{"gene":"HERC4","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2007,"finding":"HERC4 E3 ubiquitin ligase is required for proper spermatozoon maturation, specifically for removal of the cytoplasmic droplet during spermiogenesis; male mice homozygous for a Herc4 mutation show ~50% reduced fertility with spermatozoa retaining cytoplasmic droplets and reduced motility.","method":"Mouse knockout/loss-of-function with phenotypic readout (fertility assay, spermatozoa morphology analysis)","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse model with specific cellular phenotype, replicated across multiple litters","pmids":["17967448"],"is_preprint":false},{"year":2016,"finding":"HERC4 interacts with the transcription factor c-Maf and catalyzes its K48-linked polyubiquitination at K85 and K297, leading to proteasome-mediated degradation of c-Maf; the deubiquitinase USP5 antagonizes this process.","method":"Affinity chromatography, mass spectrometry, Co-IP, in vitro ubiquitination assay, site-directed mutagenesis (K85R, K297R), xenograft tumor model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including affinity chromatography/MS identification, Co-IP, mutagenesis, and in vivo validation","pmids":["26825710"],"is_preprint":false},{"year":2019,"finding":"HERC4 is a novel E3 ubiquitin ligase for the tumor suppressor LATS1; HERC4 promotes LATS1 ubiquitination and proteasomal degradation, thereby inactivating LATS1 and promoting breast cancer tumorigenesis.","method":"Co-IP, ubiquitination assay, knockdown/overexpression with proliferation/migration/tumor growth readouts, in vivo xenograft","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP and ubiquitination assay with functional KD/OE phenotypes; single lab","pmids":["30710319"],"is_preprint":false},{"year":2015,"finding":"In Drosophila, the HECT-domain E3 ligase Herc4 ubiquitylates and destabilizes Salvador (Sav), a scaffold protein in the Hippo pathway; Hippo kinase activity antagonizes Sav/Herc4 interaction in a kinase-dependent manner, creating a positive feedback loop that stabilizes Sav.","method":"Cell-based RNAi screen, Co-IP, ubiquitination assay, genetic epistasis in Drosophila","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi screen plus Co-IP and ubiquitination assay with mechanistic epistasis; single lab","pmids":["26125558"],"is_preprint":false},{"year":2019,"finding":"Herc4 (Drosophila ortholog) binds to Smoothened (Smo) and promotes its ubiquitination and proteasomal degradation to regulate Hedgehog signaling; this degradation is regulated by Hedgehog in PKA-primed phosphorylation-dependent and independent manners.","method":"Co-IP, ubiquitination assay, Drosophila genetic modifier screen, Hedgehog reporter assay","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and in vivo ubiquitination assay with Drosophila genetic validation; single lab","pmids":["30925584"],"is_preprint":false},{"year":2019,"finding":"HERC4 interacts with Smoothened (Smo) in non-small cell lung cancer cells and promotes its ubiquitination and degradation, suppressing Hedgehog pathway activation; depletion of HERC4 increases Smo protein levels and promotes cancer cell proliferation.","method":"Drosophila modifier screen, Co-IP in mammalian cells, ubiquitination assay, knockdown with Hedgehog pathway reporter and proliferation readout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and ubiquitination assay plus functional KD phenotype; single lab","pmids":["31010679"],"is_preprint":false},{"year":2023,"finding":"HERC4 interacts with MafA and mediates K63-linked polyubiquitination at K33, which does not lead to degradation but instead suppresses MafA phosphorylation by GSK3β and thereby inhibits MafA transcriptional activity and downstream STAT3 signaling.","method":"Co-IP, ubiquitination assay specifying K63-linkage, site-directed mutagenesis (K33R), transcriptional reporter assay, xenograft tumor model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro ubiquitination with linkage specificity, mutagenesis, transcriptional assays, and in vivo validation; multiple orthogonal methods in single paper","pmids":["37028761"],"is_preprint":false},{"year":2024,"finding":"HERC4, a HECT-domain E3 ligase, mediates the targeted proteasomal degradation of STING induced by the small molecule AK59; AK59 acts as a molecular glue/degrader that hijacks HERC4 to ubiquitinate STING, and is effective on common pathological STING mutations.","method":"Targeted protein degradation screen, proteomic identification of HERC4 as the responsible E3 ligase, functional ubiquitination assay, cell-based STING degradation assay with AK59","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — mechanistic identification of HERC4 as the E3 ligase with multiple validation experiments and compound-induced degradation mechanistic follow-up","pmids":["38811577"],"is_preprint":false},{"year":2021,"finding":"HERC4 directly downregulates SAV1 (Salvador 1) protein levels in hepatocellular carcinoma cells, as demonstrated by co-immunoprecipitation; overexpression of HERC4 reverses the inhibitory effects of SAV1 on HCC cell proliferation, migration, and invasion.","method":"Co-IP, Western blot, RT-qPCR, immunofluorescence, functional overexpression/knockdown assays, xenograft tumor model","journal":"Translational cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus functional assays; single lab, mechanism of SAV1 regulation (ubiquitination) not fully shown","pmids":["35116265"],"is_preprint":false},{"year":2025,"finding":"HERC4 promotes ubiquitination of KRT19 (Keratin 19), leading to its downregulation and promoting epithelial-mesenchymal transition and metastasis in lung adenocarcinoma.","method":"Co-IP, GST pull-down assay, ubiquitination assay, knockdown/overexpression with EMT and migration/invasion readouts, mouse model","journal":"Translational oncology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, GST pull-down, and ubiquitination assay with functional phenotype; single lab","pmids":["41192149"],"is_preprint":false}],"current_model":"HERC4 is a HECT-domain E3 ubiquitin ligase that targets multiple substrates for proteasomal degradation (c-Maf via K48-linked polyubiquitination, LATS1, Smoothened, SAV1, KRT19, STING) or for non-degradative K63-linked polyubiquitination (MafA at K33 to suppress its phosphorylation and transcriptional activity), and is required in vivo for spermatozoon maturation and cytoplasmic droplet removal during spermiogenesis."},"narrative":{"teleology":[{"year":2007,"claim":"The first in vivo function of HERC4 was established: it is essential for spermatozoon maturation, resolving the question of whether this HECT-domain ligase has a physiological role beyond generic ubiquitin conjugation.","evidence":"Herc4-mutant male mice showed ~50% reduced fertility with cytoplasmic droplet retention and reduced sperm motility","pmids":["17967448"],"confidence":"High","gaps":["The spermatogenesis substrate(s) of HERC4 remain unidentified","Whether HERC4 catalytic activity (versus scaffolding) is required for droplet removal is untested","Female reproductive or other developmental phenotypes not examined"]},{"year":2015,"claim":"HERC4 was linked to Hippo pathway regulation through ubiquitination and destabilization of the scaffold protein Salvador (Sav), revealing that Hippo kinase activity creates a positive feedback loop by antagonizing the Sav–Herc4 interaction.","evidence":"RNAi screen, Co-IP, ubiquitination assay, and genetic epistasis in Drosophila","pmids":["26125558"],"confidence":"Medium","gaps":["Mammalian conservation of Hippo-dependent regulation of HERC4–SAV1 interaction not demonstrated at this time","Ubiquitin chain type on Sav not determined","Direct HECT-domain catalytic requirement not shown via catalytic-dead mutant"]},{"year":2016,"claim":"HERC4 was identified as the E3 ligase responsible for K48-linked polyubiquitination and proteasomal degradation of the myeloma oncoprotein c-Maf, establishing a direct tumor-suppressive mechanism and identifying specific ubiquitination sites (K85, K297).","evidence":"Affinity chromatography/MS identification of HERC4–c-Maf interaction, Co-IP, in vitro ubiquitination, site-directed mutagenesis, and xenograft validation","pmids":["26825710"],"confidence":"High","gaps":["How HERC4 activity toward c-Maf is regulated remains unclear","Whether USP5-mediated antagonism is the sole deubiquitinase counterbalancing HERC4 is unresolved"]},{"year":2019,"claim":"HERC4 was shown to ubiquitinate and degrade Smoothened (Smo), establishing it as a negative regulator of Hedgehog signaling in both Drosophila and mammalian (NSCLC) contexts, with regulation by PKA-primed phosphorylation.","evidence":"Drosophila genetic modifier screen, Co-IP and ubiquitination assays in fly and mammalian cells, Hedgehog reporter and proliferation readouts","pmids":["30925584","31010679"],"confidence":"Medium","gaps":["Whether HERC4-mediated Smo degradation is relevant in non-cancer Hedgehog-dependent tissues (e.g. limb, neural tube) is untested","Ubiquitin chain linkage type on Smo not characterized"]},{"year":2019,"claim":"HERC4 was identified as an E3 ligase for the Hippo pathway kinase LATS1, broadening its role as a multi-substrate regulator of Hippo signaling beyond Salvador.","evidence":"Co-IP, ubiquitination assay, knockdown/overexpression in breast cancer cells, xenograft","pmids":["30710319"],"confidence":"Medium","gaps":["Whether HERC4 targets LATS1 and SAV1 simultaneously or in distinct contexts is unknown","The ubiquitin chain type on LATS1 was not determined"]},{"year":2021,"claim":"HERC4-mediated SAV1 downregulation was confirmed in mammalian hepatocellular carcinoma cells, extending the Drosophila Salvador finding to a human cancer context.","evidence":"Co-IP, western blot, functional overexpression/knockdown assays in HCC cells, xenograft","pmids":["35116265"],"confidence":"Medium","gaps":["Direct ubiquitination of SAV1 by HERC4 was not formally demonstrated in this study","Whether Hippo kinase-dependent feedback seen in Drosophila operates in HCC is unknown"]},{"year":2023,"claim":"HERC4 was shown to catalyze a non-degradative K63-linked polyubiquitination of MafA at K33, establishing that HERC4 can regulate substrate activity without promoting degradation — by blocking GSK3β phosphorylation and suppressing STAT3 signaling.","evidence":"Co-IP, K63-linkage-specific ubiquitination assay, K33R mutagenesis, transcriptional reporter, xenograft","pmids":["37028761"],"confidence":"High","gaps":["Structural basis for how K63-ubiquitination at K33 sterically blocks GSK3β access is unknown","Whether HERC4 uses K63-linkage on other substrates beyond MafA is untested"]},{"year":2024,"claim":"HERC4 was shown to be co-optable by a molecular glue (AK59) for targeted degradation of STING, demonstrating its pharmacological tractability as an E3 ligase for induced proximity approaches.","evidence":"Targeted protein degradation screen, proteomic identification, functional ubiquitination and STING degradation assays","pmids":["38811577"],"confidence":"High","gaps":["Whether HERC4 ubiquitinates STING under physiological (non-drug) conditions is unknown","Structural basis for AK59-mediated HERC4–STING proximity not resolved"]},{"year":2025,"claim":"HERC4 was identified as the E3 ligase for KRT19, linking its ubiquitin ligase activity to epithelial-mesenchymal transition and metastasis in lung adenocarcinoma.","evidence":"Co-IP, GST pull-down, ubiquitination assay, EMT/invasion readouts, mouse model","pmids":["41192149"],"confidence":"Medium","gaps":["Whether KRT19 ubiquitination by HERC4 is K48-linked and proteasomal was not explicitly determined","Regulation of HERC4 expression or activity in the EMT context is not characterized"]},{"year":null,"claim":"Key unresolved questions include the structural basis for HERC4's broad substrate selectivity, the identity of its spermatogenesis substrate(s), and whether its K63-linked ubiquitination activity extends beyond MafA.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure of HERC4 or HERC4–substrate complex exists","The spermatogenesis substrate(s) remain unknown despite clear in vivo phenotype","Mechanisms regulating HERC4 expression, stability, or catalytic activation are largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,3,4,5,6,7,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,6,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,3,4,5,6,7,8,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,5,8]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0]}],"complexes":[],"partners":["MAF","MAFA","LATS1","SAV1","SMO","STING1","KRT19","USP5"],"other_free_text":[]},"mechanistic_narrative":"HERC4 is a HECT-domain E3 ubiquitin ligase that targets a diverse set of substrates for proteasomal degradation and non-degradative ubiquitin signaling, functioning in spermatogenesis, developmental signaling, and oncogenic pathways. HERC4 catalyzes K48-linked polyubiquitination of c-Maf (at K85/K297), LATS1, Smoothened, SAV1, and KRT19, directing these substrates to proteasomal degradation, thereby modulating Hippo, Hedgehog, and myeloma-relevant transcriptional programs [PMID:26825710, PMID:30710319, PMID:30925584, PMID:26125558, PMID:41192149]. HERC4 also mediates K63-linked polyubiquitination of MafA at K33, which does not trigger degradation but instead blocks GSK3β-dependent phosphorylation and suppresses MafA transcriptional activity and downstream STAT3 signaling [PMID:37028761]. In vivo, HERC4 is required for cytoplasmic droplet removal during spermiogenesis, and male mice lacking functional Herc4 exhibit approximately 50% reduced fertility with morphologically abnormal spermatozoa [PMID:17967448]."},"prefetch_data":{"uniprot":{"accession":"Q5GLZ8","full_name":"Probable E3 ubiquitin-protein ligase HERC4","aliases":["HECT domain and RCC1-like domain-containing protein 4","HECT-type E3 ubiquitin transferase HERC4"],"length_aa":1057,"mass_kda":118.6,"function":"Probable E3 ubiquitin-protein ligase involved in either protein trafficking or in the distribution of cellular structures. Required for spermatozoon maturation and fertility, and for the removal of the cytoplasmic droplet of the spermatozoon. E3 ubiquitin-protein ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfer it to targeted substrates","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q5GLZ8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HERC4","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HERC4","total_profiled":1310},"omim":[{"mim_id":"609248","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 4; HERC4","url":"https://www.omim.org/entry/609248"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli fibrillar center","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HERC4"},"hgnc":{"alias_symbol":["DKFZP564G092","KIAA1593"],"prev_symbol":[]},"alphafold":{"accession":"Q5GLZ8","domains":[{"cath_id":"2.130.10.30","chopping":"3-380","consensus_level":"medium","plddt":95.0758,"start":3,"end":380},{"cath_id":"-","chopping":"403-617","consensus_level":"medium","plddt":94.3609,"start":403,"end":617},{"cath_id":"3.30.2410.10","chopping":"946-1051","consensus_level":"high","plddt":88.9025,"start":946,"end":1051}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5GLZ8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5GLZ8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5GLZ8-F1-predicted_aligned_error_v6.png","plddt_mean":91.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HERC4","jax_strain_url":"https://www.jax.org/strain/search?query=HERC4"},"sequence":{"accession":"Q5GLZ8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5GLZ8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5GLZ8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5GLZ8"}},"corpus_meta":[{"pmid":"17967448","id":"PMC_17967448","title":"Disruption of the ubiquitin ligase HERC4 causes defects in spermatozoon maturation and impaired fertility.","date":"2007","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17967448","citation_count":60,"is_preprint":false},{"pmid":"26825710","id":"PMC_26825710","title":"The ubiquitin ligase HERC4 mediates c-Maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice.","date":"2016","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/26825710","citation_count":59,"is_preprint":false},{"pmid":"28856724","id":"PMC_28856724","title":"SIRT1/HERC4 Locus Associated With Bisphosphonate-Induced Osteonecrosis of the Jaw: An Exome-Wide Association Analysis.","date":"2017","source":"Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research","url":"https://pubmed.ncbi.nlm.nih.gov/28856724","citation_count":27,"is_preprint":false},{"pmid":"30710319","id":"PMC_30710319","title":"A miRNA-HERC4 pathway promotes breast tumorigenesis by inactivating tumor suppressor LATS1.","date":"2019","source":"Protein & cell","url":"https://pubmed.ncbi.nlm.nih.gov/30710319","citation_count":23,"is_preprint":false},{"pmid":"26125558","id":"PMC_26125558","title":"Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26125558","citation_count":22,"is_preprint":false},{"pmid":"24225229","id":"PMC_24225229","title":"The expression and clinical significance of HERC4 in breast cancer.","date":"2013","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/24225229","citation_count":21,"is_preprint":false},{"pmid":"30925584","id":"PMC_30925584","title":"E3 ligase Herc4 regulates Hedgehog signalling through promoting Smoothened degradation.","date":"2019","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30925584","citation_count":20,"is_preprint":false},{"pmid":"38811577","id":"PMC_38811577","title":"Small molecule induced STING degradation facilitated by the HECT ligase HERC4.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38811577","citation_count":18,"is_preprint":false},{"pmid":"31010679","id":"PMC_31010679","title":"HERC4 exerts an anti-tumor role through destabilizing the oncoprotein Smo.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31010679","citation_count":16,"is_preprint":false},{"pmid":"25684480","id":"PMC_25684480","title":"Expression of HERC4 in lung cancer and its correlation with clinicopathological parameters.","date":"2015","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/25684480","citation_count":13,"is_preprint":false},{"pmid":"28430527","id":"PMC_28430527","title":"HERC4 Is Overexpressed in Hepatocellular Carcinoma and Contributes to the Proliferation and Migration of Hepatocellular Carcinoma Cells.","date":"2017","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28430527","citation_count":8,"is_preprint":false},{"pmid":"19819847","id":"PMC_19819847","title":"[Influence of Tripterygium wilfordii on the expression of spermiogenesis related genes Herc4, Ipo11 and Mrto4 in mice].","date":"2009","source":"Yi chuan = Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/19819847","citation_count":8,"is_preprint":false},{"pmid":"35116265","id":"PMC_35116265","title":"SAV1, regulated by HERC4, inhibits the proliferation, migration, and invasion of hepatocellular carcinoma.","date":"2021","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35116265","citation_count":7,"is_preprint":false},{"pmid":"37028761","id":"PMC_37028761","title":"The ubiquitin ligase HERC4 suppresses MafA transcriptional activity triggered by GSK3β in myeloma by atypical K63-linked polyubiquitination.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37028761","citation_count":5,"is_preprint":false},{"pmid":"38181892","id":"PMC_38181892","title":"HERC4 modulates ovarian cancer cell proliferation by regulating SMO-elicited hedgehog signaling.","date":"2024","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/38181892","citation_count":4,"is_preprint":false},{"pmid":"28219869","id":"PMC_28219869","title":"[RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].","date":"2016","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/28219869","citation_count":2,"is_preprint":false},{"pmid":"25176076","id":"PMC_25176076","title":"[Expression of E3 ligase HERC4 in breast cancer and its clinical implications].","date":"2014","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/25176076","citation_count":1,"is_preprint":false},{"pmid":"39611657","id":"PMC_39611657","title":"METTL3 and HERC4: Elevated Expression and Impact on Hepatocellular Carcinoma Progression.","date":"2024","source":"Cancer biotherapy & radiopharmaceuticals","url":"https://pubmed.ncbi.nlm.nih.gov/39611657","citation_count":1,"is_preprint":false},{"pmid":"41192149","id":"PMC_41192149","title":"HERC4 downregulates KRT19 to promote lung adenocarcinoma, migration, invasion and EMT.","date":"2025","source":"Translational oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41192149","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9971,"output_tokens":2354,"usd":0.032612},"stage2":{"model":"claude-opus-4-6","input_tokens":5674,"output_tokens":2719,"usd":0.144517},"total_usd":0.177129,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"HERC4 E3 ubiquitin ligase is required for proper spermatozoon maturation, specifically for removal of the cytoplasmic droplet during spermiogenesis; male mice homozygous for a Herc4 mutation show ~50% reduced fertility with spermatozoa retaining cytoplasmic droplets and reduced motility.\",\n      \"method\": \"Mouse knockout/loss-of-function with phenotypic readout (fertility assay, spermatozoa morphology analysis)\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse model with specific cellular phenotype, replicated across multiple litters\",\n      \"pmids\": [\"17967448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HERC4 interacts with the transcription factor c-Maf and catalyzes its K48-linked polyubiquitination at K85 and K297, leading to proteasome-mediated degradation of c-Maf; the deubiquitinase USP5 antagonizes this process.\",\n      \"method\": \"Affinity chromatography, mass spectrometry, Co-IP, in vitro ubiquitination assay, site-directed mutagenesis (K85R, K297R), xenograft tumor model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including affinity chromatography/MS identification, Co-IP, mutagenesis, and in vivo validation\",\n      \"pmids\": [\"26825710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HERC4 is a novel E3 ubiquitin ligase for the tumor suppressor LATS1; HERC4 promotes LATS1 ubiquitination and proteasomal degradation, thereby inactivating LATS1 and promoting breast cancer tumorigenesis.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown/overexpression with proliferation/migration/tumor growth readouts, in vivo xenograft\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and ubiquitination assay with functional KD/OE phenotypes; single lab\",\n      \"pmids\": [\"30710319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Drosophila, the HECT-domain E3 ligase Herc4 ubiquitylates and destabilizes Salvador (Sav), a scaffold protein in the Hippo pathway; Hippo kinase activity antagonizes Sav/Herc4 interaction in a kinase-dependent manner, creating a positive feedback loop that stabilizes Sav.\",\n      \"method\": \"Cell-based RNAi screen, Co-IP, ubiquitination assay, genetic epistasis in Drosophila\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi screen plus Co-IP and ubiquitination assay with mechanistic epistasis; single lab\",\n      \"pmids\": [\"26125558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Herc4 (Drosophila ortholog) binds to Smoothened (Smo) and promotes its ubiquitination and proteasomal degradation to regulate Hedgehog signaling; this degradation is regulated by Hedgehog in PKA-primed phosphorylation-dependent and independent manners.\",\n      \"method\": \"Co-IP, ubiquitination assay, Drosophila genetic modifier screen, Hedgehog reporter assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and in vivo ubiquitination assay with Drosophila genetic validation; single lab\",\n      \"pmids\": [\"30925584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HERC4 interacts with Smoothened (Smo) in non-small cell lung cancer cells and promotes its ubiquitination and degradation, suppressing Hedgehog pathway activation; depletion of HERC4 increases Smo protein levels and promotes cancer cell proliferation.\",\n      \"method\": \"Drosophila modifier screen, Co-IP in mammalian cells, ubiquitination assay, knockdown with Hedgehog pathway reporter and proliferation readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and ubiquitination assay plus functional KD phenotype; single lab\",\n      \"pmids\": [\"31010679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC4 interacts with MafA and mediates K63-linked polyubiquitination at K33, which does not lead to degradation but instead suppresses MafA phosphorylation by GSK3β and thereby inhibits MafA transcriptional activity and downstream STAT3 signaling.\",\n      \"method\": \"Co-IP, ubiquitination assay specifying K63-linkage, site-directed mutagenesis (K33R), transcriptional reporter assay, xenograft tumor model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro ubiquitination with linkage specificity, mutagenesis, transcriptional assays, and in vivo validation; multiple orthogonal methods in single paper\",\n      \"pmids\": [\"37028761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC4, a HECT-domain E3 ligase, mediates the targeted proteasomal degradation of STING induced by the small molecule AK59; AK59 acts as a molecular glue/degrader that hijacks HERC4 to ubiquitinate STING, and is effective on common pathological STING mutations.\",\n      \"method\": \"Targeted protein degradation screen, proteomic identification of HERC4 as the responsible E3 ligase, functional ubiquitination assay, cell-based STING degradation assay with AK59\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mechanistic identification of HERC4 as the E3 ligase with multiple validation experiments and compound-induced degradation mechanistic follow-up\",\n      \"pmids\": [\"38811577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HERC4 directly downregulates SAV1 (Salvador 1) protein levels in hepatocellular carcinoma cells, as demonstrated by co-immunoprecipitation; overexpression of HERC4 reverses the inhibitory effects of SAV1 on HCC cell proliferation, migration, and invasion.\",\n      \"method\": \"Co-IP, Western blot, RT-qPCR, immunofluorescence, functional overexpression/knockdown assays, xenograft tumor model\",\n      \"journal\": \"Translational cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus functional assays; single lab, mechanism of SAV1 regulation (ubiquitination) not fully shown\",\n      \"pmids\": [\"35116265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC4 promotes ubiquitination of KRT19 (Keratin 19), leading to its downregulation and promoting epithelial-mesenchymal transition and metastasis in lung adenocarcinoma.\",\n      \"method\": \"Co-IP, GST pull-down assay, ubiquitination assay, knockdown/overexpression with EMT and migration/invasion readouts, mouse model\",\n      \"journal\": \"Translational oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, GST pull-down, and ubiquitination assay with functional phenotype; single lab\",\n      \"pmids\": [\"41192149\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HERC4 is a HECT-domain E3 ubiquitin ligase that targets multiple substrates for proteasomal degradation (c-Maf via K48-linked polyubiquitination, LATS1, Smoothened, SAV1, KRT19, STING) or for non-degradative K63-linked polyubiquitination (MafA at K33 to suppress its phosphorylation and transcriptional activity), and is required in vivo for spermatozoon maturation and cytoplasmic droplet removal during spermiogenesis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HERC4 is a HECT-domain E3 ubiquitin ligase that targets a diverse set of substrates for proteasomal degradation and non-degradative ubiquitin signaling, functioning in spermatogenesis, developmental signaling, and oncogenic pathways. HERC4 catalyzes K48-linked polyubiquitination of c-Maf (at K85/K297), LATS1, Smoothened, SAV1, and KRT19, directing these substrates to proteasomal degradation, thereby modulating Hippo, Hedgehog, and myeloma-relevant transcriptional programs [PMID:26825710, PMID:30710319, PMID:30925584, PMID:26125558, PMID:41192149]. HERC4 also mediates K63-linked polyubiquitination of MafA at K33, which does not trigger degradation but instead blocks GSK3β-dependent phosphorylation and suppresses MafA transcriptional activity and downstream STAT3 signaling [PMID:37028761]. In vivo, HERC4 is required for cytoplasmic droplet removal during spermiogenesis, and male mice lacking functional Herc4 exhibit approximately 50% reduced fertility with morphologically abnormal spermatozoa [PMID:17967448].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"The first in vivo function of HERC4 was established: it is essential for spermatozoon maturation, resolving the question of whether this HECT-domain ligase has a physiological role beyond generic ubiquitin conjugation.\",\n      \"evidence\": \"Herc4-mutant male mice showed ~50% reduced fertility with cytoplasmic droplet retention and reduced sperm motility\",\n      \"pmids\": [\"17967448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The spermatogenesis substrate(s) of HERC4 remain unidentified\",\n        \"Whether HERC4 catalytic activity (versus scaffolding) is required for droplet removal is untested\",\n        \"Female reproductive or other developmental phenotypes not examined\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"HERC4 was linked to Hippo pathway regulation through ubiquitination and destabilization of the scaffold protein Salvador (Sav), revealing that Hippo kinase activity creates a positive feedback loop by antagonizing the Sav–Herc4 interaction.\",\n      \"evidence\": \"RNAi screen, Co-IP, ubiquitination assay, and genetic epistasis in Drosophila\",\n      \"pmids\": [\"26125558\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mammalian conservation of Hippo-dependent regulation of HERC4–SAV1 interaction not demonstrated at this time\",\n        \"Ubiquitin chain type on Sav not determined\",\n        \"Direct HECT-domain catalytic requirement not shown via catalytic-dead mutant\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"HERC4 was identified as the E3 ligase responsible for K48-linked polyubiquitination and proteasomal degradation of the myeloma oncoprotein c-Maf, establishing a direct tumor-suppressive mechanism and identifying specific ubiquitination sites (K85, K297).\",\n      \"evidence\": \"Affinity chromatography/MS identification of HERC4–c-Maf interaction, Co-IP, in vitro ubiquitination, site-directed mutagenesis, and xenograft validation\",\n      \"pmids\": [\"26825710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How HERC4 activity toward c-Maf is regulated remains unclear\",\n        \"Whether USP5-mediated antagonism is the sole deubiquitinase counterbalancing HERC4 is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"HERC4 was shown to ubiquitinate and degrade Smoothened (Smo), establishing it as a negative regulator of Hedgehog signaling in both Drosophila and mammalian (NSCLC) contexts, with regulation by PKA-primed phosphorylation.\",\n      \"evidence\": \"Drosophila genetic modifier screen, Co-IP and ubiquitination assays in fly and mammalian cells, Hedgehog reporter and proliferation readouts\",\n      \"pmids\": [\"30925584\", \"31010679\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether HERC4-mediated Smo degradation is relevant in non-cancer Hedgehog-dependent tissues (e.g. limb, neural tube) is untested\",\n        \"Ubiquitin chain linkage type on Smo not characterized\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"HERC4 was identified as an E3 ligase for the Hippo pathway kinase LATS1, broadening its role as a multi-substrate regulator of Hippo signaling beyond Salvador.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, knockdown/overexpression in breast cancer cells, xenograft\",\n      \"pmids\": [\"30710319\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether HERC4 targets LATS1 and SAV1 simultaneously or in distinct contexts is unknown\",\n        \"The ubiquitin chain type on LATS1 was not determined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"HERC4-mediated SAV1 downregulation was confirmed in mammalian hepatocellular carcinoma cells, extending the Drosophila Salvador finding to a human cancer context.\",\n      \"evidence\": \"Co-IP, western blot, functional overexpression/knockdown assays in HCC cells, xenograft\",\n      \"pmids\": [\"35116265\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct ubiquitination of SAV1 by HERC4 was not formally demonstrated in this study\",\n        \"Whether Hippo kinase-dependent feedback seen in Drosophila operates in HCC is unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"HERC4 was shown to catalyze a non-degradative K63-linked polyubiquitination of MafA at K33, establishing that HERC4 can regulate substrate activity without promoting degradation — by blocking GSK3β phosphorylation and suppressing STAT3 signaling.\",\n      \"evidence\": \"Co-IP, K63-linkage-specific ubiquitination assay, K33R mutagenesis, transcriptional reporter, xenograft\",\n      \"pmids\": [\"37028761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for how K63-ubiquitination at K33 sterically blocks GSK3β access is unknown\",\n        \"Whether HERC4 uses K63-linkage on other substrates beyond MafA is untested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"HERC4 was shown to be co-optable by a molecular glue (AK59) for targeted degradation of STING, demonstrating its pharmacological tractability as an E3 ligase for induced proximity approaches.\",\n      \"evidence\": \"Targeted protein degradation screen, proteomic identification, functional ubiquitination and STING degradation assays\",\n      \"pmids\": [\"38811577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether HERC4 ubiquitinates STING under physiological (non-drug) conditions is unknown\",\n        \"Structural basis for AK59-mediated HERC4–STING proximity not resolved\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HERC4 was identified as the E3 ligase for KRT19, linking its ubiquitin ligase activity to epithelial-mesenchymal transition and metastasis in lung adenocarcinoma.\",\n      \"evidence\": \"Co-IP, GST pull-down, ubiquitination assay, EMT/invasion readouts, mouse model\",\n      \"pmids\": [\"41192149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether KRT19 ubiquitination by HERC4 is K48-linked and proteasomal was not explicitly determined\",\n        \"Regulation of HERC4 expression or activity in the EMT context is not characterized\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for HERC4's broad substrate selectivity, the identity of its spermatogenesis substrate(s), and whether its K63-linked ubiquitination activity extends beyond MafA.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of HERC4 or HERC4–substrate complex exists\",\n        \"The spermatogenesis substrate(s) remain unknown despite clear in vivo phenotype\",\n        \"Mechanisms regulating HERC4 expression, stability, or catalytic activation are largely uncharacterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 3, 4, 5, 6, 7, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 3, 4, 5, 6, 7, 8, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 5, 8]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MAF\",\n      \"MAFA\",\n      \"LATS1\",\n      \"SAV1\",\n      \"SMO\",\n      \"STING1\",\n      \"KRT19\",\n      \"USP5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}