{"gene":"HERC4","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2007,"finding":"HERC4 E3 ubiquitin ligase is required for proper maturation of spermatozoa and removal of the cytoplasmic droplet; male mice homozygous for a Herc4 mutation show ~50% reduced fertility with abnormal spermatozoa retaining cytoplasmic droplets at tail angulations, establishing a role for HERC4 in the ubiquitin-proteasome-dependent remodeling during spermiogenesis.","method":"Knockout mouse model (homozygous Herc4 mutation), histological analysis of testis and sperm morphology, fertility assays","journal":"Developmental Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with specific cellular phenotype (cytoplasmic droplet retention), replicated in vivo fertility assay","pmids":["17967448"],"is_preprint":false},{"year":2016,"finding":"HERC4 interacts with the transcription factor c-Maf and catalyzes its K48-linked polyubiquitination at residues K85 and K297, leading to proteasome-mediated degradation of c-Maf; this ubiquitination is counteracted by the deubiquitinase USP5.","method":"Affinity chromatography, mass spectrometry, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (K85R, K297R), in vitro and in vivo xenograft models","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including MS identification, Co-IP, mutagenesis of ubiquitination sites, and USP5 eraser identified","pmids":["26825710"],"is_preprint":false},{"year":2015,"finding":"Drosophila Herc4 (HECT domain E3 ligase) directly ubiquitylates and destabilizes the Hippo pathway scaffold protein Salvador (Sav); Hippo kinase activity antagonizes Herc4-mediated Sav degradation by reducing the Sav/Herc4 interaction in a kinase-dependent manner, creating a positive feedback loop.","method":"Cell-based RNAi screen, ubiquitylation assay, co-immunoprecipitation, genetic epistasis in Drosophila, kinase-dead Hippo mutants","journal":"PLoS ONE","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi screen plus reciprocal Co-IP plus kinase-dependency established with kinase-dead mutant, multiple orthogonal methods","pmids":["26125558"],"is_preprint":false},{"year":2019,"finding":"HERC4 acts as an E3 ligase for the tumor suppressor LATS1, promoting its ubiquitination and proteasomal degradation, thereby inactivating the Hippo pathway and promoting breast tumorigenesis; miR-136-5p and miR-1285-5p suppress HERC4 expression.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression in breast cancer cell lines, in vivo xenograft tumor growth assay","journal":"Protein & Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay in a single lab, supported by in vivo xenograft data","pmids":["30710319"],"is_preprint":false},{"year":2019,"finding":"HERC4 binds to Smoothened (Smo) and promotes its ubiquitination and proteasomal degradation, thereby negatively regulating Hedgehog signaling; this degradation is modulated by Hh signaling through PKA-primed phosphorylation-dependent and -independent mechanisms.","method":"Co-immunoprecipitation, ubiquitination assay, Drosophila modifier screen, Hh pathway reporter assays, knockdown/overexpression in mammalian cells","journal":"Journal of Molecular Cell Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Drosophila genetic screen plus Co-IP plus ubiquitination assay plus mechanistic phosphorylation analysis across two independent studies (PMID 30925584 and 31010679)","pmids":["30925584","31010679"],"is_preprint":false},{"year":2019,"finding":"Drosophila dHerc4 degrades dSmo (Smoothened) and depletion of dherc4 increases dSmo protein levels and activates the Hedgehog pathway; human HERC4 reciprocally interacts with Smo in NSCLC cells and knockdown of HERC4 activates Hh pathway and promotes NSCLC cell proliferation.","method":"Drosophila genetic modifier screen, co-immunoprecipitation (reciprocal), knockdown in NSCLC cell lines, Hh pathway reporter assay","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and Drosophila genetics, single lab","pmids":["31010679"],"is_preprint":false},{"year":2021,"finding":"HERC4 directly downregulates SAV1 (Salvador 1) protein levels in hepatocellular carcinoma cells, as demonstrated by co-immunoprecipitation and the ability of HERC4 overexpression to reverse the tumor-suppressive effects of SAV1.","method":"Co-immunoprecipitation, RT-qPCR, Western blot, immunofluorescence, functional assays (EdU, colony formation, Transwell), xenograft model","journal":"Translational Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus multiple functional assays and in vivo xenograft, single lab","pmids":["35116265"],"is_preprint":false},{"year":2023,"finding":"HERC4 interacts with MafA and mediates atypical K63-linked polyubiquitination at K33, which inhibits GSK3β-triggered MafA phosphorylation and transcriptional activity (instead of promoting degradation); a K33R MafA variant prevents HERC4 from inhibiting MafA phosphorylation. HERC4 also suppresses MafA-activated STAT3 signaling.","method":"Co-immunoprecipitation, ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K33R), phosphorylation assay, STAT3 reporter assay, xenograft model","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — K63-linkage specificity determined, mutagenesis of ubiquitination site, phosphorylation and transcriptional activity assays, in vivo validation","pmids":["37028761"],"is_preprint":false},{"year":2024,"finding":"The small molecule AK59 induces STING degradation by recruiting the E3 ligase HERC4 (a HECT-domain protein) to ubiquitinate and degrade STING via the ubiquitin-proteasome system; AK59 is effective on common pathological STING mutations.","method":"Targeted protein degradation (TPD) assay, compound-induced ubiquitination, proteasome inhibitor rescue, mechanistic characterization with HERC4 knockdown/knockout","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct small molecule-induced ubiquitination via HERC4 demonstrated with proteasome rescue and HERC4 genetic ablation, published in high-impact peer-reviewed journal","pmids":["38811577"],"is_preprint":false},{"year":2025,"finding":"HERC4 interacts with KRT19 (cytokeratin 19) and promotes its ubiquitination, leading to KRT19 downregulation in lung adenocarcinoma; KRT19 loss drives EMT and increases metastatic potential.","method":"Co-immunoprecipitation, GST pull-down assay, ubiquitination assay, CCK-8, wound healing, Transwell assay, mouse model","journal":"Translational Oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and GST pull-down plus ubiquitination assay confirming substrate relationship, single lab","pmids":["41192149"],"is_preprint":false},{"year":2024,"finding":"HERC4 overexpression in ovarian cancer cells reduces SMO protein levels and inhibits downstream Hedgehog signaling pathway components, suppressing cell proliferation in vitro and tumor growth in vivo.","method":"Lentiviral overexpression, CCK-8 cell viability assay, Western blot, RT-PCR, xenograft tumor model","journal":"Biochimica et Biophysica Acta - General Subjects","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression with pathway readout, no direct ubiquitination assay or Co-IP for SMO in this study, single lab","pmids":["38181892"],"is_preprint":false},{"year":2024,"finding":"METTL3 interacts with HERC4 (demonstrated by co-immunoprecipitation) in hepatocellular carcinoma cells; METTL3 overexpression upregulates HERC4 expression, and HERC4 overexpression can reverse the effects of METTL3 silencing on HCC cell proliferation and migration.","method":"Co-immunoprecipitation, m6A quantification, immunofluorescence, proliferation/migration assays, immunoblotting","journal":"Cancer Biotherapy & Radiopharmaceuticals","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP, no direct mechanistic link between m6A modification and HERC4 regulation established, single lab","pmids":["39611657"],"is_preprint":false}],"current_model":"HERC4 is a HECT-domain E3 ubiquitin ligase that targets multiple substrates for proteasomal degradation (c-Maf, LATS1, Smoothened/SMO, KRT19, STING) or for non-degradative K63-linked ubiquitination (MafA at K33, inhibiting its phosphorylation and transcriptional activity), and plays a physiological role in spermiogenesis by promoting cytoplasmic droplet removal; it also negatively regulates the Hippo scaffold Salvador and can be recruited by small molecules (e.g., AK59) for targeted protein degradation."},"narrative":{"mechanistic_narrative":"HERC4 is a HECT-domain E3 ubiquitin ligase that controls the abundance and activity of diverse signaling proteins by catalyzing their ubiquitination, with physiological functions in spermiogenesis and recurrent roles in cancer-associated signaling [PMID:17967448, PMID:26825710]. In male germ cells, HERC4 is required for proper sperm maturation and removal of the cytoplasmic droplet, with loss-of-function reducing fertility and producing morphologically abnormal spermatozoa [PMID:17967448]. As a degradative ligase, HERC4 assembles K48-linked polyubiquitin chains on the transcription factor c-Maf at K85 and K297 to drive its proteasomal destruction, a reaction opposed by the deubiquitinase USP5 [PMID:26825710], and similarly targets the Hippo tumor suppressor LATS1, Smoothened, and KRT19 for degradation [PMID:30710319, PMID:30925584, PMID:31010679, PMID:41192149]. Beyond degradation, HERC4 can also catalyze non-degradative K63-linked ubiquitination of MafA at K33, which blocks GSK3β-triggered MafA phosphorylation and suppresses its transcriptional and STAT3-activating activity [PMID:37028761]. Through degradation of Smoothened, HERC4 negatively regulates Hedgehog signaling, an activity conserved from Drosophila to human cells, where dSmo/SMO levels rise upon HERC4 depletion [PMID:30925584, PMID:31010679]. HERC4 also engages the Hippo pathway scaffold Salvador/SAV1, ubiquitylating and destabilizing it; in Drosophila, Hippo kinase activity antagonizes this degradation to form a positive feedback loop [PMID:26125558, PMID:35116265]. The ligase is pharmacologically tractable: the small molecule AK59 recruits HERC4 to ubiquitinate and degrade STING, including common pathological STING mutants [PMID:38811577].","teleology":[{"year":2007,"claim":"Established the first in vivo physiological role for HERC4, showing it is required for ubiquitin-proteasome-dependent remodeling of maturing sperm rather than being merely a biochemical curiosity.","evidence":"Homozygous Herc4 mutant mouse with testis/sperm histology and fertility assays","pmids":["17967448"],"confidence":"High","gaps":["No direct substrate in spermiogenesis identified","Molecular target whose ubiquitination drives cytoplasmic droplet removal unknown"]},{"year":2015,"claim":"Identified the first direct substrate by showing Drosophila Herc4 ubiquitylates and destabilizes the Hippo scaffold Salvador, placing HERC4 within Hippo pathway regulation and revealing a kinase-controlled feedback loop.","evidence":"RNAi screen, ubiquitylation and reciprocal Co-IP assays, kinase-dead Hippo mutants in Drosophila","pmids":["26125558"],"confidence":"High","gaps":["Ubiquitin chain linkage on Sav not defined","Mammalian SAV1 regulation not addressed in this study"]},{"year":2016,"claim":"Defined HERC4 as a degradative ligase in human cancer by mapping K48-linked ubiquitination of c-Maf to specific lysines and identifying USP5 as the opposing eraser.","evidence":"Affinity-MS, Co-IP, ubiquitination assays, K85R/K297R mutagenesis, xenografts","pmids":["26825710"],"confidence":"High","gaps":["Regulation of HERC4 activity toward c-Maf not defined","Substrate recognition determinants unknown"]},{"year":2019,"claim":"Expanded the substrate repertoire to Smoothened and LATS1, linking HERC4 to negative regulation of Hedgehog signaling and inactivation of Hippo signaling in tumorigenesis.","evidence":"Co-IP, ubiquitination assays, Drosophila modifier screens, Hh reporter assays, knockdown/overexpression and xenografts in cancer cell lines","pmids":["30710319","30925584","31010679"],"confidence":"High","gaps":["Ubiquitin linkage types not fully resolved for all substrates","How signaling context selects between substrates unknown","LATS1 finding from a single lab"]},{"year":2021,"claim":"Extended Salvador regulation to mammalian SAV1, showing HERC4 downregulates SAV1 to reverse its tumor-suppressive effect in hepatocellular carcinoma.","evidence":"Co-IP, functional assays, and xenograft in HCC cells","pmids":["35116265"],"confidence":"Medium","gaps":["Direct ubiquitination of SAV1 not demonstrated in this study","Single lab"]},{"year":2023,"claim":"Revealed that HERC4 is not exclusively degradative, catalyzing atypical K63-linked ubiquitination of MafA at K33 to block its phosphorylation and transcriptional output instead of destroying it.","evidence":"Linkage-specific ubiquitination assays, K33R mutagenesis, phosphorylation and STAT3 reporter assays, xenografts","pmids":["37028761"],"confidence":"High","gaps":["Structural basis for K63 vs K48 chain selection unknown","How HERC4 distinguishes MafA from c-Maf mechanistically unclear"]},{"year":2024,"claim":"Demonstrated HERC4 is pharmacologically harnessable for targeted protein degradation, as the small molecule AK59 recruits HERC4 to ubiquitinate and degrade STING.","evidence":"Compound-induced ubiquitination, proteasome rescue, HERC4 knockdown/knockout","pmids":["38811577"],"confidence":"High","gaps":["Structural basis of AK59-HERC4-STING ternary complex not defined","Generalizability to other neosubstrates unknown"]},{"year":2025,"claim":"Added KRT19 as a HERC4 substrate, connecting HERC4 activity to EMT and metastatic potential in lung adenocarcinoma.","evidence":"Co-IP, GST pull-down, ubiquitination assay, migration assays, mouse model","pmids":["41192149"],"confidence":"Medium","gaps":["Ubiquitin linkage and degradation pathway for KRT19 not fully resolved","Single lab"]},{"year":null,"claim":"How HERC4 selects among its many substrates and switches between degradative K48 and non-degradative K63 ubiquitin chains remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of HERC4 substrate recognition","Determinants of chain-type selection unknown","Regulation of HERC4 catalytic activity in different tissues uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,4,7]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,7,8]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4,7,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,3,9]}],"complexes":[],"partners":["C-MAF","MAFA","LATS1","SMO","SAV1","KRT19","STING","USP5"],"other_free_text":[]}},"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":23,"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":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":"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":9,"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":"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":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":"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":10740,"output_tokens":3027,"usd":0.038812,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10331,"output_tokens":2934,"usd":0.062503,"stage2_stop_reason":"end_turn"},"total_usd":0.101315,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","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 maturation of spermatozoa and removal of the cytoplasmic droplet; male mice homozygous for a Herc4 mutation show ~50% reduced fertility with abnormal spermatozoa retaining cytoplasmic droplets at tail angulations, establishing a role for HERC4 in the ubiquitin-proteasome-dependent remodeling during spermiogenesis.\",\n      \"method\": \"Knockout mouse model (homozygous Herc4 mutation), histological analysis of testis and sperm morphology, fertility assays\",\n      \"journal\": \"Developmental Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with specific cellular phenotype (cytoplasmic droplet retention), replicated in vivo fertility assay\",\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 residues K85 and K297, leading to proteasome-mediated degradation of c-Maf; this ubiquitination is counteracted by the deubiquitinase USP5.\",\n      \"method\": \"Affinity chromatography, mass spectrometry, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (K85R, K297R), in vitro and in vivo xenograft models\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including MS identification, Co-IP, mutagenesis of ubiquitination sites, and USP5 eraser identified\",\n      \"pmids\": [\"26825710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila Herc4 (HECT domain E3 ligase) directly ubiquitylates and destabilizes the Hippo pathway scaffold protein Salvador (Sav); Hippo kinase activity antagonizes Herc4-mediated Sav degradation by reducing the Sav/Herc4 interaction in a kinase-dependent manner, creating a positive feedback loop.\",\n      \"method\": \"Cell-based RNAi screen, ubiquitylation assay, co-immunoprecipitation, genetic epistasis in Drosophila, kinase-dead Hippo mutants\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi screen plus reciprocal Co-IP plus kinase-dependency established with kinase-dead mutant, multiple orthogonal methods\",\n      \"pmids\": [\"26125558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HERC4 acts as an E3 ligase for the tumor suppressor LATS1, promoting its ubiquitination and proteasomal degradation, thereby inactivating the Hippo pathway and promoting breast tumorigenesis; miR-136-5p and miR-1285-5p suppress HERC4 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression in breast cancer cell lines, in vivo xenograft tumor growth assay\",\n      \"journal\": \"Protein & Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay in a single lab, supported by in vivo xenograft data\",\n      \"pmids\": [\"30710319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HERC4 binds to Smoothened (Smo) and promotes its ubiquitination and proteasomal degradation, thereby negatively regulating Hedgehog signaling; this degradation is modulated by Hh signaling through PKA-primed phosphorylation-dependent and -independent mechanisms.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Drosophila modifier screen, Hh pathway reporter assays, knockdown/overexpression in mammalian cells\",\n      \"journal\": \"Journal of Molecular Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Drosophila genetic screen plus Co-IP plus ubiquitination assay plus mechanistic phosphorylation analysis across two independent studies (PMID 30925584 and 31010679)\",\n      \"pmids\": [\"30925584\", \"31010679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Drosophila dHerc4 degrades dSmo (Smoothened) and depletion of dherc4 increases dSmo protein levels and activates the Hedgehog pathway; human HERC4 reciprocally interacts with Smo in NSCLC cells and knockdown of HERC4 activates Hh pathway and promotes NSCLC cell proliferation.\",\n      \"method\": \"Drosophila genetic modifier screen, co-immunoprecipitation (reciprocal), knockdown in NSCLC cell lines, Hh pathway reporter assay\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and Drosophila genetics, single lab\",\n      \"pmids\": [\"31010679\"],\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 and the ability of HERC4 overexpression to reverse the tumor-suppressive effects of SAV1.\",\n      \"method\": \"Co-immunoprecipitation, RT-qPCR, Western blot, immunofluorescence, functional assays (EdU, colony formation, Transwell), xenograft model\",\n      \"journal\": \"Translational Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus multiple functional assays and in vivo xenograft, single lab\",\n      \"pmids\": [\"35116265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC4 interacts with MafA and mediates atypical K63-linked polyubiquitination at K33, which inhibits GSK3β-triggered MafA phosphorylation and transcriptional activity (instead of promoting degradation); a K33R MafA variant prevents HERC4 from inhibiting MafA phosphorylation. HERC4 also suppresses MafA-activated STAT3 signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K33R), phosphorylation assay, STAT3 reporter assay, xenograft model\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — K63-linkage specificity determined, mutagenesis of ubiquitination site, phosphorylation and transcriptional activity assays, in vivo validation\",\n      \"pmids\": [\"37028761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The small molecule AK59 induces STING degradation by recruiting the E3 ligase HERC4 (a HECT-domain protein) to ubiquitinate and degrade STING via the ubiquitin-proteasome system; AK59 is effective on common pathological STING mutations.\",\n      \"method\": \"Targeted protein degradation (TPD) assay, compound-induced ubiquitination, proteasome inhibitor rescue, mechanistic characterization with HERC4 knockdown/knockout\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct small molecule-induced ubiquitination via HERC4 demonstrated with proteasome rescue and HERC4 genetic ablation, published in high-impact peer-reviewed journal\",\n      \"pmids\": [\"38811577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC4 interacts with KRT19 (cytokeratin 19) and promotes its ubiquitination, leading to KRT19 downregulation in lung adenocarcinoma; KRT19 loss drives EMT and increases metastatic potential.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down assay, ubiquitination assay, CCK-8, wound healing, Transwell assay, mouse model\",\n      \"journal\": \"Translational Oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and GST pull-down plus ubiquitination assay confirming substrate relationship, single lab\",\n      \"pmids\": [\"41192149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC4 overexpression in ovarian cancer cells reduces SMO protein levels and inhibits downstream Hedgehog signaling pathway components, suppressing cell proliferation in vitro and tumor growth in vivo.\",\n      \"method\": \"Lentiviral overexpression, CCK-8 cell viability assay, Western blot, RT-PCR, xenograft tumor model\",\n      \"journal\": \"Biochimica et Biophysica Acta - General Subjects\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression with pathway readout, no direct ubiquitination assay or Co-IP for SMO in this study, single lab\",\n      \"pmids\": [\"38181892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL3 interacts with HERC4 (demonstrated by co-immunoprecipitation) in hepatocellular carcinoma cells; METTL3 overexpression upregulates HERC4 expression, and HERC4 overexpression can reverse the effects of METTL3 silencing on HCC cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, m6A quantification, immunofluorescence, proliferation/migration assays, immunoblotting\",\n      \"journal\": \"Cancer Biotherapy & Radiopharmaceuticals\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP, no direct mechanistic link between m6A modification and HERC4 regulation established, single lab\",\n      \"pmids\": [\"39611657\"],\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, LATS1, Smoothened/SMO, KRT19, STING) or for non-degradative K63-linked ubiquitination (MafA at K33, inhibiting its phosphorylation and transcriptional activity), and plays a physiological role in spermiogenesis by promoting cytoplasmic droplet removal; it also negatively regulates the Hippo scaffold Salvador and can be recruited by small molecules (e.g., AK59) for targeted protein degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HERC4 is a HECT-domain E3 ubiquitin ligase that controls the abundance and activity of diverse signaling proteins by catalyzing their ubiquitination, with physiological functions in spermiogenesis and recurrent roles in cancer-associated signaling [#0, #1]. In male germ cells, HERC4 is required for proper sperm maturation and removal of the cytoplasmic droplet, with loss-of-function reducing fertility and producing morphologically abnormal spermatozoa [#0]. As a degradative ligase, HERC4 assembles K48-linked polyubiquitin chains on the transcription factor c-Maf at K85 and K297 to drive its proteasomal destruction, a reaction opposed by the deubiquitinase USP5 [#1], and similarly targets the Hippo tumor suppressor LATS1, Smoothened, and KRT19 for degradation [#3, #4, #9]. Beyond degradation, HERC4 can also catalyze non-degradative K63-linked ubiquitination of MafA at K33, which blocks GSK3\\u03b2-triggered MafA phosphorylation and suppresses its transcriptional and STAT3-activating activity [#7]. Through degradation of Smoothened, HERC4 negatively regulates Hedgehog signaling, an activity conserved from Drosophila to human cells, where dSmo/SMO levels rise upon HERC4 depletion [#4, #5]. HERC4 also engages the Hippo pathway scaffold Salvador/SAV1, ubiquitylating and destabilizing it; in Drosophila, Hippo kinase activity antagonizes this degradation to form a positive feedback loop [#2, #6]. The ligase is pharmacologically tractable: the small molecule AK59 recruits HERC4 to ubiquitinate and degrade STING, including common pathological STING mutants [#8].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the first in vivo physiological role for HERC4, showing it is required for ubiquitin-proteasome-dependent remodeling of maturing sperm rather than being merely a biochemical curiosity.\",\n      \"evidence\": \"Homozygous Herc4 mutant mouse with testis/sperm histology and fertility assays\",\n      \"pmids\": [\"17967448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct substrate in spermiogenesis identified\", \"Molecular target whose ubiquitination drives cytoplasmic droplet removal unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the first direct substrate by showing Drosophila Herc4 ubiquitylates and destabilizes the Hippo scaffold Salvador, placing HERC4 within Hippo pathway regulation and revealing a kinase-controlled feedback loop.\",\n      \"evidence\": \"RNAi screen, ubiquitylation and reciprocal Co-IP assays, kinase-dead Hippo mutants in Drosophila\",\n      \"pmids\": [\"26125558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage on Sav not defined\", \"Mammalian SAV1 regulation not addressed in this study\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined HERC4 as a degradative ligase in human cancer by mapping K48-linked ubiquitination of c-Maf to specific lysines and identifying USP5 as the opposing eraser.\",\n      \"evidence\": \"Affinity-MS, Co-IP, ubiquitination assays, K85R/K297R mutagenesis, xenografts\",\n      \"pmids\": [\"26825710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of HERC4 activity toward c-Maf not defined\", \"Substrate recognition determinants unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded the substrate repertoire to Smoothened and LATS1, linking HERC4 to negative regulation of Hedgehog signaling and inactivation of Hippo signaling in tumorigenesis.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, Drosophila modifier screens, Hh reporter assays, knockdown/overexpression and xenografts in cancer cell lines\",\n      \"pmids\": [\"30710319\", \"30925584\", \"31010679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage types not fully resolved for all substrates\", \"How signaling context selects between substrates unknown\", \"LATS1 finding from a single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended Salvador regulation to mammalian SAV1, showing HERC4 downregulates SAV1 to reverse its tumor-suppressive effect in hepatocellular carcinoma.\",\n      \"evidence\": \"Co-IP, functional assays, and xenograft in HCC cells\",\n      \"pmids\": [\"35116265\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of SAV1 not demonstrated in this study\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed that HERC4 is not exclusively degradative, catalyzing atypical K63-linked ubiquitination of MafA at K33 to block its phosphorylation and transcriptional output instead of destroying it.\",\n      \"evidence\": \"Linkage-specific ubiquitination assays, K33R mutagenesis, phosphorylation and STAT3 reporter assays, xenografts\",\n      \"pmids\": [\"37028761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for K63 vs K48 chain selection unknown\", \"How HERC4 distinguishes MafA from c-Maf mechanistically unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated HERC4 is pharmacologically harnessable for targeted protein degradation, as the small molecule AK59 recruits HERC4 to ubiquitinate and degrade STING.\",\n      \"evidence\": \"Compound-induced ubiquitination, proteasome rescue, HERC4 knockdown/knockout\",\n      \"pmids\": [\"38811577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of AK59-HERC4-STING ternary complex not defined\", \"Generalizability to other neosubstrates unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added KRT19 as a HERC4 substrate, connecting HERC4 activity to EMT and metastatic potential in lung adenocarcinoma.\",\n      \"evidence\": \"Co-IP, GST pull-down, ubiquitination assay, migration assays, mouse model\",\n      \"pmids\": [\"41192149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage and degradation pathway for KRT19 not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HERC4 selects among its many substrates and switches between degradative K48 and non-degradative K63 ubiquitin chains remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of HERC4 substrate recognition\", \"Determinants of chain-type selection unknown\", \"Regulation of HERC4 catalytic activity in different tissues uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 4, 7]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 7, 8]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4, 7, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 3, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"c-Maf\", \"MafA\", \"LATS1\", \"SMO\", \"SAV1\", \"KRT19\", \"STING\", \"USP5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}