{"gene":"ZDHHC3","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2002,"finding":"GODZ (ZDHHC3) is a Golgi apparatus-specific protein with a DHHC zinc finger domain and four putative transmembrane regions; overexpression in COS7 cells suppressed sorting of the glutamate receptor GluRα1 from the Golgi apparatus, implicating ZDHHC3 in membrane protein trafficking.","method":"Overexpression in COS7 cells, subcellular localization by immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, cell overexpression with defined trafficking phenotype, no reconstitution or mutagenesis of catalytic site","pmids":["12163046"],"is_preprint":false},{"year":2004,"finding":"ZDHHC3 (GODZ) palmitoylates the γ2 subunit of GABA(A) receptors via a cytoplasmic loop cysteine-rich 14-amino acid domain conserved in γ1-3 subunits; ZDHHC3 is localized asymmetrically in the neuronal Golgi complex and interacts with γ2 through the SOS-recruitment (yeast two-hybrid) system.","method":"SOS-recruitment yeast two-hybrid, coexpression in heterologous cells with palmitoylation assay, subcellular localization by immunofluorescence","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction assay plus cell-based palmitoylation assay, replicated in subsequent studies","pmids":["15229235"],"is_preprint":false},{"year":2006,"finding":"ZDHHC3 and its paralog SERZ-β (DHHC7) form homomultimers and heteromultimers; ZDHHC3 is the primary enzyme palmitoylating the GABA(A) receptor γ2 subunit, and dominant-negative ZDHHC3 (C157S) or ZDHHC3 RNAi reduces GABA(A) receptor accumulation at inhibitory synapses and impairs GABAergic synaptic function without affecting AMPA receptor-mediated transmission.","method":"Co-immunoprecipitation, in vivo cross-linking, RNAi knockdown in neurons, dominant-negative overexpression, whole-cell and synaptic electrophysiology","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, cross-linking, RNAi, electrophysiology), replicated across labs","pmids":["17151279"],"is_preprint":false},{"year":2009,"finding":"ZDHHC3 mediates Ca²⁺ transport when expressed in Xenopus oocytes; this transport is dependent on palmitoylation activity (abolished by 2-bromopalmitate or DHHC→DHHS active-site mutation by ~80%), but a separate V61R mutation abolishes Ca²⁺ transport without affecting palmitoyl acyltransferase activity, indicating the two functions are separable.","method":"Two-electrode voltage clamp, fluorescence Ca²⁺ imaging, ⁴⁵Ca²⁺ isotopic uptake in Xenopus oocytes, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro functional assay with mutagenesis but single lab, no replication","pmids":["19955568"],"is_preprint":false},{"year":2012,"finding":"ZDHHC3 is the palmitoyltransferase responsible for palmitoylation of integrin β4 and α6 subunits; DHHC3 ablation accelerates lysosomal degradation of α6β4 via increased cathepsin D exposure, impairs integrin signaling through Src, reduces β4 phosphorylation at S1356 and S1424, and blocks integrin-dependent cable formation on Matrigel, while ~10 other cell-surface proteins including α3β1 are unaffected.","method":"RNAi knockdown, overexpression in multiple cell lines, palmitoylation assay, Src signaling/phosphorylation assays, cathepsin D inhibitor rescue (Pepstatin A), cell-surface biotinylation","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (RNAi, OE, inhibitor rescue, signaling assays) in single lab","pmids":["22314500"],"is_preprint":false},{"year":2012,"finding":"ZDHHC3 interacts with the death domain of TRAIL receptor DR4 (but not DR5) through its DHHC and C-terminal transmembrane domains, and promotes localization of DR4 to the plasma membrane via the DHHC motif, thereby sensitizing tumor cells to TRAIL-induced apoptosis.","method":"SOS protein-recruitment yeast two-hybrid, co-immunoprecipitation, subcellular localization assay, apoptosis assays, cysteine mutagenesis of DR4","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid plus Co-IP plus functional apoptosis rescue, single lab","pmids":["22240897"],"is_preprint":false},{"year":2015,"finding":"ZDHHC3 undergoes autoacylation (palmitoylation) at the cysteine within the DHHC motif; conserved cysteines outside the DHHC motif coordinate two zinc ions per ZDHHC3 molecule, and mutation of these cysteines or chelation of zinc by EDTA causes structural perturbation and loss of palmitoyl acyltransferase activity.","method":"Mass spectrometry identification of palmitoylation site, site-directed mutagenesis of conserved CRD cysteines, limited proteolysis, metal chelation with EDTA, zinc quantification using fluorescent indicator mag-fura-2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mass spectrometry + mutagenesis + metal quantification, multiple orthogonal methods in single lab","pmids":["26487721"],"is_preprint":false},{"year":2016,"finding":"In GODZ (ZDHHC3) knockout mice, palmitoylation of γ2 subunit of GABA(A) receptors and GAP-43 is significantly reduced; GABA(A) receptor synaptic accumulation and GABAergic innervation are decreased in GODZ KO neurons competing with wild-type neurons; total cell-surface GABA(A) receptor expression and whole-cell GABAergic currents are unaltered in isolated DKO neurons, indicating GODZ-mediated palmitoylation selectively controls the synaptic pool of receptors. SERZ-β (DHHC7) KO alone does not affect γ2 palmitoylation.","method":"Knockout mice (GODZ KO, SERZ-β KO, double KO), palmitoylation assay, electrophysiology, immunofluorescence, subcellular fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple orthogonal readouts, replicated across GODZ, SERZ-β, and DKO genotypes","pmids":["27875292"],"is_preprint":false},{"year":2016,"finding":"ZDHHC3 is phosphorylated by FGFR1 at Tyr18 and by Src kinase at Tyr295 and Tyr297; abrogation of these tyrosine phosphorylation sites increases ZDHHC3 autopalmitoylation, enhances interaction with NCAM, upregulates NCAM palmitoylation, and promotes neurite outgrowth in hippocampal neurons.","method":"Pharmacological inhibition and overexpression of FGFR/Src, site-directed mutagenesis of ZDHHC3 tyrosines, cell-free and cell-based kinase assays, palmitoylation assay, co-immunoprecipitation, neurite outgrowth assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis + cell-free kinase assay + cell-based functional rescue in neurons, multiple orthogonal methods single lab","pmids":["27247265"],"is_preprint":false},{"year":2016,"finding":"ZDHHC3 functions downstream of acsl1b to palmitoylate Gsα at mapped cysteine residues in Xenopus oocytes, maintaining meiotic G2/prophase I arrest; depletion of ZDHHC3 reduces palmitoylated Gsα levels and lowers the progesterone threshold for G2/M transition from 2 μM to 0.01 μM.","method":"RNA depletion (antisense morpholino) in Xenopus oocytes, palmitoylation assay, site-directed mutagenesis of Gsα palmitoylation sites and ZDHHC3 active site, progesterone dose-response assay","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic depletion + mutagenesis + biochemical palmitoylation assay, single lab, Xenopus ortholog model","pmids":["27512151"],"is_preprint":false},{"year":2017,"finding":"ZDHHC3 palmitoylates ERGIC3 protein; loss of ZDHHC3-dependent palmitoylation of ERGIC3 leads to upregulation of TXNIP, increased oxidative stress, and cellular senescence in breast cancer cells; these antitumor effects are reversed by wild-type but not enzyme-active-site-deficient ZDHHC3, and are substantially negated by co-depletion of TXNIP.","method":"RNAi ablation, wild-type vs. catalytic mutant ZDHHC3 reconstitution, gene array, fluorescence dye oxidative stress assays, xenograft tumor model, flow cytometry for immune cell recruitment","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic mutant reconstitution + gene array + functional in vivo rescue, single lab","pmids":["29055014"],"is_preprint":false},{"year":2017,"finding":"ZDHHC3 binds specifically to HSV-1 UL20 (but not other HSV-1 proteins) in the Golgi apparatus via yeast two-hybrid and pulldown assays, palmitoylates UL20 (blocked by dominant-negative ZDHHC3 C157S or 2-bromopalmitate), and is required for proper localization of UL20 and glycoprotein K (gK) and for HSV-1 replication in vitro.","method":"Yeast two-hybrid, pulldown assay, dominant-negative ZDHHC3 overexpression, palmitoylation assay (acyl-RAC), 2-bromopalmitate inhibition, immunofluorescence localization, viral titer assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction assays plus palmitoylation assay plus functional viral replication readout, replicated in GODZ KO mice paper","pmids":["28724772"],"is_preprint":false},{"year":2018,"finding":"In ZDHHC3 (GODZ) knockout MEFs, HSV-1 replication is compromised; ZDHHC3 absence blocks UL20 palmitoylation, alters localization and expression of UL20 and gK, affects expression of gB and gC, and disrupts tegument/capsid protein localization; in vivo, GODZ KO mice show reduced corneal HSV-1 replication, lower corneal scarring, and reduced latency reactivation.","method":"GODZ KO mouse-derived MEFs, palmitoylation assay, electron microscopy, immunofluorescence, viral titer, in vivo ocular infection model","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO model + multiple orthogonal mechanistic readouts + in vivo validation, replicates findings of PMID:28724772","pmids":["29187538"],"is_preprint":false},{"year":2020,"finding":"Comparative mass spectrometry-based palmitoyl-proteomics of breast and prostate cancer cells ± ZDHHC3 ablation identified 22–28 antioxidant/redox-regulatory proteins as candidate ZDHHC3 substrates; ZDHHC3 ablation elevated oxidative stress, which synergized with chemotherapeutic agents to enhance anti-growth effects.","method":"Comparative mass spectrometry palmitoyl-proteomics, RNAi ablation, fluorescence dye oxidative stress assays, cell proliferation assays","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry substrate identification + functional oxidative stress assays, single lab","pmids":["32986127"],"is_preprint":false},{"year":2021,"finding":"A high-throughput Acyl-cLIP assay was developed and validated for ZDHHC3/7/20 enzymatic activity; in vitro results from this assay correlated with cell-based palmitoylation assays, confirming ZDHHC3 catalytic activity as amenable to quantitative screening.","method":"Acyl-cLIP (acylation-coupled lipophilic induction of polarization) high-throughput assay, cell-based palmitoylation assay","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with cell-based validation, single lab, assay development paper","pmids":["34374518"],"is_preprint":false},{"year":2023,"finding":"ZDHHC3 palmitoylates IRHOM2 at C476 within the iRhom homology domain via its DHHC (C157) catalytic domain; palmitoylation promotes IRHOM2 cytomembrane translocation and stabilization by blocking TRIM31-mediated ubiquitination and proteasomal degradation; hepatocyte-specific ZDHHC3 knockout suppresses IRHOM2 accumulation and attenuates NASH pathology in rodent and rabbit models.","method":"Co-immunoprecipitation, site-directed mutagenesis (C476 of IRHOM2, C157 of ZDHHC3), acyl-RAC palmitoylation assay, ubiquitination assay, hepatocyte-specific KO mice, in vivo NASH diet models","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of both substrate and enzyme active site + ubiquitination assay + genetic KO in vivo, multiple orthogonal methods single lab","pmids":["37544908"],"is_preprint":false},{"year":2024,"finding":"ZDHHC3 inhibits PD-L1 lysosomal degradation by palmitoylating PD-L1; the natural compound benzosceptrin C (BC) inhibits ZDHHC3 enzymatic activity, causing PD-L1 to relocate from the membrane to the cytoplasm, preventing recycling endosome-mediated return to the membrane, and triggering lysosomal degradation of PD-L1.","method":"Palmitoylation assay, ZDHHC3 enzymatic inhibition assay, subcellular localization imaging, lysosomal inhibitor rescue, in vivo MC38 tumor model, T cell cytotoxicity assay","journal":"Cell reports. Medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — enzymatic inhibition + localization imaging + lysosomal rescue + in vivo model, multiple orthogonal methods single lab","pmids":["38237597"],"is_preprint":false},{"year":2024,"finding":"ZDHHC3 S-acylates SCAP at C264, antagonizing HACE1-mediated ubiquitination of SCAP and preventing its proteasomal degradation; SREBP2 transcriptionally upregulates ZDHHC3, forming a positive feedback loop that sustains cholesterol biosynthesis in HCC; depalmitoylase ABHD17A reverses this modification.","method":"Site-directed mutagenesis (SCAP C264), palmitoylation assay, ubiquitination assay, ChIP/transcription factor binding assay, CRISPR/siRNA knockdown, in vivo DEN/CCl4 HCC mouse model, ZDHHC3 small-molecule inhibitor","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of palmitoylation site + ubiquitination assay + transcriptional feedback validation + in vivo KO model, multiple orthogonal methods single lab","pmids":["39522165"],"is_preprint":false},{"year":2024,"finding":"ZDHHC3 palmitoylates Cadm4 at cysteine-347 (C347) to stabilize its plasma membrane localization in oligodendrocytes; genetic deletion of ZDHHC3 reduces Cadm4 palmitoylation, causes Cadm4 internalization and degradation, and leads to CNS myelination defects and impaired neuronal transmission/cognitive behavior phenocopying Cadm4-C347A knock-in mice.","method":"Site-directed mutagenesis (Cadm4 C347A knock-in), ZDHHC3 knockout mice, palmitoylation assay, subcellular fractionation, confocal imaging, electrophysiology, behavioral assays","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO + substrate knock-in phenocopy + palmitoylation assay + functional neurophysiology, multiple orthogonal methods single lab","pmids":["39327467"],"is_preprint":false},{"year":2024,"finding":"ZDHHC3 catalyzes palmitoylation of the PML/RARα oncofusion protein, which is required for its oncogenic transcriptional activity; ZDHHC3 knockdown or overexpression respectively suppresses or promotes APL cell proliferation and blocks differentiation, and ZDHHC3 inhibition arrests malignant progression including in drug-resistant APL.","method":"RNAi knockdown, overexpression, palmitoylation assay, gene expression profiling (proliferation/differentiation markers), in vivo APL mouse model","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — palmitoylation assay plus bidirectional genetic manipulation plus in vivo model, single lab","pmids":["39227737"],"is_preprint":false},{"year":2025,"finding":"ZDHHC3 palmitoylates PRRSV nucleocapsid (N) protein at cysteine 90, which prevents N protein interaction with Nsp9 and inhibits viral RNA synthesis; the depalmitoylase LYPLA1 reverses this modification, counteracting ZDHHC3 activity and thereby promoting PRRSV replication.","method":"Palmitoylation assay, site-directed mutagenesis (N protein C90), co-immunoprecipitation (Nsp9-N interaction), viral RNA synthesis assay, siRNA knockdown, LYPLA1 inhibitor ML348","journal":"Veterinary microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of palmitoylation site + interaction assay + viral replication readout, single lab, porcine virus system","pmids":["39787744"],"is_preprint":false},{"year":2026,"finding":"ZDHHC3 (together with ZDHHC7) mediates S-acylation of the small GTPase ARL15 at three conserved N-terminal cysteine residues (Cys17, Cys22, Cys23); loss of S-acylation disrupts ARL15 membrane association; dual siRNA knockdown and CRISPR knockout of both ZDHHC3 and ZDHHC7 markedly reduces ARL15 S-acylation and redistributes ARL15 from membranes to cytosol.","method":"Acyl-PEGyl exchange gel-shift assay (APEGS), site-directed mutagenesis of ARL15 cysteines, siRNA knockdown, CRISPR/Cas9 gene disruption, confocal imaging, subcellular fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — APEGS stoichiometry assay + mutagenesis + CRISPR validation, multiple methods single lab","pmids":["41999893"],"is_preprint":false},{"year":2026,"finding":"ZDHHC3 S-acylates CRY1 (the core circadian transcriptional repressor) at four cysteine residues; this S-acylation is required for CRY1 nuclear import and interaction with key clock components; loss of CRY1 S-acylation via cysteine mutagenesis or genetic deletion of DHHC3 impairs CRY1 repressor function and disrupts cellular circadian rhythms.","method":"Unbiased palmitoylation screen of clock proteins, site-directed mutagenesis of CRY1 cysteines, DHHC3 genetic deletion, nuclear import assay, co-immunoprecipitation of clock components, circadian rhythm bioluminescence reporter assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis + genetic KO + functional circadian reporter, single lab, preprint not yet peer-reviewed","pmids":["42239456"],"is_preprint":true}],"current_model":"ZDHHC3 (GODZ) is a Golgi-localized DHHC-family palmitoyl acyltransferase that catalyzes S-palmitoylation of a broad set of substrates—including GABA(A) receptor γ2 subunit, integrin α6β4, NCAM, IRHOM2, SCAP, Cadm4, PD-L1, PML/RARα, HSV-1 UL20, CRY1, and ARL15—via a two-step autoacylation–transacylation mechanism requiring the DHHC catalytic motif and zinc coordination by conserved CRD cysteines; its activity is regulated by FGFR1/Src-mediated tyrosine phosphorylation, and it controls substrate membrane targeting, stability (by antagonizing ubiquitin-proteasomal degradation), synaptic clustering, viral replication, cholesterol biosynthesis, circadian rhythms, and antitumor immunity."},"narrative":{"mechanistic_narrative":"ZDHHC3 (GODZ) is a Golgi-resident DHHC-family palmitoyl acyltransferase that S-acylates a broad set of membrane and signaling proteins to control their membrane targeting, stability, and downstream function [PMID:12163046, PMID:15229235]. Catalysis proceeds through a two-step mechanism: ZDHHC3 first autoacylates the cysteine within its DHHC motif, and conserved cysteines outside this motif coordinate two zinc ions required for structural integrity and enzymatic activity [PMID:26487721]. Its activity is tuned by FGFR1/Src-mediated tyrosine phosphorylation at Tyr18, Tyr295, and Tyr297, which when abolished increases autopalmitoylation and substrate modification [PMID:27247265]. In the nervous system ZDHHC3 palmitoylates the GABA(A) receptor γ2 subunit through a cytoplasmic cysteine-rich loop, selectively controlling the synaptic receptor pool and GABAergic innervation [PMID:15229235, PMID:17151279, PMID:27875292], and palmitoylates Cadm4 to stabilize its plasma-membrane localization in oligodendrocytes, supporting CNS myelination [PMID:39327467]. A recurrent mechanistic theme is that ZDHHC3-mediated palmitoylation stabilizes substrates by antagonizing their degradation: it protects integrin α6β4 from lysosomal turnover [PMID:22314500], blocks TRIM31-mediated ubiquitination of IRHOM2 [PMID:37544908], antagonizes HACE1-mediated ubiquitination of SCAP within an SREBP2 positive-feedback loop sustaining cholesterol biosynthesis [PMID:39522165], and prevents lysosomal degradation of PD-L1 to modulate antitumor immunity [PMID:38237597]. ZDHHC3 also palmitoylates viral proteins required for replication, including HSV-1 UL20 [PMID:28724772, PMID:29187538] and the PRRSV nucleocapsid protein [PMID:39787744], and modifies the PML/RARα oncofusion protein and the circadian repressor CRY1 [PMID:39227737, PMID:42239456]. Substrate breadth is supported by palmitoyl-proteomic surveys identifying numerous redox-regulatory candidate substrates [PMID:32986127].","teleology":[{"year":2002,"claim":"Established ZDHHC3 as a Golgi-specific DHHC-domain protein involved in membrane protein trafficking, framing where and on what class of proteins it acts.","evidence":"Overexpression and immunofluorescence localization in COS7 cells with a GluRα1 sorting phenotype","pmids":["12163046"],"confidence":"Medium","gaps":["No catalytic-site mutagenesis or reconstitution","Enzymatic palmitoylation activity not yet demonstrated"]},{"year":2004,"claim":"Identified the first defined substrate, the GABA(A) receptor γ2 subunit, establishing ZDHHC3 as a bona fide palmitoyl acyltransferase acting through a substrate cysteine-rich domain.","evidence":"Yeast two-hybrid interaction plus cell-based palmitoylation assay and Golgi localization in neurons","pmids":["15229235"],"confidence":"High","gaps":["In vitro reconstitution with purified components absent","Physiological consequence at synapses not yet tested"]},{"year":2006,"claim":"Demonstrated that ZDHHC3 oligomerizes and is the primary enzyme controlling synaptic GABA(A) receptor accumulation, linking palmitoylation to inhibitory synaptic function.","evidence":"Co-IP, cross-linking, neuronal RNAi, dominant-negative C157S, and electrophysiology","pmids":["17151279"],"confidence":"High","gaps":["Functional role of homo/heteromultimerization unresolved","Redundancy with DHHC7 not fully delineated"]},{"year":2009,"claim":"Showed a palmitoylation-independent Ca2+ transport function separable from acyltransferase activity, raising the possibility of a second molecular role.","evidence":"Voltage clamp, Ca2+ imaging and 45Ca uptake in Xenopus oocytes with V61R vs DHHS mutants","pmids":["19955568"],"confidence":"Medium","gaps":["Single lab, no replication","Physiological relevance in mammalian cells unknown"]},{"year":2012,"claim":"Generalized ZDHHC3 substrate scope beyond neurons (integrin α6β4, TRAIL receptor DR4) and revealed that palmitoylation protects substrates from degradation and shapes their membrane localization and signaling.","evidence":"RNAi, overexpression, palmitoylation and Src signaling assays, cathepsin D rescue, Co-IP, apoptosis assays","pmids":["22314500","22240897"],"confidence":"High","gaps":["Whether DR4 effect requires palmitoylation per se vs interaction not fully separated","Selectivity determinants among integrin subunits unexplained"]},{"year":2015,"claim":"Defined the catalytic chemistry: autoacylation at the DHHC cysteine plus zinc coordination by conserved CRD cysteines required for activity and structure.","evidence":"Mass spectrometry, CRD cysteine mutagenesis, limited proteolysis, EDTA chelation, zinc quantification","pmids":["26487721"],"confidence":"High","gaps":["No high-resolution structure of the enzyme","Transacylation step kinetics not resolved"]},{"year":2016,"claim":"In vivo genetics confirmed substrate selectivity (synaptic GABA(A) receptor pool, GAP-43) and uncovered tyrosine phosphorylation by FGFR1/Src as a regulatory input controlling autopalmitoylation and NCAM modification.","evidence":"GODZ/SERZ-β knockout mice with palmitoylation, electrophysiology, and fractionation; kinase assays and tyrosine-site mutagenesis with neurite outgrowth","pmids":["27875292","27247265"],"confidence":"High","gaps":["How phosphorylation alters enzyme conformation unknown","Upstream signals coupling FGFR/Src to ZDHHC3 in vivo unmapped"]},{"year":2016,"claim":"Extended the substrate repertoire to Gsα in a developmental context, linking ZDHHC3 to meiotic arrest control downstream of acsl1b.","evidence":"Morpholino depletion, mutagenesis, palmitoylation assay and progesterone dose-response in Xenopus oocytes","pmids":["27512151"],"confidence":"Medium","gaps":["Single lab, Xenopus model","Mammalian relevance untested"]},{"year":2017,"claim":"Established ZDHHC3 as a host factor for HSV-1 by palmitoylating UL20, required for viral glycoprotein localization and replication, validated genetically and in vivo.","evidence":"Yeast two-hybrid, pulldown, acyl-RAC, dominant-negative and KO-MEF/mouse ocular infection models","pmids":["28724772","29187538"],"confidence":"High","gaps":["UL20 palmitoylation site not pinpointed in the narrative","Whether targeting ZDHHC3 is therapeutically tractable against HSV-1 untested"]},{"year":2017,"claim":"Connected ZDHHC3 palmitoylation (of ERGIC3) to redox homeostasis and tumor suppression via a TXNIP-dependent oxidative-stress axis.","evidence":"Catalytic-mutant reconstitution, gene array, oxidative stress assays, xenograft, TXNIP co-depletion","pmids":["29055014"],"confidence":"Medium","gaps":["Single lab","Direct ERGIC3 palmitoylation site not mapped"]},{"year":2020,"claim":"Palmitoyl-proteomics broadened the candidate substrate landscape toward antioxidant/redox proteins and linked ZDHHC3 loss to chemosensitization.","evidence":"Comparative MS palmitoyl-proteomics with RNAi and proliferation assays in cancer cells","pmids":["32986127"],"confidence":"Medium","gaps":["Candidate substrates not individually validated as direct","Mechanism of synergy with chemotherapy unresolved"]},{"year":2021,"claim":"Provided a quantitative high-throughput assay enabling enzymatic screening of ZDHHC3, supporting drug-discovery efforts.","evidence":"Acyl-cLIP assay validated against cell-based palmitoylation for ZDHHC3/7/20","pmids":["34374518"],"confidence":"Medium","gaps":["Assay development, not a biological discovery","Selectivity over other DHHC enzymes limited"]},{"year":2023,"claim":"Defined a stability-control paradigm in metabolic disease: ZDHHC3 palmitoylates IRHOM2 at C476 to block TRIM31 ubiquitination, driving NASH pathology.","evidence":"Co-IP, dual enzyme/substrate mutagenesis, acyl-RAC, ubiquitination assay, hepatocyte-specific KO in rodent and rabbit NASH models","pmids":["37544908"],"confidence":"High","gaps":["Structural basis of palmitoyl-ubiquitin antagonism unknown","Generality of the ubiquitination-antagonism mechanism across substrates not formalized"]},{"year":2024,"claim":"Consolidated the degradation-antagonism mechanism across diverse pathways: PD-L1 (immunity), SCAP (cholesterol biosynthesis feedback), Cadm4 (myelination), and PML/RARα (leukemia oncogenicity).","evidence":"Site mutagenesis, palmitoylation/ubiquitination assays, ChIP, lysosomal rescue, KO and knock-in mice, and in vivo tumor/myelination models","pmids":["38237597","39522165","39327467","39227737"],"confidence":"High","gaps":["Determinants of substrate selectivity across these targets not unified","Whether a single inhibitor can disentangle these roles therapeutically untested"]},{"year":2026,"claim":"Expanded substrates to membrane-targeting of the GTPase ARL15 (with ZDHHC7) and to nuclear import and repressor function of circadian CRY1, broadening ZDHHC3 roles into trafficking and clock regulation.","evidence":"APEGS, cysteine mutagenesis, siRNA/CRISPR for ARL15; palmitoylation screen, mutagenesis, DHHC3 deletion and circadian reporter for CRY1 (preprint)","pmids":["41999893","42239456"],"confidence":"Medium","gaps":["CRY1 finding is a preprint awaiting peer review","How a Golgi enzyme controls nuclear CRY1 import mechanistically unclear"]},{"year":null,"claim":"How ZDHHC3 achieves substrate selectivity among its many targets, and whether its various disease-relevant roles can be separated pharmacologically, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution enzyme-substrate structure","Substrate recognition code undefined","Selectivity of available small-molecule inhibitors across DHHC enzymes incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,4,6,11,15,17,18]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,4,15,17,18]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,1,11]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,15,17,16]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[1,2,7,18]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[17]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[22]}],"complexes":[],"partners":["GABRG2","ITGB4","ITGA6","NCAM1","IRHOM2","SCAP","CADM4","ZDHHC7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NYG2","full_name":"Palmitoyltransferase ZDHHC3","aliases":["Acyltransferase ZDHHC3","Protein DHHC1","Zinc finger DHHC domain-containing protein 3","DHHC-3"],"length_aa":299,"mass_kda":34.2,"function":"Golgi-localized palmitoyltransferase that catalyzes the addition of palmitate onto various protein substrates (PubMed:19001095, PubMed:21926431, PubMed:22240897, PubMed:22314500, PubMed:23034182). Has no stringent fatty acid selectivity and in addition to palmitate can also transfer onto target proteins myristate from tetradecanoyl-CoA and stearate from octadecanoyl-CoA (By similarity). Plays an important role in G protein-coupled receptor signaling pathways involving GNAQ and potentially other heterotrimeric G proteins by regulating their dynamic association with the plasma membrane (PubMed:19001095). Palmitoylates ITGA6 and ITGB4, thereby regulating the alpha-6/beta-4 integrin localization, expression and function in cell adhesion to laminin (PubMed:22314500). Plays a role in the TRAIL-activated apoptotic signaling pathway most probably through the palmitoylation and localization to the plasma membrane of TNFRSF10A (PubMed:22240897). In the brain, by palmitoylating the gamma subunit GABRG2 of GABA(A) receptors and regulating their postsynaptic accumulation, plays a role in synaptic GABAergic inhibitory function and GABAergic innervation (By similarity). Palmitoylates the neuronal protein GAP43 which is also involved in the formation of GABAergic synapses (By similarity). Palmitoylates NCDN thereby regulating its association with endosome membranes (By similarity). Probably palmitoylates PRCD and is involved in its proper localization within the photoreceptor (By similarity). Could mediate the palmitoylation of NCAM1 and regulate neurite outgrowth (By similarity). Could palmitoylate DNAJC5 and regulate its localization to Golgi membranes (By similarity). Also constitutively palmitoylates DLG4 (By similarity). May also palmitoylate SNAP25 (By similarity). Could palmitoylate the glutamate receptors GRIA1 and GRIA2 but this has not been confirmed in vivo (By similarity). Could also palmitoylate the D(2) dopamine receptor DRD2 (PubMed:26535572). May also palmitoylate LAMTOR1, promoting its localization to lysosomal membranes (PubMed:35893977). Palmitoylates the Toll-like receptor 9/TLR9 in the Golgi and thereby regulates TLR9 trafficking to endosomes (PubMed:38169466). May palmitoylate CALHM1 and CALHM3 subunits of gustatory voltage-gated ion channels and modulate channel gating and kinetics May also function as a calcium transporter","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q9NYG2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZDHHC3","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"COPB2","stoichiometry":0.2},{"gene":"GORASP2","stoichiometry":0.2},{"gene":"TMED10","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ZDHHC3","total_profiled":1310},"omim":[{"mim_id":"621547","title":"ZDHHC PALMITOYLTRANSFERASE 4; ZDHHC4","url":"https://www.omim.org/entry/621547"},{"mim_id":"617334","title":"ZDHHC PALMITOYLTRANSFERASE 23; ZDHHC23","url":"https://www.omim.org/entry/617334"},{"mim_id":"617150","title":"ZDHHC PALMITOYLTRANSFERASE 3; ZDHHC3","url":"https://www.omim.org/entry/617150"},{"mim_id":"608784","title":"ZDHHC PALMITOYLTRANSFERASE 8; ZDHHC8","url":"https://www.omim.org/entry/608784"},{"mim_id":"190090","title":"SRC PROTOONCOGENE, NONRECEPTOR TYROSINE KINASE; SRC","url":"https://www.omim.org/entry/190090"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZDHHC3"},"hgnc":{"alias_symbol":["ZNF373","GODZ","DHHC3"],"prev_symbol":[]},"alphafold":{"accession":"Q9NYG2","domains":[{"cath_id":"1.20.140","chopping":"46-103_167-280","consensus_level":"high","plddt":94.3001,"start":46,"end":280}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NYG2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NYG2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NYG2-F1-predicted_aligned_error_v6.png","plddt_mean":85.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZDHHC3","jax_strain_url":"https://www.jax.org/strain/search?query=ZDHHC3"},"sequence":{"accession":"Q9NYG2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NYG2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NYG2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NYG2"}},"corpus_meta":[{"pmid":"15229235","id":"PMC_15229235","title":"The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ.","date":"2004","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/15229235","citation_count":203,"is_preprint":false},{"pmid":"17151279","id":"PMC_17151279","title":"GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses.","date":"2006","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/17151279","citation_count":138,"is_preprint":false},{"pmid":"38237597","id":"PMC_38237597","title":"Benzosceptrin C induces lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting DHHC3.","date":"2024","source":"Cell reports. 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in COS7 cells, subcellular localization by immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, cell overexpression with defined trafficking phenotype, no reconstitution or mutagenesis of catalytic site\",\n      \"pmids\": [\"12163046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ZDHHC3 (GODZ) palmitoylates the γ2 subunit of GABA(A) receptors via a cytoplasmic loop cysteine-rich 14-amino acid domain conserved in γ1-3 subunits; ZDHHC3 is localized asymmetrically in the neuronal Golgi complex and interacts with γ2 through the SOS-recruitment (yeast two-hybrid) system.\",\n      \"method\": \"SOS-recruitment yeast two-hybrid, coexpression in heterologous cells with palmitoylation assay, subcellular localization by immunofluorescence\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction assay plus cell-based palmitoylation assay, replicated in subsequent studies\",\n      \"pmids\": [\"15229235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZDHHC3 and its paralog SERZ-β (DHHC7) form homomultimers and heteromultimers; ZDHHC3 is the primary enzyme palmitoylating the GABA(A) receptor γ2 subunit, and dominant-negative ZDHHC3 (C157S) or ZDHHC3 RNAi reduces GABA(A) receptor accumulation at inhibitory synapses and impairs GABAergic synaptic function without affecting AMPA receptor-mediated transmission.\",\n      \"method\": \"Co-immunoprecipitation, in vivo cross-linking, RNAi knockdown in neurons, dominant-negative overexpression, whole-cell and synaptic electrophysiology\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, cross-linking, RNAi, electrophysiology), replicated across labs\",\n      \"pmids\": [\"17151279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ZDHHC3 mediates Ca²⁺ transport when expressed in Xenopus oocytes; this transport is dependent on palmitoylation activity (abolished by 2-bromopalmitate or DHHC→DHHS active-site mutation by ~80%), but a separate V61R mutation abolishes Ca²⁺ transport without affecting palmitoyl acyltransferase activity, indicating the two functions are separable.\",\n      \"method\": \"Two-electrode voltage clamp, fluorescence Ca²⁺ imaging, ⁴⁵Ca²⁺ isotopic uptake in Xenopus oocytes, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro functional assay with mutagenesis but single lab, no replication\",\n      \"pmids\": [\"19955568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ZDHHC3 is the palmitoyltransferase responsible for palmitoylation of integrin β4 and α6 subunits; DHHC3 ablation accelerates lysosomal degradation of α6β4 via increased cathepsin D exposure, impairs integrin signaling through Src, reduces β4 phosphorylation at S1356 and S1424, and blocks integrin-dependent cable formation on Matrigel, while ~10 other cell-surface proteins including α3β1 are unaffected.\",\n      \"method\": \"RNAi knockdown, overexpression in multiple cell lines, palmitoylation assay, Src signaling/phosphorylation assays, cathepsin D inhibitor rescue (Pepstatin A), cell-surface biotinylation\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (RNAi, OE, inhibitor rescue, signaling assays) in single lab\",\n      \"pmids\": [\"22314500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ZDHHC3 interacts with the death domain of TRAIL receptor DR4 (but not DR5) through its DHHC and C-terminal transmembrane domains, and promotes localization of DR4 to the plasma membrane via the DHHC motif, thereby sensitizing tumor cells to TRAIL-induced apoptosis.\",\n      \"method\": \"SOS protein-recruitment yeast two-hybrid, co-immunoprecipitation, subcellular localization assay, apoptosis assays, cysteine mutagenesis of DR4\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid plus Co-IP plus functional apoptosis rescue, single lab\",\n      \"pmids\": [\"22240897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ZDHHC3 undergoes autoacylation (palmitoylation) at the cysteine within the DHHC motif; conserved cysteines outside the DHHC motif coordinate two zinc ions per ZDHHC3 molecule, and mutation of these cysteines or chelation of zinc by EDTA causes structural perturbation and loss of palmitoyl acyltransferase activity.\",\n      \"method\": \"Mass spectrometry identification of palmitoylation site, site-directed mutagenesis of conserved CRD cysteines, limited proteolysis, metal chelation with EDTA, zinc quantification using fluorescent indicator mag-fura-2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mass spectrometry + mutagenesis + metal quantification, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"26487721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In GODZ (ZDHHC3) knockout mice, palmitoylation of γ2 subunit of GABA(A) receptors and GAP-43 is significantly reduced; GABA(A) receptor synaptic accumulation and GABAergic innervation are decreased in GODZ KO neurons competing with wild-type neurons; total cell-surface GABA(A) receptor expression and whole-cell GABAergic currents are unaltered in isolated DKO neurons, indicating GODZ-mediated palmitoylation selectively controls the synaptic pool of receptors. SERZ-β (DHHC7) KO alone does not affect γ2 palmitoylation.\",\n      \"method\": \"Knockout mice (GODZ KO, SERZ-β KO, double KO), palmitoylation assay, electrophysiology, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple orthogonal readouts, replicated across GODZ, SERZ-β, and DKO genotypes\",\n      \"pmids\": [\"27875292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZDHHC3 is phosphorylated by FGFR1 at Tyr18 and by Src kinase at Tyr295 and Tyr297; abrogation of these tyrosine phosphorylation sites increases ZDHHC3 autopalmitoylation, enhances interaction with NCAM, upregulates NCAM palmitoylation, and promotes neurite outgrowth in hippocampal neurons.\",\n      \"method\": \"Pharmacological inhibition and overexpression of FGFR/Src, site-directed mutagenesis of ZDHHC3 tyrosines, cell-free and cell-based kinase assays, palmitoylation assay, co-immunoprecipitation, neurite outgrowth assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis + cell-free kinase assay + cell-based functional rescue in neurons, multiple orthogonal methods single lab\",\n      \"pmids\": [\"27247265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZDHHC3 functions downstream of acsl1b to palmitoylate Gsα at mapped cysteine residues in Xenopus oocytes, maintaining meiotic G2/prophase I arrest; depletion of ZDHHC3 reduces palmitoylated Gsα levels and lowers the progesterone threshold for G2/M transition from 2 μM to 0.01 μM.\",\n      \"method\": \"RNA depletion (antisense morpholino) in Xenopus oocytes, palmitoylation assay, site-directed mutagenesis of Gsα palmitoylation sites and ZDHHC3 active site, progesterone dose-response assay\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic depletion + mutagenesis + biochemical palmitoylation assay, single lab, Xenopus ortholog model\",\n      \"pmids\": [\"27512151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZDHHC3 palmitoylates ERGIC3 protein; loss of ZDHHC3-dependent palmitoylation of ERGIC3 leads to upregulation of TXNIP, increased oxidative stress, and cellular senescence in breast cancer cells; these antitumor effects are reversed by wild-type but not enzyme-active-site-deficient ZDHHC3, and are substantially negated by co-depletion of TXNIP.\",\n      \"method\": \"RNAi ablation, wild-type vs. catalytic mutant ZDHHC3 reconstitution, gene array, fluorescence dye oxidative stress assays, xenograft tumor model, flow cytometry for immune cell recruitment\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant reconstitution + gene array + functional in vivo rescue, single lab\",\n      \"pmids\": [\"29055014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZDHHC3 binds specifically to HSV-1 UL20 (but not other HSV-1 proteins) in the Golgi apparatus via yeast two-hybrid and pulldown assays, palmitoylates UL20 (blocked by dominant-negative ZDHHC3 C157S or 2-bromopalmitate), and is required for proper localization of UL20 and glycoprotein K (gK) and for HSV-1 replication in vitro.\",\n      \"method\": \"Yeast two-hybrid, pulldown assay, dominant-negative ZDHHC3 overexpression, palmitoylation assay (acyl-RAC), 2-bromopalmitate inhibition, immunofluorescence localization, viral titer assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction assays plus palmitoylation assay plus functional viral replication readout, replicated in GODZ KO mice paper\",\n      \"pmids\": [\"28724772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In ZDHHC3 (GODZ) knockout MEFs, HSV-1 replication is compromised; ZDHHC3 absence blocks UL20 palmitoylation, alters localization and expression of UL20 and gK, affects expression of gB and gC, and disrupts tegument/capsid protein localization; in vivo, GODZ KO mice show reduced corneal HSV-1 replication, lower corneal scarring, and reduced latency reactivation.\",\n      \"method\": \"GODZ KO mouse-derived MEFs, palmitoylation assay, electron microscopy, immunofluorescence, viral titer, in vivo ocular infection model\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO model + multiple orthogonal mechanistic readouts + in vivo validation, replicates findings of PMID:28724772\",\n      \"pmids\": [\"29187538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Comparative mass spectrometry-based palmitoyl-proteomics of breast and prostate cancer cells ± ZDHHC3 ablation identified 22–28 antioxidant/redox-regulatory proteins as candidate ZDHHC3 substrates; ZDHHC3 ablation elevated oxidative stress, which synergized with chemotherapeutic agents to enhance anti-growth effects.\",\n      \"method\": \"Comparative mass spectrometry palmitoyl-proteomics, RNAi ablation, fluorescence dye oxidative stress assays, cell proliferation assays\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry substrate identification + functional oxidative stress assays, single lab\",\n      \"pmids\": [\"32986127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A high-throughput Acyl-cLIP assay was developed and validated for ZDHHC3/7/20 enzymatic activity; in vitro results from this assay correlated with cell-based palmitoylation assays, confirming ZDHHC3 catalytic activity as amenable to quantitative screening.\",\n      \"method\": \"Acyl-cLIP (acylation-coupled lipophilic induction of polarization) high-throughput assay, cell-based palmitoylation assay\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with cell-based validation, single lab, assay development paper\",\n      \"pmids\": [\"34374518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZDHHC3 palmitoylates IRHOM2 at C476 within the iRhom homology domain via its DHHC (C157) catalytic domain; palmitoylation promotes IRHOM2 cytomembrane translocation and stabilization by blocking TRIM31-mediated ubiquitination and proteasomal degradation; hepatocyte-specific ZDHHC3 knockout suppresses IRHOM2 accumulation and attenuates NASH pathology in rodent and rabbit models.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (C476 of IRHOM2, C157 of ZDHHC3), acyl-RAC palmitoylation assay, ubiquitination assay, hepatocyte-specific KO mice, in vivo NASH diet models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of both substrate and enzyme active site + ubiquitination assay + genetic KO in vivo, multiple orthogonal methods single lab\",\n      \"pmids\": [\"37544908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC3 inhibits PD-L1 lysosomal degradation by palmitoylating PD-L1; the natural compound benzosceptrin C (BC) inhibits ZDHHC3 enzymatic activity, causing PD-L1 to relocate from the membrane to the cytoplasm, preventing recycling endosome-mediated return to the membrane, and triggering lysosomal degradation of PD-L1.\",\n      \"method\": \"Palmitoylation assay, ZDHHC3 enzymatic inhibition assay, subcellular localization imaging, lysosomal inhibitor rescue, in vivo MC38 tumor model, T cell cytotoxicity assay\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic inhibition + localization imaging + lysosomal rescue + in vivo model, multiple orthogonal methods single lab\",\n      \"pmids\": [\"38237597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC3 S-acylates SCAP at C264, antagonizing HACE1-mediated ubiquitination of SCAP and preventing its proteasomal degradation; SREBP2 transcriptionally upregulates ZDHHC3, forming a positive feedback loop that sustains cholesterol biosynthesis in HCC; depalmitoylase ABHD17A reverses this modification.\",\n      \"method\": \"Site-directed mutagenesis (SCAP C264), palmitoylation assay, ubiquitination assay, ChIP/transcription factor binding assay, CRISPR/siRNA knockdown, in vivo DEN/CCl4 HCC mouse model, ZDHHC3 small-molecule inhibitor\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of palmitoylation site + ubiquitination assay + transcriptional feedback validation + in vivo KO model, multiple orthogonal methods single lab\",\n      \"pmids\": [\"39522165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC3 palmitoylates Cadm4 at cysteine-347 (C347) to stabilize its plasma membrane localization in oligodendrocytes; genetic deletion of ZDHHC3 reduces Cadm4 palmitoylation, causes Cadm4 internalization and degradation, and leads to CNS myelination defects and impaired neuronal transmission/cognitive behavior phenocopying Cadm4-C347A knock-in mice.\",\n      \"method\": \"Site-directed mutagenesis (Cadm4 C347A knock-in), ZDHHC3 knockout mice, palmitoylation assay, subcellular fractionation, confocal imaging, electrophysiology, behavioral assays\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO + substrate knock-in phenocopy + palmitoylation assay + functional neurophysiology, multiple orthogonal methods single lab\",\n      \"pmids\": [\"39327467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZDHHC3 catalyzes palmitoylation of the PML/RARα oncofusion protein, which is required for its oncogenic transcriptional activity; ZDHHC3 knockdown or overexpression respectively suppresses or promotes APL cell proliferation and blocks differentiation, and ZDHHC3 inhibition arrests malignant progression including in drug-resistant APL.\",\n      \"method\": \"RNAi knockdown, overexpression, palmitoylation assay, gene expression profiling (proliferation/differentiation markers), in vivo APL mouse model\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — palmitoylation assay plus bidirectional genetic manipulation plus in vivo model, single lab\",\n      \"pmids\": [\"39227737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZDHHC3 palmitoylates PRRSV nucleocapsid (N) protein at cysteine 90, which prevents N protein interaction with Nsp9 and inhibits viral RNA synthesis; the depalmitoylase LYPLA1 reverses this modification, counteracting ZDHHC3 activity and thereby promoting PRRSV replication.\",\n      \"method\": \"Palmitoylation assay, site-directed mutagenesis (N protein C90), co-immunoprecipitation (Nsp9-N interaction), viral RNA synthesis assay, siRNA knockdown, LYPLA1 inhibitor ML348\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of palmitoylation site + interaction assay + viral replication readout, single lab, porcine virus system\",\n      \"pmids\": [\"39787744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZDHHC3 (together with ZDHHC7) mediates S-acylation of the small GTPase ARL15 at three conserved N-terminal cysteine residues (Cys17, Cys22, Cys23); loss of S-acylation disrupts ARL15 membrane association; dual siRNA knockdown and CRISPR knockout of both ZDHHC3 and ZDHHC7 markedly reduces ARL15 S-acylation and redistributes ARL15 from membranes to cytosol.\",\n      \"method\": \"Acyl-PEGyl exchange gel-shift assay (APEGS), site-directed mutagenesis of ARL15 cysteines, siRNA knockdown, CRISPR/Cas9 gene disruption, confocal imaging, subcellular fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — APEGS stoichiometry assay + mutagenesis + CRISPR validation, multiple methods single lab\",\n      \"pmids\": [\"41999893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZDHHC3 S-acylates CRY1 (the core circadian transcriptional repressor) at four cysteine residues; this S-acylation is required for CRY1 nuclear import and interaction with key clock components; loss of CRY1 S-acylation via cysteine mutagenesis or genetic deletion of DHHC3 impairs CRY1 repressor function and disrupts cellular circadian rhythms.\",\n      \"method\": \"Unbiased palmitoylation screen of clock proteins, site-directed mutagenesis of CRY1 cysteines, DHHC3 genetic deletion, nuclear import assay, co-immunoprecipitation of clock components, circadian rhythm bioluminescence reporter assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis + genetic KO + functional circadian reporter, single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [\"42239456\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ZDHHC3 (GODZ) is a Golgi-localized DHHC-family palmitoyl acyltransferase that catalyzes S-palmitoylation of a broad set of substrates—including GABA(A) receptor γ2 subunit, integrin α6β4, NCAM, IRHOM2, SCAP, Cadm4, PD-L1, PML/RARα, HSV-1 UL20, CRY1, and ARL15—via a two-step autoacylation–transacylation mechanism requiring the DHHC catalytic motif and zinc coordination by conserved CRD cysteines; its activity is regulated by FGFR1/Src-mediated tyrosine phosphorylation, and it controls substrate membrane targeting, stability (by antagonizing ubiquitin-proteasomal degradation), synaptic clustering, viral replication, cholesterol biosynthesis, circadian rhythms, and antitumor immunity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZDHHC3 (GODZ) is a Golgi-resident DHHC-family palmitoyl acyltransferase that S-acylates a broad set of membrane and signaling proteins to control their membrane targeting, stability, and downstream function [#0, #1]. Catalysis proceeds through a two-step mechanism: ZDHHC3 first autoacylates the cysteine within its DHHC motif, and conserved cysteines outside this motif coordinate two zinc ions required for structural integrity and enzymatic activity [#6]. Its activity is tuned by FGFR1/Src-mediated tyrosine phosphorylation at Tyr18, Tyr295, and Tyr297, which when abolished increases autopalmitoylation and substrate modification [#8]. In the nervous system ZDHHC3 palmitoylates the GABA(A) receptor \\u03b32 subunit through a cytoplasmic cysteine-rich loop, selectively controlling the synaptic receptor pool and GABAergic innervation [#1, #2, #7], and palmitoylates Cadm4 to stabilize its plasma-membrane localization in oligodendrocytes, supporting CNS myelination [#18]. A recurrent mechanistic theme is that ZDHHC3-mediated palmitoylation stabilizes substrates by antagonizing their degradation: it protects integrin \\u03b16\\u03b24 from lysosomal turnover [#4], blocks TRIM31-mediated ubiquitination of IRHOM2 [#15], antagonizes HACE1-mediated ubiquitination of SCAP within an SREBP2 positive-feedback loop sustaining cholesterol biosynthesis [#17], and prevents lysosomal degradation of PD-L1 to modulate antitumor immunity [#16]. ZDHHC3 also palmitoylates viral proteins required for replication, including HSV-1 UL20 [#11, #12] and the PRRSV nucleocapsid protein [#20], and modifies the PML/RAR\\u03b1 oncofusion protein and the circadian repressor CRY1 [#19, #22]. Substrate breadth is supported by palmitoyl-proteomic surveys identifying numerous redox-regulatory candidate substrates [#13].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established ZDHHC3 as a Golgi-specific DHHC-domain protein involved in membrane protein trafficking, framing where and on what class of proteins it acts.\",\n      \"evidence\": \"Overexpression and immunofluorescence localization in COS7 cells with a GluR\\u03b11 sorting phenotype\",\n      \"pmids\": [\"12163046\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic-site mutagenesis or reconstitution\", \"Enzymatic palmitoylation activity not yet demonstrated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified the first defined substrate, the GABA(A) receptor \\u03b32 subunit, establishing ZDHHC3 as a bona fide palmitoyl acyltransferase acting through a substrate cysteine-rich domain.\",\n      \"evidence\": \"Yeast two-hybrid interaction plus cell-based palmitoylation assay and Golgi localization in neurons\",\n      \"pmids\": [\"15229235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro reconstitution with purified components absent\", \"Physiological consequence at synapses not yet tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated that ZDHHC3 oligomerizes and is the primary enzyme controlling synaptic GABA(A) receptor accumulation, linking palmitoylation to inhibitory synaptic function.\",\n      \"evidence\": \"Co-IP, cross-linking, neuronal RNAi, dominant-negative C157S, and electrophysiology\",\n      \"pmids\": [\"17151279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of homo/heteromultimerization unresolved\", \"Redundancy with DHHC7 not fully delineated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed a palmitoylation-independent Ca2+ transport function separable from acyltransferase activity, raising the possibility of a second molecular role.\",\n      \"evidence\": \"Voltage clamp, Ca2+ imaging and 45Ca uptake in Xenopus oocytes with V61R vs DHHS mutants\",\n      \"pmids\": [\"19955568\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, no replication\", \"Physiological relevance in mammalian cells unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Generalized ZDHHC3 substrate scope beyond neurons (integrin \\u03b16\\u03b24, TRAIL receptor DR4) and revealed that palmitoylation protects substrates from degradation and shapes their membrane localization and signaling.\",\n      \"evidence\": \"RNAi, overexpression, palmitoylation and Src signaling assays, cathepsin D rescue, Co-IP, apoptosis assays\",\n      \"pmids\": [\"22314500\", \"22240897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DR4 effect requires palmitoylation per se vs interaction not fully separated\", \"Selectivity determinants among integrin subunits unexplained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the catalytic chemistry: autoacylation at the DHHC cysteine plus zinc coordination by conserved CRD cysteines required for activity and structure.\",\n      \"evidence\": \"Mass spectrometry, CRD cysteine mutagenesis, limited proteolysis, EDTA chelation, zinc quantification\",\n      \"pmids\": [\"26487721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of the enzyme\", \"Transacylation step kinetics not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"In vivo genetics confirmed substrate selectivity (synaptic GABA(A) receptor pool, GAP-43) and uncovered tyrosine phosphorylation by FGFR1/Src as a regulatory input controlling autopalmitoylation and NCAM modification.\",\n      \"evidence\": \"GODZ/SERZ-\\u03b2 knockout mice with palmitoylation, electrophysiology, and fractionation; kinase assays and tyrosine-site mutagenesis with neurite outgrowth\",\n      \"pmids\": [\"27875292\", \"27247265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How phosphorylation alters enzyme conformation unknown\", \"Upstream signals coupling FGFR/Src to ZDHHC3 in vivo unmapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended the substrate repertoire to Gs\\u03b1 in a developmental context, linking ZDHHC3 to meiotic arrest control downstream of acsl1b.\",\n      \"evidence\": \"Morpholino depletion, mutagenesis, palmitoylation assay and progesterone dose-response in Xenopus oocytes\",\n      \"pmids\": [\"27512151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, Xenopus model\", \"Mammalian relevance untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established ZDHHC3 as a host factor for HSV-1 by palmitoylating UL20, required for viral glycoprotein localization and replication, validated genetically and in vivo.\",\n      \"evidence\": \"Yeast two-hybrid, pulldown, acyl-RAC, dominant-negative and KO-MEF/mouse ocular infection models\",\n      \"pmids\": [\"28724772\", \"29187538\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"UL20 palmitoylation site not pinpointed in the narrative\", \"Whether targeting ZDHHC3 is therapeutically tractable against HSV-1 untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected ZDHHC3 palmitoylation (of ERGIC3) to redox homeostasis and tumor suppression via a TXNIP-dependent oxidative-stress axis.\",\n      \"evidence\": \"Catalytic-mutant reconstitution, gene array, oxidative stress assays, xenograft, TXNIP co-depletion\",\n      \"pmids\": [\"29055014\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct ERGIC3 palmitoylation site not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Palmitoyl-proteomics broadened the candidate substrate landscape toward antioxidant/redox proteins and linked ZDHHC3 loss to chemosensitization.\",\n      \"evidence\": \"Comparative MS palmitoyl-proteomics with RNAi and proliferation assays in cancer cells\",\n      \"pmids\": [\"32986127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Candidate substrates not individually validated as direct\", \"Mechanism of synergy with chemotherapy unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a quantitative high-throughput assay enabling enzymatic screening of ZDHHC3, supporting drug-discovery efforts.\",\n      \"evidence\": \"Acyl-cLIP assay validated against cell-based palmitoylation for ZDHHC3/7/20\",\n      \"pmids\": [\"34374518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Assay development, not a biological discovery\", \"Selectivity over other DHHC enzymes limited\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined a stability-control paradigm in metabolic disease: ZDHHC3 palmitoylates IRHOM2 at C476 to block TRIM31 ubiquitination, driving NASH pathology.\",\n      \"evidence\": \"Co-IP, dual enzyme/substrate mutagenesis, acyl-RAC, ubiquitination assay, hepatocyte-specific KO in rodent and rabbit NASH models\",\n      \"pmids\": [\"37544908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of palmitoyl-ubiquitin antagonism unknown\", \"Generality of the ubiquitination-antagonism mechanism across substrates not formalized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Consolidated the degradation-antagonism mechanism across diverse pathways: PD-L1 (immunity), SCAP (cholesterol biosynthesis feedback), Cadm4 (myelination), and PML/RAR\\u03b1 (leukemia oncogenicity).\",\n      \"evidence\": \"Site mutagenesis, palmitoylation/ubiquitination assays, ChIP, lysosomal rescue, KO and knock-in mice, and in vivo tumor/myelination models\",\n      \"pmids\": [\"38237597\", \"39522165\", \"39327467\", \"39227737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of substrate selectivity across these targets not unified\", \"Whether a single inhibitor can disentangle these roles therapeutically untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Expanded substrates to membrane-targeting of the GTPase ARL15 (with ZDHHC7) and to nuclear import and repressor function of circadian CRY1, broadening ZDHHC3 roles into trafficking and clock regulation.\",\n      \"evidence\": \"APEGS, cysteine mutagenesis, siRNA/CRISPR for ARL15; palmitoylation screen, mutagenesis, DHHC3 deletion and circadian reporter for CRY1 (preprint)\",\n      \"pmids\": [\"41999893\", \"42239456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CRY1 finding is a preprint awaiting peer review\", \"How a Golgi enzyme controls nuclear CRY1 import mechanistically unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ZDHHC3 achieves substrate selectivity among its many targets, and whether its various disease-relevant roles can be separated pharmacologically, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution enzyme-substrate structure\", \"Substrate recognition code undefined\", \"Selectivity of available small-molecule inhibitors across DHHC enzymes incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 4, 6, 11, 15, 17, 18]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4, 15, 17, 18]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 1, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 15, 17, 16]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [1, 2, 7, 18]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GABRG2\", \"ITGB4\", \"ITGA6\", \"NCAM1\", \"IRHOM2\", \"SCAP\", \"CADM4\", \"ZDHHC7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}