{"gene":"HERC3","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2001,"finding":"HERC3 protein localizes to the cytosol and vesicular-like structures containing β-COP, ARF, and Rab5, suggesting a role in vesicular trafficking. HERC3 non-covalently binds ubiquitin, and this binding does not require the conserved catalytic cysteine in the HECT domain. HERC3 itself is a substrate of ubiquitination and is degraded by the proteasome.","method":"Subcellular fractionation, immunofluorescence co-localization, co-immunoprecipitation, mutagenesis of catalytic cysteine, proteasome inhibitor treatment","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal methods (localization, binding, mutagenesis, proteasome inhibition) in a single study; foundational characterization paper","pmids":["11163799"],"is_preprint":false},{"year":2015,"finding":"HERC3 acts as a negative regulator of NF-κB by binding (indirectly) to the RelA subunit after its liberation from IκBα, facilitating RelA ubiquitination and proteasomal degradation. HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Remarkably, this regulatory activity is independent of HERC3's intrinsic E3 ubiquitin ligase activity. HERC3, RelA, ubiquilin-1 (UBQLN1), and the 26S proteasome form a multi-protein complex, and HERC3/UBQLN1 provide a bridge between RelA and the proteasome.","method":"Co-immunoprecipitation, ubiquitination assays, catalytic-dead mutant analysis, NF-κB reporter assays, nuclear fractionation, DNA-binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, catalytic-dead mutant, multiple functional readouts (nuclear import, DNA binding, protein stability) in a single rigorous study","pmids":["26476452"],"is_preprint":false},{"year":2018,"finding":"ΔNp63α transcriptionally upregulates HERC3, and HERC3 then mediates K48-linked ubiquitination and proteasomal degradation of MM1 (a c-Myc modulator). Knockdown of HERC3 abrogates ΔNp63α-induced MM1 downregulation and induces cell senescence, placing HERC3 in a ΔNp63α→HERC3→MM1→c-Myc axis controlling cell senescence.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, overexpression, reporter/senescence assays, epistasis experiments","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal methods (Co-IP, ubiquitination assay, epistasis by KD) in a single lab study","pmids":["29880857"],"is_preprint":false},{"year":2019,"finding":"HERC3 promotes ubiquitination-mediated degradation of SMAD7 in an autolysosome-dependent manner. This leads to increased p-SMAD2/3 levels and TGFβ pathway activation, driving EMT. Autophagy inducers upregulate HERC3 expression, establishing a mechanistic link between autophagy and TGFβ/SMAD signaling via HERC3-mediated SMAD7 degradation.","method":"iTRAQ proteomics, co-immunoprecipitation, ubiquitination assays, siRNA/overexpression, intracranial xenograft, immunohistochemistry, tissue microarray","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — proteomics identification followed by Co-IP and ubiquitination assays and in vivo validation, single lab","pmids":["30862693"],"is_preprint":false},{"year":2022,"finding":"HERC3 directly interacts with EIF5A2 via its RCC1 domain and promotes K27- and K48-linked ubiquitination degradation of EIF5A2 via its HECT domain. Specific ubiquitination sites on EIF5A2 were identified as K47, K67, K85, and K121. This interaction inhibits EMT and metastasis in colorectal cancer through the EIF5A2/TGFβ/Smad2/3 signaling axis.","method":"Co-immunoprecipitation, GST-pulldown, in vivo ubiquitination assays, domain-deletion mutants, transwell/wound healing assays, mass spectrometry","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, GST-pulldown, ubiquitination site mapping by MS, domain mutants; single lab","pmids":["35064108"],"is_preprint":false},{"year":2022,"finding":"HERC3 directly interacts with RPL23A and acts as an E3 ligase to ubiquitinate and degrade RPL23A via K48-linked polyubiquitination through its HECT domain. HERC3-mediated RPL23A degradation modulates the c-Myc/p21 axis and regulates CRC cell proliferation and cell-cycle arrest at G0/G1.","method":"Co-immunoprecipitation, mass spectrometry, GST-pulldown, in vivo ubiquitination assays, cycloheximide chase, loss/gain-of-function experiments, rescue experiments","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, MS substrate identification, ubiquitination assays, rescue epistasis; single lab","pmids":["35637966"],"is_preprint":false},{"year":2022,"finding":"HERC3 directly interacts with ERK2 through its HECT domain and promotes ERK2 ubiquitination. HERC3 also modulates p53 protein levels and phosphorylation, linking ERK2 degradation to p53-mediated apoptosis.","method":"Co-immunoprecipitation, GST-pulldown, ubiquitination assays, xenograft model","journal":"Molecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and GST-pulldown only, limited mechanistic detail on ubiquitination mechanism, single study","pmids":["35889210"],"is_preprint":false},{"year":2023,"finding":"HERC3 promotes YAP/TAZ protein stability and Hippo-independent tumorigenesis independently of its E3 ubiquitin ligase enzymatic activity. HERC3 directly binds to β-TrCP (the YAP/TAZ ubiquitin E3 ligase adaptor), blocking β-TrCP interaction with YAP/TAZ and thereby preventing YAP/TAZ ubiquitination and proteasomal degradation.","method":"Co-immunoprecipitation, catalytic-dead mutant analysis, ubiquitination assays, knockdown, protein stability assays, breast tumor cell/tissue correlation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, catalytic-dead mutant establishing ligase-independent mechanism, ubiquitination assays, multiple functional readouts; rigorous single-lab study published in high-tier journal","pmids":["36598329"],"is_preprint":false},{"year":2023,"finding":"HERC3 ubiquitinates NCOA1, targeting it for proteasomal degradation. HERC3 deficiency leads to NCOA1 accumulation, which assembles a NCOA1-p300-Runx2 complex that transactivates matrix metallopeptidase (MMP) gene expression, promoting extracellular matrix degradation in intervertebral disc degeneration.","method":"Immunoprecipitation, mass spectrometry, ubiquitination assays, loss-of-function (HERC3-deficient model), immunoblot, aged mouse IDD model","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP/MS substrate identification, ubiquitination assay, in vivo mouse model; single lab","pmids":["36878279"],"is_preprint":false},{"year":2024,"finding":"HERC3 facilitates ERAD of select membrane proteins by directly recognizing exposed (cytoplasmic) membrane-spanning domains (MSDs) of misfolded CFTR, but not MSDs embedded in liposomes. HERC3 operates independently of the ER-embedded E3 ligases RNF5 and RNF185 to mediate retrotranslocation and ERAD of misfolded CFTR. This identifies a distinct ERAD branch for cytoplasmic quality control of membrane-spanning domains.","method":"Multiplex knockdown/knockout experiments, real-time kinetic ERAD assays, in vitro binding assay with liposome-embedded vs. exposed MSDs, CFTR/ABCB1 misfolded substrate models","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution binding assay distinguishing MSD topology, multiplex KD/KO with kinetic measurements, epistasis with RNF5/185; rigorous mechanistic dissection in a single study","pmids":["38722278"],"is_preprint":false},{"year":2024,"finding":"CRISPR-generated Herc3-/- mice develop accumulation of activated subretinal microglia (Iba1+/CD16+), retinal thinning, and functional deficits, demonstrating a non-redundant role for HERC3 in retinal homeostasis and microglial activation suppression in vivo.","method":"CRISPR knockout mouse generation, fundus imaging, OCT, histology, optomotor testing, electrophysiology, bulk RNA sequencing","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with multiple orthogonal phenotypic readouts and transcriptomic profiling; mechanism (specific substrate) not fully resolved","pmids":["38321224"],"is_preprint":false}],"current_model":"HERC3 is a HECT-domain E3 ubiquitin ligase that localizes to the cytosol and vesicular compartments; it ubiquitinates and promotes proteasomal degradation of substrates including SMAD7, EIF5A2, RPL23A, MM1, NCOA1, and ERK2, thereby regulating TGFβ/EMT, NF-κB, Hippo/YAP-TAZ, cell senescence, and cell-cycle pathways—while in certain contexts (NF-κB RelA destabilization, YAP/TAZ stabilization) its regulatory activity is independent of its catalytic E3 ligase function, instead acting as a scaffold linking substrates to the proteasome or blocking E3 adaptor access; additionally, HERC3 mediates a distinct branch of ER-associated degradation by recognizing exposed cytoplasmic membrane-spanning domains of misfolded membrane proteins such as CFTR."},"narrative":{"mechanistic_narrative":"HERC3 is a HECT-domain E3 ubiquitin ligase that functions as a hub for targeted proteasomal protein degradation, controlling signaling pathways that govern proliferation, senescence, epithelial-mesenchymal transition, and protein quality control [PMID:26476452, PMID:35637966]. Through its HECT domain it catalyzes K48-linked (and in some substrates K27-linked) polyubiquitination of multiple targets—including MM1 in a ΔNp63α→HERC3→MM1→c-Myc senescence axis [PMID:29880857], the eukaryotic translation factor EIF5A2 [PMID:35064108], the ribosomal protein RPL23A acting on the c-Myc/p21 axis to arrest cells at G0/G1 [PMID:35637966], and NCOA1 to restrain a NCOA1-p300-Runx2 complex that drives matrix metalloproteinase expression [PMID:36878279]; substrate engagement can occur through its RCC1 domain, as shown for EIF5A2 [PMID:35064108]. HERC3 also drives degradation of SMAD7 in an autolysosome-dependent manner, relieving inhibition of TGFβ/SMAD2/3 signaling and promoting EMT [PMID:30862693]. Notably, a subset of HERC3's regulatory functions is independent of its catalytic ligase activity: it restrains NF-κB by bridging liberated RelA to the 26S proteasome together with UBQLN1 [PMID:26476452], and it stabilizes YAP/TAZ by directly binding β-TrCP to block adaptor access [PMID:36598329]. In ER-associated degradation, HERC3 defines a distinct branch that recognizes exposed cytoplasmic membrane-spanning domains of misfolded membrane proteins such as CFTR, operating independently of the ER-embedded ligases RNF5 and RNF185 [PMID:38722278]. In vivo, loss of HERC3 in mice causes subretinal microglial activation and retinal degeneration, establishing a non-redundant homeostatic role [PMID:38321224].","teleology":[{"year":2001,"claim":"Established HERC3 as a cytosolic/vesicular HECT-family protein that non-covalently binds ubiquitin and is itself a proteasome substrate, framing its biology around the ubiquitin system before any substrate was known.","evidence":"Subcellular fractionation, immunofluorescence co-localization with β-COP/ARF/Rab5, Co-IP, catalytic-cysteine mutagenesis, and proteasome inhibition","pmids":["11163799"],"confidence":"Medium","gaps":["No physiological substrate identified","Functional consequence of vesicular localization untested","Ubiquitin-binding interface not mapped"]},{"year":2015,"claim":"Showed HERC3 negatively regulates NF-κB by bridging RelA to the proteasome via UBQLN1, and that this activity is independent of its catalytic ligase function, revealing a scaffolding mode of action.","evidence":"Co-IP, ubiquitination assays, catalytic-dead mutant, NF-κB reporter, nuclear fractionation, and DNA-binding assays","pmids":["26476452"],"confidence":"High","gaps":["Indirect RelA binding partner not fully defined","Whether the same complex regulates other transcription factors unknown"]},{"year":2018,"claim":"Placed HERC3 in a senescence-controlling axis by demonstrating it is transcriptionally induced by ΔNp63α and degrades MM1 via K48-linked ubiquitination to modulate c-Myc.","evidence":"Co-IP, ubiquitination assays, siRNA knockdown, overexpression, and epistasis/senescence assays","pmids":["29880857"],"confidence":"Medium","gaps":["Direct vs. indirect MM1 binding not resolved","Ubiquitination sites on MM1 not mapped"]},{"year":2019,"claim":"Connected HERC3 to TGFβ/EMT by showing autophagy-induced HERC3 degrades SMAD7 in an autolysosome-dependent manner, enhancing SMAD2/3 signaling.","evidence":"iTRAQ proteomics, Co-IP, ubiquitination assays, knockdown/overexpression, and intracranial xenograft with tissue analysis","pmids":["30862693"],"confidence":"Medium","gaps":["Mechanism linking ubiquitination to autolysosomal rather than proteasomal degradation unclear","Ubiquitin linkage type on SMAD7 not defined"]},{"year":2022,"claim":"Defined direct substrates and degradation chemistry: HERC3 binds EIF5A2 via its RCC1 domain to drive K27/K48 ubiquitination, and binds RPL23A to drive K48 ubiquitination, linking these to TGFβ/EMT suppression and c-Myc/p21-dependent cell-cycle arrest respectively.","evidence":"Reciprocal Co-IP, GST-pulldown, domain-deletion mutants, MS-based ubiquitination site mapping, cycloheximide chase, and rescue experiments","pmids":["35064108","35637966"],"confidence":"Medium","gaps":["Context determining anti- vs. pro-EMT outcomes across substrates unresolved","In vitro reconstitution of ligase activity not performed"]},{"year":2022,"claim":"Proposed ERK2 as a HERC3 substrate linked to p53-mediated apoptosis, extending HERC3 into MAPK signaling.","evidence":"Co-IP, GST-pulldown, ubiquitination assays, and xenograft model","pmids":["35889210"],"confidence":"Low","gaps":["Single low-confidence study with limited mechanistic detail on ubiquitination","Ubiquitin linkage and sites on ERK2 not defined","p53 link correlative"]},{"year":2023,"claim":"Demonstrated a ligase-independent stabilizing function in which HERC3 binds β-TrCP to block YAP/TAZ ubiquitination, driving Hippo-independent tumorigenesis.","evidence":"Reciprocal Co-IP, catalytic-dead mutant, ubiquitination/protein-stability assays, knockdown, and breast tumor tissue correlation","pmids":["36598329"],"confidence":"High","gaps":["Structural basis of β-TrCP competition unknown","Whether HERC3 blocks other β-TrCP substrates untested"]},{"year":2023,"claim":"Identified NCOA1 as a HERC3 substrate whose loss derepresses a NCOA1-p300-Runx2 transcriptional complex driving MMP-mediated matrix degradation in disc degeneration.","evidence":"Co-IP/MS, ubiquitination assays, HERC3-deficient model, and aged mouse IDD model","pmids":["36878279"],"confidence":"Medium","gaps":["Ubiquitin linkage and sites on NCOA1 not mapped","Direct vs. indirect engagement not fully resolved"]},{"year":2024,"claim":"Defined a distinct ERAD branch in which HERC3 directly recognizes exposed cytoplasmic membrane-spanning domains of misfolded CFTR, acting independently of ER-embedded ligases RNF5/RNF185.","evidence":"Multiplex KD/KO, real-time kinetic ERAD assays, in vitro binding to liposome-embedded vs. exposed MSDs, and CFTR/ABCB1 substrate models","pmids":["38722278"],"confidence":"High","gaps":["Structural determinants of MSD recognition unresolved","Full substrate repertoire of this ERAD branch unknown"]},{"year":2024,"claim":"Established a non-redundant in vivo role for HERC3 in retinal homeostasis, with knockout causing subretinal microglial activation and retinal degeneration.","evidence":"CRISPR knockout mice with fundus imaging, OCT, histology, optomotor testing, electrophysiology, and bulk RNA-seq","pmids":["38321224"],"confidence":"Medium","gaps":["Causal substrate driving the retinal phenotype not identified","Cell-autonomous vs. non-autonomous microglial effects unresolved"]},{"year":null,"claim":"It remains unknown how HERC3 selects between catalytic degradation and ligase-independent scaffolding modes, and which substrate(s) account for its in vivo developmental and homeostatic phenotypes.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for substrate recognition across diverse targets","No structural data on HECT/RCC1 substrate engagement","In vivo substrate(s) linking molecular activity to organismal phenotype undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,5,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[4,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,5,8,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5]}],"complexes":["RelA-UBQLN1-26S proteasome complex"],"partners":["RELA","UBQLN1","EIF5A2","RPL23A","MAPK1","NCOA1","BTRC","CFTR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15034","full_name":"Probable E3 ubiquitin-protein ligase HERC3","aliases":["HECT domain and RCC1-like domain-containing protein 3","HECT-type E3 ubiquitin transferase HERC3"],"length_aa":1050,"mass_kda":117.2,"function":"E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates","subcellular_location":"Cytoplasm; Cytoplasmic vesicle","url":"https://www.uniprot.org/uniprotkb/Q15034/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HERC3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HERC3","total_profiled":1310},"omim":[{"mim_id":"611399","title":"SODIUM CHANNEL AND CLATHRIN LINKER 1; SCLT1","url":"https://www.omim.org/entry/611399"},{"mim_id":"609249","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 6; HERC6","url":"https://www.omim.org/entry/609249"},{"mim_id":"608242","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 5; HERC5","url":"https://www.omim.org/entry/608242"},{"mim_id":"605837","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 2; HERC2","url":"https://www.omim.org/entry/605837"},{"mim_id":"605200","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 3; HERC3","url":"https://www.omim.org/entry/605200"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"retina","ntpm":7.9}],"url":"https://www.proteinatlas.org/search/HERC3"},"hgnc":{"alias_symbol":["KIAA0032"],"prev_symbol":[]},"alphafold":{"accession":"Q15034","domains":[{"cath_id":"3.30.2160.10","chopping":"820-903","consensus_level":"medium","plddt":91.0401,"start":820,"end":903},{"cath_id":"3.30.2410.10","chopping":"937-1042","consensus_level":"high","plddt":83.3009,"start":937,"end":1042},{"cath_id":"1.25.40","chopping":"403-621","consensus_level":"medium","plddt":91.0807,"start":403,"end":621}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15034","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15034-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15034-F1-predicted_aligned_error_v6.png","plddt_mean":89.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HERC3","jax_strain_url":"https://www.jax.org/strain/search?query=HERC3"},"sequence":{"accession":"Q15034","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15034.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15034/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15034"}},"corpus_meta":[{"pmid":"30862693","id":"PMC_30862693","title":"HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma.","date":"2019","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/30862693","citation_count":89,"is_preprint":false},{"pmid":"22790983","id":"PMC_22790983","title":"Epigenetic control of alternative mRNA processing at the imprinted Herc3/Nap1l5 locus.","date":"2012","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/22790983","citation_count":40,"is_preprint":false},{"pmid":"11163799","id":"PMC_11163799","title":"HERC3 binding to and regulation by ubiquitin.","date":"2001","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/11163799","citation_count":34,"is_preprint":false},{"pmid":"26476452","id":"PMC_26476452","title":"The ubiquitin ligase HERC3 attenuates NF-κB-dependent transcription independently of its enzymatic activity by delivering the RelA subunit for degradation.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/26476452","citation_count":30,"is_preprint":false},{"pmid":"35064108","id":"PMC_35064108","title":"HERC3 regulates epithelial-mesenchymal transition by directly ubiquitination degradation EIF5A2 and inhibits metastasis of colorectal cancer.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35064108","citation_count":28,"is_preprint":false},{"pmid":"29880857","id":"PMC_29880857","title":"ΔNp63α down-regulates c-Myc modulator MM1 via E3 ligase HERC3 in the regulation of cell senescence.","date":"2018","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/29880857","citation_count":28,"is_preprint":false},{"pmid":"36598329","id":"PMC_36598329","title":"HERC3 promotes YAP/TAZ stability and tumorigenesis independently of its ubiquitin ligase activity.","date":"2023","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/36598329","citation_count":24,"is_preprint":false},{"pmid":"35637966","id":"PMC_35637966","title":"HERC3 directly targets RPL23A for ubiquitination degradation and further regulates Colorectal Cancer proliferation and the cell cycle.","date":"2022","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35637966","citation_count":17,"is_preprint":false},{"pmid":"38722278","id":"PMC_38722278","title":"HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.","date":"2024","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38722278","citation_count":10,"is_preprint":false},{"pmid":"36878279","id":"PMC_36878279","title":"Accumulation of NCOA1 dependent on HERC3 deficiency transactivates matrix metallopeptidases and promotes extracellular matrix degradation in intervertebral disc degeneration.","date":"2023","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36878279","citation_count":10,"is_preprint":false},{"pmid":"38321224","id":"PMC_38321224","title":"E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38321224","citation_count":7,"is_preprint":false},{"pmid":"35889210","id":"PMC_35889210","title":"A Novel ERK2 Degrader Z734 Induces Apoptosis of MCF-7 Cells via the HERC3/p53 Signaling Pathway.","date":"2022","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35889210","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8350,"output_tokens":3199,"usd":0.036518,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10658,"output_tokens":3822,"usd":0.07442,"stage2_stop_reason":"end_turn"},"total_usd":0.110938,"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\": 2001,\n      \"finding\": \"HERC3 protein localizes to the cytosol and vesicular-like structures containing β-COP, ARF, and Rab5, suggesting a role in vesicular trafficking. HERC3 non-covalently binds ubiquitin, and this binding does not require the conserved catalytic cysteine in the HECT domain. HERC3 itself is a substrate of ubiquitination and is degraded by the proteasome.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence co-localization, co-immunoprecipitation, mutagenesis of catalytic cysteine, proteasome inhibitor treatment\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal methods (localization, binding, mutagenesis, proteasome inhibition) in a single study; foundational characterization paper\",\n      \"pmids\": [\"11163799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HERC3 acts as a negative regulator of NF-κB by binding (indirectly) to the RelA subunit after its liberation from IκBα, facilitating RelA ubiquitination and proteasomal degradation. HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Remarkably, this regulatory activity is independent of HERC3's intrinsic E3 ubiquitin ligase activity. HERC3, RelA, ubiquilin-1 (UBQLN1), and the 26S proteasome form a multi-protein complex, and HERC3/UBQLN1 provide a bridge between RelA and the proteasome.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, catalytic-dead mutant analysis, NF-κB reporter assays, nuclear fractionation, DNA-binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, catalytic-dead mutant, multiple functional readouts (nuclear import, DNA binding, protein stability) in a single rigorous study\",\n      \"pmids\": [\"26476452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ΔNp63α transcriptionally upregulates HERC3, and HERC3 then mediates K48-linked ubiquitination and proteasomal degradation of MM1 (a c-Myc modulator). Knockdown of HERC3 abrogates ΔNp63α-induced MM1 downregulation and induces cell senescence, placing HERC3 in a ΔNp63α→HERC3→MM1→c-Myc axis controlling cell senescence.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, overexpression, reporter/senescence assays, epistasis experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal methods (Co-IP, ubiquitination assay, epistasis by KD) in a single lab study\",\n      \"pmids\": [\"29880857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HERC3 promotes ubiquitination-mediated degradation of SMAD7 in an autolysosome-dependent manner. This leads to increased p-SMAD2/3 levels and TGFβ pathway activation, driving EMT. Autophagy inducers upregulate HERC3 expression, establishing a mechanistic link between autophagy and TGFβ/SMAD signaling via HERC3-mediated SMAD7 degradation.\",\n      \"method\": \"iTRAQ proteomics, co-immunoprecipitation, ubiquitination assays, siRNA/overexpression, intracranial xenograft, immunohistochemistry, tissue microarray\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — proteomics identification followed by Co-IP and ubiquitination assays and in vivo validation, single lab\",\n      \"pmids\": [\"30862693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HERC3 directly interacts with EIF5A2 via its RCC1 domain and promotes K27- and K48-linked ubiquitination degradation of EIF5A2 via its HECT domain. Specific ubiquitination sites on EIF5A2 were identified as K47, K67, K85, and K121. This interaction inhibits EMT and metastasis in colorectal cancer through the EIF5A2/TGFβ/Smad2/3 signaling axis.\",\n      \"method\": \"Co-immunoprecipitation, GST-pulldown, in vivo ubiquitination assays, domain-deletion mutants, transwell/wound healing assays, mass spectrometry\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, GST-pulldown, ubiquitination site mapping by MS, domain mutants; single lab\",\n      \"pmids\": [\"35064108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HERC3 directly interacts with RPL23A and acts as an E3 ligase to ubiquitinate and degrade RPL23A via K48-linked polyubiquitination through its HECT domain. HERC3-mediated RPL23A degradation modulates the c-Myc/p21 axis and regulates CRC cell proliferation and cell-cycle arrest at G0/G1.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, GST-pulldown, in vivo ubiquitination assays, cycloheximide chase, loss/gain-of-function experiments, rescue experiments\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, MS substrate identification, ubiquitination assays, rescue epistasis; single lab\",\n      \"pmids\": [\"35637966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HERC3 directly interacts with ERK2 through its HECT domain and promotes ERK2 ubiquitination. HERC3 also modulates p53 protein levels and phosphorylation, linking ERK2 degradation to p53-mediated apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, GST-pulldown, ubiquitination assays, xenograft model\",\n      \"journal\": \"Molecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and GST-pulldown only, limited mechanistic detail on ubiquitination mechanism, single study\",\n      \"pmids\": [\"35889210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC3 promotes YAP/TAZ protein stability and Hippo-independent tumorigenesis independently of its E3 ubiquitin ligase enzymatic activity. HERC3 directly binds to β-TrCP (the YAP/TAZ ubiquitin E3 ligase adaptor), blocking β-TrCP interaction with YAP/TAZ and thereby preventing YAP/TAZ ubiquitination and proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, catalytic-dead mutant analysis, ubiquitination assays, knockdown, protein stability assays, breast tumor cell/tissue correlation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, catalytic-dead mutant establishing ligase-independent mechanism, ubiquitination assays, multiple functional readouts; rigorous single-lab study published in high-tier journal\",\n      \"pmids\": [\"36598329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC3 ubiquitinates NCOA1, targeting it for proteasomal degradation. HERC3 deficiency leads to NCOA1 accumulation, which assembles a NCOA1-p300-Runx2 complex that transactivates matrix metallopeptidase (MMP) gene expression, promoting extracellular matrix degradation in intervertebral disc degeneration.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, ubiquitination assays, loss-of-function (HERC3-deficient model), immunoblot, aged mouse IDD model\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP/MS substrate identification, ubiquitination assay, in vivo mouse model; single lab\",\n      \"pmids\": [\"36878279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC3 facilitates ERAD of select membrane proteins by directly recognizing exposed (cytoplasmic) membrane-spanning domains (MSDs) of misfolded CFTR, but not MSDs embedded in liposomes. HERC3 operates independently of the ER-embedded E3 ligases RNF5 and RNF185 to mediate retrotranslocation and ERAD of misfolded CFTR. This identifies a distinct ERAD branch for cytoplasmic quality control of membrane-spanning domains.\",\n      \"method\": \"Multiplex knockdown/knockout experiments, real-time kinetic ERAD assays, in vitro binding assay with liposome-embedded vs. exposed MSDs, CFTR/ABCB1 misfolded substrate models\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution binding assay distinguishing MSD topology, multiplex KD/KO with kinetic measurements, epistasis with RNF5/185; rigorous mechanistic dissection in a single study\",\n      \"pmids\": [\"38722278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CRISPR-generated Herc3-/- mice develop accumulation of activated subretinal microglia (Iba1+/CD16+), retinal thinning, and functional deficits, demonstrating a non-redundant role for HERC3 in retinal homeostasis and microglial activation suppression in vivo.\",\n      \"method\": \"CRISPR knockout mouse generation, fundus imaging, OCT, histology, optomotor testing, electrophysiology, bulk RNA sequencing\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with multiple orthogonal phenotypic readouts and transcriptomic profiling; mechanism (specific substrate) not fully resolved\",\n      \"pmids\": [\"38321224\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HERC3 is a HECT-domain E3 ubiquitin ligase that localizes to the cytosol and vesicular compartments; it ubiquitinates and promotes proteasomal degradation of substrates including SMAD7, EIF5A2, RPL23A, MM1, NCOA1, and ERK2, thereby regulating TGFβ/EMT, NF-κB, Hippo/YAP-TAZ, cell senescence, and cell-cycle pathways—while in certain contexts (NF-κB RelA destabilization, YAP/TAZ stabilization) its regulatory activity is independent of its catalytic E3 ligase function, instead acting as a scaffold linking substrates to the proteasome or blocking E3 adaptor access; additionally, HERC3 mediates a distinct branch of ER-associated degradation by recognizing exposed cytoplasmic membrane-spanning domains of misfolded membrane proteins such as CFTR.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HERC3 is a HECT-domain E3 ubiquitin ligase that functions as a hub for targeted proteasomal protein degradation, controlling signaling pathways that govern proliferation, senescence, epithelial-mesenchymal transition, and protein quality control [#1, #5]. Through its HECT domain it catalyzes K48-linked (and in some substrates K27-linked) polyubiquitination of multiple targets—including MM1 in a \\u0394Np63\\u03b1\\u2192HERC3\\u2192MM1\\u2192c-Myc senescence axis [#2], the eukaryotic translation factor EIF5A2 [#4], the ribosomal protein RPL23A acting on the c-Myc/p21 axis to arrest cells at G0/G1 [#5], and NCOA1 to restrain a NCOA1-p300-Runx2 complex that drives matrix metalloproteinase expression [#8]; substrate engagement can occur through its RCC1 domain, as shown for EIF5A2 [#4]. HERC3 also drives degradation of SMAD7 in an autolysosome-dependent manner, relieving inhibition of TGF\\u03b2/SMAD2/3 signaling and promoting EMT [#3]. Notably, a subset of HERC3's regulatory functions is independent of its catalytic ligase activity: it restrains NF-\\u03baB by bridging liberated RelA to the 26S proteasome together with UBQLN1 [#1], and it stabilizes YAP/TAZ by directly binding \\u03b2-TrCP to block adaptor access [#7]. In ER-associated degradation, HERC3 defines a distinct branch that recognizes exposed cytoplasmic membrane-spanning domains of misfolded membrane proteins such as CFTR, operating independently of the ER-embedded ligases RNF5 and RNF185 [#9]. In vivo, loss of HERC3 in mice causes subretinal microglial activation and retinal degeneration, establishing a non-redundant homeostatic role [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established HERC3 as a cytosolic/vesicular HECT-family protein that non-covalently binds ubiquitin and is itself a proteasome substrate, framing its biology around the ubiquitin system before any substrate was known.\",\n      \"evidence\": \"Subcellular fractionation, immunofluorescence co-localization with \\u03b2-COP/ARF/Rab5, Co-IP, catalytic-cysteine mutagenesis, and proteasome inhibition\",\n      \"pmids\": [\"11163799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No physiological substrate identified\", \"Functional consequence of vesicular localization untested\", \"Ubiquitin-binding interface not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed HERC3 negatively regulates NF-\\u03baB by bridging RelA to the proteasome via UBQLN1, and that this activity is independent of its catalytic ligase function, revealing a scaffolding mode of action.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, catalytic-dead mutant, NF-\\u03baB reporter, nuclear fractionation, and DNA-binding assays\",\n      \"pmids\": [\"26476452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Indirect RelA binding partner not fully defined\", \"Whether the same complex regulates other transcription factors unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed HERC3 in a senescence-controlling axis by demonstrating it is transcriptionally induced by \\u0394Np63\\u03b1 and degrades MM1 via K48-linked ubiquitination to modulate c-Myc.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, siRNA knockdown, overexpression, and epistasis/senescence assays\",\n      \"pmids\": [\"29880857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect MM1 binding not resolved\", \"Ubiquitination sites on MM1 not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected HERC3 to TGF\\u03b2/EMT by showing autophagy-induced HERC3 degrades SMAD7 in an autolysosome-dependent manner, enhancing SMAD2/3 signaling.\",\n      \"evidence\": \"iTRAQ proteomics, Co-IP, ubiquitination assays, knockdown/overexpression, and intracranial xenograft with tissue analysis\",\n      \"pmids\": [\"30862693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking ubiquitination to autolysosomal rather than proteasomal degradation unclear\", \"Ubiquitin linkage type on SMAD7 not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined direct substrates and degradation chemistry: HERC3 binds EIF5A2 via its RCC1 domain to drive K27/K48 ubiquitination, and binds RPL23A to drive K48 ubiquitination, linking these to TGF\\u03b2/EMT suppression and c-Myc/p21-dependent cell-cycle arrest respectively.\",\n      \"evidence\": \"Reciprocal Co-IP, GST-pulldown, domain-deletion mutants, MS-based ubiquitination site mapping, cycloheximide chase, and rescue experiments\",\n      \"pmids\": [\"35064108\", \"35637966\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Context determining anti- vs. pro-EMT outcomes across substrates unresolved\", \"In vitro reconstitution of ligase activity not performed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Proposed ERK2 as a HERC3 substrate linked to p53-mediated apoptosis, extending HERC3 into MAPK signaling.\",\n      \"evidence\": \"Co-IP, GST-pulldown, ubiquitination assays, and xenograft model\",\n      \"pmids\": [\"35889210\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single low-confidence study with limited mechanistic detail on ubiquitination\", \"Ubiquitin linkage and sites on ERK2 not defined\", \"p53 link correlative\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a ligase-independent stabilizing function in which HERC3 binds \\u03b2-TrCP to block YAP/TAZ ubiquitination, driving Hippo-independent tumorigenesis.\",\n      \"evidence\": \"Reciprocal Co-IP, catalytic-dead mutant, ubiquitination/protein-stability assays, knockdown, and breast tumor tissue correlation\",\n      \"pmids\": [\"36598329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of \\u03b2-TrCP competition unknown\", \"Whether HERC3 blocks other \\u03b2-TrCP substrates untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified NCOA1 as a HERC3 substrate whose loss derepresses a NCOA1-p300-Runx2 transcriptional complex driving MMP-mediated matrix degradation in disc degeneration.\",\n      \"evidence\": \"Co-IP/MS, ubiquitination assays, HERC3-deficient model, and aged mouse IDD model\",\n      \"pmids\": [\"36878279\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage and sites on NCOA1 not mapped\", \"Direct vs. indirect engagement not fully resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a distinct ERAD branch in which HERC3 directly recognizes exposed cytoplasmic membrane-spanning domains of misfolded CFTR, acting independently of ER-embedded ligases RNF5/RNF185.\",\n      \"evidence\": \"Multiplex KD/KO, real-time kinetic ERAD assays, in vitro binding to liposome-embedded vs. exposed MSDs, and CFTR/ABCB1 substrate models\",\n      \"pmids\": [\"38722278\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural determinants of MSD recognition unresolved\", \"Full substrate repertoire of this ERAD branch unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a non-redundant in vivo role for HERC3 in retinal homeostasis, with knockout causing subretinal microglial activation and retinal degeneration.\",\n      \"evidence\": \"CRISPR knockout mice with fundus imaging, OCT, histology, optomotor testing, electrophysiology, and bulk RNA-seq\",\n      \"pmids\": [\"38321224\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal substrate driving the retinal phenotype not identified\", \"Cell-autonomous vs. non-autonomous microglial effects unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how HERC3 selects between catalytic degradation and ligase-independent scaffolding modes, and which substrate(s) account for its in vivo developmental and homeostatic phenotypes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for substrate recognition across diverse targets\", \"No structural data on HECT/RCC1 substrate engagement\", \"In vivo substrate(s) linking molecular activity to organismal phenotype undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 5, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 5, 8, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"RelA-UBQLN1-26S proteasome complex\"],\n    \"partners\": [\"RELA\", \"UBQLN1\", \"EIF5A2\", \"RPL23A\", \"MAPK1\", \"NCOA1\", \"BTRC\", \"CFTR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}