T cell activation to oncogenic pathway activation in cancer."},"narrative":{"teleology":[{"year":2016,"claim":"The first substrate and physiological function of ZDHHC20 were established: palmitoylation of EGFR C-terminal cysteines pins its tail to the plasma membrane, restraining signaling—revealing that loss of ZDHHC20-mediated palmitoylation sensitizes cancer cells to EGFR inhibitors.","evidence":"Mass spectrometry, Cys-to-Ala mutagenesis, DHHC20 knockdown, and transformation/migration assays in cancer cell lines","pmids":["27153536"],"confidence":"High","gaps":["Whether EGFR palmitoylation by ZDHHC20 is regulated by upstream signals was not addressed","The stoichiometry of palmitoylation on individual EGFR cysteines was not determined"]},{"year":2017,"claim":"ZDHHC20 was shown to palmitoylate the antiviral effector IFITM3 and uniquely colocalize with it at lysosomes, distinguishing it from other DHHC enzymes and linking its activity to innate antiviral defense.","evidence":"Systematic ZDHHC library screen across 23 family members, ZDHHC KO cell lines, colocalization imaging, and influenza infection assays","pmids":["29079573"],"confidence":"High","gaps":["Whether ZDHHC20 lysosomal localization is dynamically regulated was not resolved","The precise palmitoylation sites on IFITM3 modified by ZDHHC20 specifically (versus other ZDHHCs) were not delineated"]},{"year":2019,"claim":"Reconstitution and simulation revealed that ZDHHC20 deforms the cytoplasmic membrane leaflet to hydrate its DHHC catalytic cysteine, establishing the structural basis for its autoacylation step.","evidence":"Biochemical reconstitution in POPC nanodiscs combined with microsecond all-atom molecular dynamics simulations","pmids":["31858978"],"confidence":"High","gaps":["No high-resolution experimental structure of ZDHHC20 was obtained","How membrane deformation couples to the subsequent substrate acylation (transacylation) step was not determined"]},{"year":2021,"claim":"ZDHHC20-mediated palmitoylation of ORAI1 at Cys143 was shown to be required for lipid-raft targeting, TCR signaling at the immune synapse, sustained Ca²⁺ entry, and IL-2 secretion, establishing ZDHHC20 as a regulator of adaptive immunity.","evidence":"Cys143 mutagenesis, patch-clamp electrophysiology, SOCE measurements, NFATC1 translocation, and IL-2 secretion in Jurkat T cells","pmids":["34913437"],"confidence":"High","gaps":["Whether ZDHHC20 palmitoylates ORAI1 in primary T cells in vivo was not tested","Whether other ZDHHCs contribute to ORAI1 palmitoylation was not fully excluded"]},{"year":2022,"claim":"Molecular dynamics and structure-activity studies defined the acyl chain length selectivity mechanism: a hydrophobic cavity in ZDHHC20's transmembrane helices accommodates C16 palmitoyl-CoA, and a specific alanine residue determines isoform-selective inhibitor binding.","evidence":"MD simulations with V185G mutant shifting to C18 selectivity; SAR of acrylamide-based inhibitors with isoform selectivity assays","pmids":["35563480","36262404"],"confidence":"Medium","gaps":["The C16 selectivity mechanism was primarily computationally derived and not validated by in vitro reconstitution with different acyl-CoA substrates","Inhibitor selectivity was tested against ZDHHC2 but not against the full ZDHHC family"]},{"year":2024,"claim":"Multiple studies converged to show that ZDHHC20-mediated palmitoylation stabilizes substrates (YTHDF3, FASN, CD80) by competing with ubiquitin-dependent degradation, establishing a general palmitoylation-versus-ubiquitination stability switch operating in cancer and immunity.","evidence":"Palmitoylation site mutagenesis, ubiquitination assays, protein half-life measurements, and in vivo models for YTHDF3/MYC in pancreatic cancer, FASN in hepatocarcinogenesis, and CD80 in T cell costimulation","pmids":["38821916","39696259","38467718"],"confidence":"High","gaps":["Whether the palmitoylation-ubiquitination competition involves direct steric occlusion of the same or proximal lysine residues was not resolved","The E3 ligases targeting YTHDF3 and CD80 for degradation upon loss of palmitoylation were not identified (only the SNX8-TRIM28 complex was identified for FASN)"]},{"year":2024,"claim":"An in vivo shRNA screen identified ZDHHC20 as essential for pancreatic cancer metastatic outgrowth, functioning through modulation of tumor–NK cell interactions rather than cell-autonomous growth.","evidence":"In vivo shRNA screen with immunocompromised and NK-cell-depleted animal models; chemical genetics substrate profiling","pmids":["38733589"],"confidence":"Medium","gaps":["The specific ZDHHC20 substrates mediating NK cell evasion were identified by chemical genetics but not individually validated","Whether the immune evasion mechanism operates in human patients was not tested"]},{"year":2024,"claim":"ZDHHC20 was shown to activate PI3K-AKT signaling in hepatocellular carcinoma, promoting proliferation that is reversible by PI3K/AKT inhibitors, though the direct palmitoylation substrate was not identified.","evidence":"ZDHHC20 knockdown/overexpression, phospho-PI3K/AKT western blots, pharmacological inhibitors (LY294002, MK2206), xenograft model","pmids":["39309302"],"confidence":"Medium","gaps":["The palmitoylation substrate linking ZDHHC20 to PI3K-AKT activation was not identified","Whether this effect is independent of ZDHHC20's known substrates (e.g., EGFR, FASN) was not tested"]},{"year":2025,"claim":"ATM-dependent phosphorylation of ZDHHC20 at Ser339 was shown to activate palmitoylation of KAP1/TRIM28, enhancing its chromatin binding and recruitment of DDR factors BRCA1 and 53BP1—revealing ZDHHC20 as a signal-regulated enzyme in the DNA damage response.","evidence":"ZDHHC family screen, acyl-biotin exchange, phospho-site mutagenesis, ZDHHC20 KO mice, chromatin fractionation, DDR factor recruitment and radiosensitivity assays","pmids":["41109928"],"confidence":"High","gaps":["Whether other DNA damage kinases phosphorylate ZDHHC20 at additional sites was not explored","The structural basis for how Ser339 phosphorylation enhances enzymatic activity was not determined"]},{"year":2026,"claim":"ZDHHC20 palmitoylation of CMPK2 at Cys137/153 was shown to maintain its mitochondrial localization and promote MAVS-dependent type I IFN responses, with PPT1 acting as the counteracting depalmitoylase—establishing a writer-eraser pair for antiviral innate immunity.","evidence":"Metabolic chemical library screen, CMPK2 Cys mutagenesis, PPT1 inhibitor DC661, IFN-I measurement, viral replication assays, in vivo diet and inhibitor studies","pmids":["42011944"],"confidence":"High","gaps":["Whether other ZDHHCs also palmitoylate CMPK2 was not fully excluded","The structural basis of ZDHHC20-CMPK2 recognition was not resolved"]},{"year":2026,"claim":"RAB11A was identified as a scaffold that recruits ZDHHC20 to palmitoylate FGFR3 for plasma membrane retention in bladder cancer, with SREBP2-driven transcription of RAB11A as an upstream regulatory node.","evidence":"Co-immunoprecipitation, promoter-reporter assays, RAB11A/ZDHHC20 knockdown, xenograft models","pmids":["41586981"],"confidence":"Medium","gaps":["The palmitoylation site(s) on FGFR3 were not validated by site-directed mutagenesis","Whether RAB11A scaffolding is specific to FGFR3 or extends to other ZDHHC20 substrates was not tested"]},{"year":2026,"claim":"MEF2A was identified as a direct transcriptional activator of ZDHHC20, and ZDHHC20 was placed upstream of NF-κB signaling in AML, promoting proliferation, glycolysis, and chemoresistance.","evidence":"ChIP assay, dual-luciferase reporter, MEF2A-KD/ZDHHC20-OE epistasis rescue, NF-κB pathway western blots, xenograft model","pmids":["41882733"],"confidence":"Medium","gaps":["The palmitoylation substrate connecting ZDHHC20 to NF-κB activation was not identified","Whether MEF2A regulation of ZDHHC20 operates in non-AML contexts was not explored"]},{"year":null,"claim":"Key unresolved questions include the high-resolution structure of ZDHHC20, the rules governing substrate recognition across its diverse substrates, whether signal-dependent regulation (as shown for ATM-Ser339 phosphorylation) is a general feature, and the therapeutic window of ZDHHC20 inhibition given its roles in both tumor promotion and immune function.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimental high-resolution structure of ZDHHC20 has been determined","Substrate recognition determinants beyond the hydrophilic ridge in TM4 remain undefined","In vivo consequences of systemic ZDHHC20 inhibition on immune homeostasis are unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,3,6,7,8,12,13,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,8,14]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,10]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,3,8,9,13]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,7,10,11,14,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,6,7,8,12,13]}],"complexes":[],"partners":["EGFR","IFITM3","ORAI1","YTHDF3","FASN","CD80","TRIM28","CMPK2"],"other_free_text":[]},"mechanistic_narrative":"ZDHHC20 is an integral membrane protein S-acyltransferase (palmitoyltransferase) that transfers palmitoyl groups from palmitoyl-CoA to cysteine residues on a broad array of substrate proteins, thereby controlling their membrane association, stability, and signaling competence across immunity, DNA damage repair, and oncogenesis. Its catalytic mechanism involves membrane deformation that hydrates the DHHC-motif catalytic cysteine for autoacylation, with a transmembrane hydrophobic cavity that confers selectivity for C16 acyl chains [PMID:31858978, PMID:35563480]. Validated substrates include EGFR (C-terminal tail pinning and signal attenuation) [PMID:27153536], IFITM3 (antiviral activity at lysosomes) [PMID:29079573], ORAI1 (lipid raft targeting for T cell Ca²⁺/NFAT signaling) [PMID:34913437], YTHDF3 (protection from chaperone-mediated autophagy, stabilizing MYC mRNA in pancreatic cancer) [PMID:38821916], FASN (competition with ubiquitin-proteasome degradation in hepatocarcinogenesis) [PMID:39696259], CD80 (membrane retention and T cell costimulation) [PMID:38467718], KAP1/TRIM28 (ATM-phosphorylation-dependent palmitoylation enhancing chromatin binding and DNA damage repair) [PMID:41109928], CMPK2 (mitochondrial localization and MAVS-dependent antiviral IFN-I responses) [PMID:42011944], and FGFR3 (RAB11A-scaffolded plasma membrane retention in bladder cancer) [PMID:41586981]. For multiple substrates, ZDHHC20-mediated palmitoylation competes with ubiquitination to protect proteins from proteasomal or lysosomal degradation, establishing palmitoylation as a stability switch [PMID:39696259, PMID:38467718, PMID:38821916]."},"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":184,"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":88,"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":50,"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":44,"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":20,"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":17,"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":13,"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":11,"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":6,"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":"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":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":"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":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":10417,"output_tokens":4414,"usd":0.048731},"stage2":{"model":"claude-opus-4-6","input_tokens":7866,"output_tokens":3800,"usd":0.201495},"total_usd":0.250226,"stage1_batch_id":"msgbatch_01BparvgRZ9S2LMidvaoo86K","stage2_batch_id":"msgbatch_01NkBfv9SVZWthhS7j6mU6G3","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 C-terminal tail; this palmitoylation 'pins' the unstructured C-terminal tail to the plasma membrane, impeding EGFR activation. Loss of palmitoylation (via DHHC20 inhibition or Cys-to-Ala mutation) increases sustained EGFR signaling and sensitizes cancer cells to EGFR tyrosine kinase inhibition.\",\n      \"method\": \"Mass spectrometry identification of palmitoylated cysteines; cysteine-to-alanine mutagenesis; DHHC20 knockdown; cell migration and transformation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (MS, mutagenesis, KD, functional readouts) in a single study; highly cited foundational paper\",\n      \"pmids\": [\"27153536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZDHHC20 palmitoylates IFITM3 and uniquely colocalizes with it at lysosomes, unlike ZDHHC3, 7, and 15, which show perinuclear localization. ZDHHC20 overexpression specifically enhances IFITM3 antiviral activity against influenza, and the lysosomal site of palmitoylation may influence IFITM3 function.\",\n      \"method\": \"ZDHHC library overexpression screen; ZDHHC knockout cell lines; colocalization imaging; influenza infection assay; siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic screen across 23 ZDHHCs with functional readout, replicated in KO lines and siRNA knockdown\",\n      \"pmids\": [\"29079573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human DHHC20 is catalytically active when reconstituted in POPC nanodiscs and induces a drastic deformation of the cytoplasmic leaflet of the lipid membrane, hydrating the catalytic cysteine of the conserved DHHC motif to enable autoacylation by acyl-CoA.\",\n      \"method\": \"Biochemical reconstitution in nanodiscs; microsecond all-atom molecular dynamics simulations\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution combined with extensive computational modeling providing structural mechanism\",\n      \"pmids\": [\"31858978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZDHHC20 S-acylates ORAI1 at Cys143, targeting it to lipid rafts/cholesterol-rich domains; this is required for TCR recruitment and signaling at the immune synapse, long-lasting Ca2+ elevation, NFATC1 translocation, and IL-2 secretion in Jurkat T cells.\",\n      \"method\": \"Cys143 mutagenesis; patch-clamp (ORAI1 currents); store-operated Ca2+ entry measurements; NFATC1 translocation assay; IL-2 secretion assay; enforced ZDHHC20 expression; immune synapse imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of palmitoylation site combined with multiple functional readouts (electrophysiology, Ca2+ imaging, cytokine secretion)\",\n      \"pmids\": [\"34913437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Molecular dynamics of hDHHC20 reveals that only C16 acyl-CoA adopts a conformation suitable for autoacylation within the hydrophobic cavity formed by four transmembrane helices; V185G mutation at the cavity ceiling shifts selectivity toward C18, and an unusual hydrophilic ridge in TM helix 4 is implicated in protein substrate association.\",\n      \"method\": \"Molecular dynamics simulations of membrane-embedded hDHHC20 with mutants; acyl-CoA binding modeling; mutagenesis of spike protein residues reducing S-acylation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 computationally, Tier 3 experimentally — primarily MD simulations with limited direct biochemical validation\",\n      \"pmids\": [\"35563480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Structure-activity relationship study of acrylamide-based inhibitors identified a specific alanine residue in zDHHC20 that accommodates inhibitor selectivity; cyanomyracrylamide and derivatives inhibit zDHHC20 acyltransferase activity with isoform selectivity over the homologous zDHHC2.\",\n      \"method\": \"SAR medicinal chemistry; enzymatic inhibition assays against zDHHC20 and zDHHC2\",\n      \"journal\": \"ACS medicinal chemistry letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic SAR with functional inhibition assays identifying selectivity determinant\",\n      \"pmids\": [\"36262404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 palmitoylates YTHDF3 at Cys474, protecting it from chaperone-mediated autophagic degradation, leading to accumulation of YTHDF3 protein, increased MYC mRNA stability, and promotion of pancreatic cancer progression. A YTHDF3-derived peptide competing for this palmitoylation site reduces MYC expression and tumor growth.\",\n      \"method\": \"KPC mouse model; acyl-biotin exchange assay; site-directed mutagenesis (Cys474); protein stability/half-life assays; lysosomal pathway inhibitors; peptide competition assay; xenograft models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of palmitoylation site with mechanistic follow-up in multiple models including in vivo\",\n      \"pmids\": [\"38821916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 palmitoylates fatty acid synthase (FASN) at Cys1471 and Cys1881, competing with ubiquitin-proteasome degradation via E3 ligase complex SNX8-TRIM28; loss of palmitoylation (ZDHHC20 KO or C1471S/C1881S mutation) accelerates FASN degradation and reduces hepatocarcinogenesis.\",\n      \"method\": \"ZDHHC20 knockout mice; chemical carcinogen HCC models; palmitoylation LC-MS; acyl-biotin exchange assay; co-immunoprecipitation; ubiquitination assays; protein half-life assays; mutagenesis (C1471S/C1881S)\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of palmitoylation sites, in vivo KO models, multiple biochemical assays identifying E3 ligase competition mechanism\",\n      \"pmids\": [\"39696259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"zDHHC20 palmitoylates CD80 at Cys261/262/266/271 in the transmembrane and cytoplasmic regions, protecting CD80 from ubiquitination and degradation, ensuring plasma membrane localization, and enabling its T cell costimulatory function; palmitoylation-deficient (4CS) CD80 loses membrane targeting and costimulatory activity.\",\n      \"method\": \"Metabolic labeling; acyl-biotin exchange assay; mutagenesis (4CS); co-immunoprecipitation; ubiquitination assay; immunofluorescence; T cell activation assay\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of all palmitoylation sites with multiple orthogonal readouts (ubiquitination, localization, function)\",\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 effect is abrogated in immunocompromised animals and NK-cell-depleted animals, indicating ZDHHC20 modulates tumor-innate immune interactions to enable metastasis. Chemical genetics substrate profiling identified multiple ZDHHC20 substrates mediating this effect.\",\n      \"method\": \"In vivo shRNA screen; NK-cell depletion; chemical genetics substrate profiling; immunocompromised animal models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic screen with mechanistic follow-up linking ZDHHC20 to innate immune evasion, but substrates not fully validated\",\n      \"pmids\": [\"38733589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC20 activates the PI3K-AKT signaling pathway in hepatocellular carcinoma; knockdown decreases phosphorylation of PI3K and AKT and reduces proliferation, while overexpression increases PI3K/AKT phosphorylation and promotes proliferation; PI3K/AKT inhibitors block ZDHHC20-driven proliferation.\",\n      \"method\": \"ZDHHC20 knockdown/overexpression; Western blot for p-PI3K and p-AKT; CCK-8, colony formation, apoptosis assays; xenograft model; pharmacological inhibitors (LY294002, MK2206)\",\n      \"journal\": \"Journal of hepatocellular carcinoma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD/OE with defined pathway readout and inhibitor confirmation, but specific palmitoylation substrate linking ZDHHC20 to PI3K/AKT not identified\",\n      \"pmids\": [\"39309302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A selective, orally bioavailable acyltransferase inhibitor SD-066-4 inhibits ZDHHC20 by interacting with a specific alanine residue providing isoform selectivity; SD-066-4 stably reduces EGFR S-acylation in KRAS-mutant cells and blocks growth of KRAS-mutant lung tumors in vivo.\",\n      \"method\": \"Small molecule inhibitor development; mutagenesis identifying selectivity-determining alanine; EGFR palmitoylation assay; KRAS-mutant cell/tumor growth assays; in vivo tumor model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — selective inhibitor with defined binding residue and in vivo validation; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.07.18.604152\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZDHHC20 palmitoylates KAP1/TRIM28 at Cys232; this palmitoylation is induced by ATM-dependent phosphorylation of ZDHHC20 at Ser339 after DNA damage. Palmitoylated KAP1 shows increased chromatin binding and promotes recruitment of DDR factors BRCA1 and 53BP1, enhancing DNA damage repair.\",\n      \"method\": \"ZDHHC family screen; acyl-biotin exchange assay; palmitoylation label-free quantitative proteomics; ZDHHC20 KO mice and xenografts; phospho-site mutagenesis; chromatin fractionation; BRCA1/53BP1 recruitment assays; radiosensitivity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-specific mutagenesis, proteomics, in vivo KO, and multiple mechanistic readouts linking ATM phosphorylation of ZDHHC20 to KAP1 palmitoylation and DDR\",\n      \"pmids\": [\"41109928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZDHHC20 catalyzes palmitoylation of CMPK2 at Cys137 and Cys153, maintaining its mitochondrial localization; this promotes ddhCTP production and MAVS stabilization for antiviral IFN-I responses. Depalmitoylation by PPT1 reverses this. Palmitic acid activates this pathway, and PPT1 inhibition restores CMPK2 palmitoylation and antiviral immunity.\",\n      \"method\": \"Metabolic chemical library screen; palmitoylation assays; CMPK2 KO; PPT1 inhibitor (DC661); IFN-I measurement; viral replication assay; mutagenesis of Cys137/153; in vivo diet and inhibitor administration\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-specific mutagenesis with writer (ZDHHC20) and eraser (PPT1) identified, functional readouts in cells and in vivo\",\n      \"pmids\": [\"42011944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RAB11A acts as a scaffold that recruits ZDHHC20 to palmitoylate FGFR3, promoting its plasma membrane retention and preventing degradation in bladder cancer. SREBP2 transcriptionally activates RAB11A (dependent on its liquid-liquid phase separation). Knockdown of RAB11A or ZDHHC20 reduces membrane FGFR3 and attenuates tumor growth in xenografts.\",\n      \"method\": \"Co-immunoprecipitation; promoter-reporter assay; SREBP2 F178A phase-separation mutant; RAB11A/ZDHHC20 knockdown; xenograft models; immunoblotting for membrane FGFR3; functional proliferation/apoptosis assays\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, reporter assay, and in vivo KD evidence but palmitoylation site on FGFR3 not directly validated by mutagenesis\",\n      \"pmids\": [\"41586981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MEF2A transcriptionally activates ZDHHC20 (validated by dual-luciferase reporter and ChIP assays), and ZDHHC20 in turn activates the NF-κB pathway to promote AML cell proliferation, glycolysis, and doxorubicin resistance; ZDHHC20 overexpression rescues antitumor effects of MEF2A silencing.\",\n      \"method\": \"Dual-luciferase reporter assay; ChIP assay; MEF2A knockdown/ZDHHC20 overexpression rescue; NF-κB pathway western blot; xenograft model; functional proliferation/apoptosis/glycolysis assays\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — ChIP and reporter assay establish transcriptional relationship; epistasis rescue places ZDHHC20 downstream of MEF2A and upstream of NF-κB, but palmitoylation substrate linking ZDHHC20 to NF-κB not identified\",\n      \"pmids\": [\"41882733\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZDHHC20 is an integral membrane palmitoyltransferase that autoacylates at its conserved DHHC catalytic cysteine—facilitated by membrane deformation it induces—and then transfers C16 palmitoyl chains to substrate cysteine residues on diverse proteins including EGFR, IFITM3, ORAI1, YTHDF3, FASN, CD80, KAP1/TRIM28, CMPK2, and FGFR3, thereby regulating their membrane localization, stability (competing with ubiquitin-proteasome degradation), and signaling activity in contexts ranging from antiviral immunity and T cell activation to oncogenic pathway activation in cancer.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ZDHHC20 is an integral membrane protein S-acyltransferase (palmitoyltransferase) that transfers palmitoyl groups from palmitoyl-CoA to cysteine residues on a broad array of substrate proteins, thereby controlling their membrane association, stability, and signaling competence across immunity, DNA damage repair, and oncogenesis. Its catalytic mechanism involves membrane deformation that hydrates the DHHC-motif catalytic cysteine for autoacylation, with a transmembrane hydrophobic cavity that confers selectivity for C16 acyl chains [PMID:31858978, PMID:35563480]. Validated substrates include EGFR (C-terminal tail pinning and signal attenuation) [PMID:27153536], IFITM3 (antiviral activity at lysosomes) [PMID:29079573], ORAI1 (lipid raft targeting for T cell Ca²⁺/NFAT signaling) [PMID:34913437], YTHDF3 (protection from chaperone-mediated autophagy, stabilizing MYC mRNA in pancreatic cancer) [PMID:38821916], FASN (competition with ubiquitin-proteasome degradation in hepatocarcinogenesis) [PMID:39696259], CD80 (membrane retention and T cell costimulation) [PMID:38467718], KAP1/TRIM28 (ATM-phosphorylation-dependent palmitoylation enhancing chromatin binding and DNA damage repair) [PMID:41109928], CMPK2 (mitochondrial localization and MAVS-dependent antiviral IFN-I responses) [PMID:42011944], and FGFR3 (RAB11A-scaffolded plasma membrane retention in bladder cancer) [PMID:41586981]. For multiple substrates, ZDHHC20-mediated palmitoylation competes with ubiquitination to protect proteins from proteasomal or lysosomal degradation, establishing palmitoylation as a stability switch [PMID:39696259, PMID:38467718, PMID:38821916].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"The first substrate and physiological function of ZDHHC20 were established: palmitoylation of EGFR C-terminal cysteines pins its tail to the plasma membrane, restraining signaling—revealing that loss of ZDHHC20-mediated palmitoylation sensitizes cancer cells to EGFR inhibitors.\",\n      \"evidence\": \"Mass spectrometry, Cys-to-Ala mutagenesis, DHHC20 knockdown, and transformation/migration assays in cancer cell lines\",\n      \"pmids\": [\"27153536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EGFR palmitoylation by ZDHHC20 is regulated by upstream signals was not addressed\", \"The stoichiometry of palmitoylation on individual EGFR cysteines was not determined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"ZDHHC20 was shown to palmitoylate the antiviral effector IFITM3 and uniquely colocalize with it at lysosomes, distinguishing it from other DHHC enzymes and linking its activity to innate antiviral defense.\",\n      \"evidence\": \"Systematic ZDHHC library screen across 23 family members, ZDHHC KO cell lines, colocalization imaging, and influenza infection assays\",\n      \"pmids\": [\"29079573\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZDHHC20 lysosomal localization is dynamically regulated was not resolved\", \"The precise palmitoylation sites on IFITM3 modified by ZDHHC20 specifically (versus other ZDHHCs) were not delineated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Reconstitution and simulation revealed that ZDHHC20 deforms the cytoplasmic membrane leaflet to hydrate its DHHC catalytic cysteine, establishing the structural basis for its autoacylation step.\",\n      \"evidence\": \"Biochemical reconstitution in POPC nanodiscs combined with microsecond all-atom molecular dynamics simulations\",\n      \"pmids\": [\"31858978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution experimental structure of ZDHHC20 was obtained\", \"How membrane deformation couples to the subsequent substrate acylation (transacylation) step was not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"ZDHHC20-mediated palmitoylation of ORAI1 at Cys143 was shown to be required for lipid-raft targeting, TCR signaling at the immune synapse, sustained Ca²⁺ entry, and IL-2 secretion, establishing ZDHHC20 as a regulator of adaptive immunity.\",\n      \"evidence\": \"Cys143 mutagenesis, patch-clamp electrophysiology, SOCE measurements, NFATC1 translocation, and IL-2 secretion in Jurkat T cells\",\n      \"pmids\": [\"34913437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZDHHC20 palmitoylates ORAI1 in primary T cells in vivo was not tested\", \"Whether other ZDHHCs contribute to ORAI1 palmitoylation was not fully excluded\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Molecular dynamics and structure-activity studies defined the acyl chain length selectivity mechanism: a hydrophobic cavity in ZDHHC20's transmembrane helices accommodates C16 palmitoyl-CoA, and a specific alanine residue determines isoform-selective inhibitor binding.\",\n      \"evidence\": \"MD simulations with V185G mutant shifting to C18 selectivity; SAR of acrylamide-based inhibitors with isoform selectivity assays\",\n      \"pmids\": [\"35563480\", \"36262404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The C16 selectivity mechanism was primarily computationally derived and not validated by in vitro reconstitution with different acyl-CoA substrates\", \"Inhibitor selectivity was tested against ZDHHC2 but not against the full ZDHHC family\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple studies converged to show that ZDHHC20-mediated palmitoylation stabilizes substrates (YTHDF3, FASN, CD80) by competing with ubiquitin-dependent degradation, establishing a general palmitoylation-versus-ubiquitination stability switch operating in cancer and immunity.\",\n      \"evidence\": \"Palmitoylation site mutagenesis, ubiquitination assays, protein half-life measurements, and in vivo models for YTHDF3/MYC in pancreatic cancer, FASN in hepatocarcinogenesis, and CD80 in T cell costimulation\",\n      \"pmids\": [\"38821916\", \"39696259\", \"38467718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the palmitoylation-ubiquitination competition involves direct steric occlusion of the same or proximal lysine residues was not resolved\", \"The E3 ligases targeting YTHDF3 and CD80 for degradation upon loss of palmitoylation were not identified (only the SNX8-TRIM28 complex was identified for FASN)\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"An in vivo shRNA screen identified ZDHHC20 as essential for pancreatic cancer metastatic outgrowth, functioning through modulation of tumor–NK cell interactions rather than cell-autonomous growth.\",\n      \"evidence\": \"In vivo shRNA screen with immunocompromised and NK-cell-depleted animal models; chemical genetics substrate profiling\",\n      \"pmids\": [\"38733589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific ZDHHC20 substrates mediating NK cell evasion were identified by chemical genetics but not individually validated\", \"Whether the immune evasion mechanism operates in human patients was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"ZDHHC20 was shown to activate PI3K-AKT signaling in hepatocellular carcinoma, promoting proliferation that is reversible by PI3K/AKT inhibitors, though the direct palmitoylation substrate was not identified.\",\n      \"evidence\": \"ZDHHC20 knockdown/overexpression, phospho-PI3K/AKT western blots, pharmacological inhibitors (LY294002, MK2206), xenograft model\",\n      \"pmids\": [\"39309302\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The palmitoylation substrate linking ZDHHC20 to PI3K-AKT activation was not identified\", \"Whether this effect is independent of ZDHHC20's known substrates (e.g., EGFR, FASN) was not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ATM-dependent phosphorylation of ZDHHC20 at Ser339 was shown to activate palmitoylation of KAP1/TRIM28, enhancing its chromatin binding and recruitment of DDR factors BRCA1 and 53BP1—revealing ZDHHC20 as a signal-regulated enzyme in the DNA damage response.\",\n      \"evidence\": \"ZDHHC family screen, acyl-biotin exchange, phospho-site mutagenesis, ZDHHC20 KO mice, chromatin fractionation, DDR factor recruitment and radiosensitivity assays\",\n      \"pmids\": [\"41109928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other DNA damage kinases phosphorylate ZDHHC20 at additional sites was not explored\", \"The structural basis for how Ser339 phosphorylation enhances enzymatic activity was not determined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"ZDHHC20 palmitoylation of CMPK2 at Cys137/153 was shown to maintain its mitochondrial localization and promote MAVS-dependent type I IFN responses, with PPT1 acting as the counteracting depalmitoylase—establishing a writer-eraser pair for antiviral innate immunity.\",\n      \"evidence\": \"Metabolic chemical library screen, CMPK2 Cys mutagenesis, PPT1 inhibitor DC661, IFN-I measurement, viral replication assays, in vivo diet and inhibitor studies\",\n      \"pmids\": [\"42011944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other ZDHHCs also palmitoylate CMPK2 was not fully excluded\", \"The structural basis of ZDHHC20-CMPK2 recognition was not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"RAB11A was identified as a scaffold that recruits ZDHHC20 to palmitoylate FGFR3 for plasma membrane retention in bladder cancer, with SREBP2-driven transcription of RAB11A as an upstream regulatory node.\",\n      \"evidence\": \"Co-immunoprecipitation, promoter-reporter assays, RAB11A/ZDHHC20 knockdown, xenograft models\",\n      \"pmids\": [\"41586981\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The palmitoylation site(s) on FGFR3 were not validated by site-directed mutagenesis\", \"Whether RAB11A scaffolding is specific to FGFR3 or extends to other ZDHHC20 substrates was not tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"MEF2A was identified as a direct transcriptional activator of ZDHHC20, and ZDHHC20 was placed upstream of NF-κB signaling in AML, promoting proliferation, glycolysis, and chemoresistance.\",\n      \"evidence\": \"ChIP assay, dual-luciferase reporter, MEF2A-KD/ZDHHC20-OE epistasis rescue, NF-κB pathway western blots, xenograft model\",\n      \"pmids\": [\"41882733\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The palmitoylation substrate connecting ZDHHC20 to NF-κB activation was not identified\", \"Whether MEF2A regulation of ZDHHC20 operates in non-AML contexts was not explored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the high-resolution structure of ZDHHC20, the rules governing substrate recognition across its diverse substrates, whether signal-dependent regulation (as shown for ATM-Ser339 phosphorylation) is a general feature, and the therapeutic window of ZDHHC20 inhibition given its roles in both tumor promotion and immune function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental high-resolution structure of ZDHHC20 has been determined\", \"Substrate recognition determinants beyond the hydrophilic ridge in TM4 remain undefined\", \"In vivo consequences of systemic ZDHHC20 inhibition on immune homeostasis are unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 7, 8, 12, 13, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 8, 14]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 10]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 3, 8, 9, 13]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 7, 10, 11, 14, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 6, 7, 8, 12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"EGFR\",\n      \"IFITM3\",\n      \"ORAI1\",\n      \"YTHDF3\",\n      \"FASN\",\n      \"CD80\",\n      \"TRIM28\",\n      \"CMPK2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}