{"gene":"HERC1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1996,"finding":"HERC1 (p619) localizes to the cytosol and Golgi apparatus in a Brefeldin A-sensitive manner; its N-terminal RCC1-like domain (RLD1) stimulates guanine nucleotide exchange on ARF1 and Rab proteins but not on Ran or R-Ras2/TC21; its C-terminal RCC1-like domain interacts specifically with myristoylated ARF1.","method":"Subcellular fractionation, immunofluorescence, Brefeldin A treatment, in vitro GEF assay with purified recombinant domains","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro GEF activity assay with domain mapping, subcellular localization with pharmacological perturbation, multiple orthogonal methods in a single foundational study","pmids":["8861955"],"is_preprint":false},{"year":2003,"finding":"The HECT domain of HERC1 physically interacts with M2-type pyruvate kinase (M2-PK); this interaction does not induce M2-PK ubiquitination nor affect its enzymatic activity.","method":"Co-immunoprecipitation, pulldown with recombinant HECT domain, immunofluorescence colocalization","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal pulldown and colocalization in a single lab; negative result on ubiquitination also experimentally established","pmids":["12650930"],"is_preprint":false},{"year":2004,"finding":"HERC1 is recruited to ARF6-induced, aluminum fluoride-stimulated actin-rich surface protrusions in HeLa cells; HERC1 overexpression alone does not stimulate protrusion formation, indicating its recruitment is downstream of ARF6 activation rather than HERC1 acting as an ARF6-GEF in this context. A phosphoinositide-binding mechanism was proposed for translocation.","method":"Immunofluorescence of transfected HeLa cells treated with AlF4⁻, overexpression experiments","journal":"FEBS letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (immunofluorescence), mechanistic conclusion is partially negative/inferred","pmids":["14960311"],"is_preprint":false},{"year":2005,"finding":"HERC1 RLD1 stimulates GDP release from ARF6 but inhibits GDP/GTP exchange under conditions where ARNO promotes it; HERC1's GEF activity requires phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) bound to RLD1, and purified HERC1 contains PI(4,5)P2 co-purifying with RLD1.","method":"In vitro guanine nucleotide exchange assay with purified recombinant RLD1, lipid co-purification/binding assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution of GEF activity with defined lipid requirement, single lab but multiple orthogonal biochemical methods","pmids":["15642342"],"is_preprint":false},{"year":2006,"finding":"HERC1 interacts with TSC2 and functions as an E3 ubiquitin ligase that destabilizes TSC2; TSC1 binding to TSC2 sterically excludes HERC1 from the TSC2 complex, thereby stabilizing TSC2. Disease-causing TSC2 mutations allow HERC1 binding even in the presence of TSC1.","method":"Co-immunoprecipitation, pulldown assays, protein stability assays with wild-type and mutant TSC2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mutant analysis, protein stability experiments; replicated in multiple subsequent studies confirming the HERC1-TSC2 axis","pmids":["16464865"],"is_preprint":false},{"year":2009,"finding":"A missense mutation Gly483Glu in the N-terminal RCC1-like domain of HERC1 causes progressive Purkinje cell degeneration in tambaleante mice via extensive autophagy, accompanied by increased mutant HERC1 protein levels and decreased mTOR activity; transgenic rescue with wild-type Herc1 BAC or human HERC1 cDNA validated the causal role.","method":"Positional cloning, transgenic rescue, histology, Western blot for mTOR pathway components, autophagy markers","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — transgenic rescue with two constructs, positional cloning, biochemical pathway analysis; independently foundational for HERC1 neurological function","pmids":["20041218"],"is_preprint":false},{"year":2014,"finding":"HPV5 E6 recruits HERC1 to ubiquitinate and degrade activated BAK (after UV-induced conformational change and phosphorylation); HERC1 physically interacts with activated BAK via a putative BH3-like domain in HERC1, and a specific lysine on BAK is required for E6-mediated proteolysis.","method":"siRNA functional screen, co-immunoprecipitation in E6-expressing UV-treated cells, site-directed mutagenesis of BAK lysine residue, domain analysis","journal":"International journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Moderate — functional siRNA screen validated by Co-IP, mutagenesis of substrate, BH3 domain mapping; multiple orthogonal methods in single lab","pmids":["25408501"],"is_preprint":false},{"year":2015,"finding":"HERC1 E3 ubiquitin ligase is required for normal neuromuscular junction morphology and evoked neurotransmitter release; the tambaleante HERC1 mutation reduces motor end-plate area and impairs presynaptic vesicle release before cerebellar degeneration occurs.","method":"Electrophysiology (evoked neurotransmitter release), morphometry of motor end-plates, in vivo motor function assays in tambaleante mice","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular phenotype with loss-of-function model, electrophysiological readout, single lab","pmids":["25746226"],"is_preprint":false},{"year":2018,"finding":"HERC1 controls C-RAF protein stability by promoting its K48-linked polyubiquitination and proteasomal degradation; HERC1 directly interacts with C-RAF as shown by Co-IP and pulldown; in vitro ubiquitylation assay confirms C-RAF is a direct substrate of HERC1. HERC1 knockdown increases ERK phosphorylation and cell proliferation via C-RAF stabilization.","method":"Co-immunoprecipitation, pulldown, confocal microscopy, in vitro ubiquitylation assay, pharmacological RAF inhibitors, siRNA, K48-linkage-specific ubiquitin detection","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitylation assay establishing direct substrate relationship, reciprocal Co-IP, linkage-specific ubiquitin analysis, and functional pathway rescue; multiple orthogonal methods","pmids":["30140388"],"is_preprint":false},{"year":2018,"finding":"HERC1 mutation in tambaleante mice causes compact myelin damage (tomacula, hypermyelination foci) in motor axons, delays action potential propagation, and alters terminal Schwann cells at the NMJ, associated with increased phospho-Akt2 in sciatic nerve.","method":"Electrophysiology (nerve conduction velocity), electron microscopy, immunohistochemistry, Western blot for pAkt2 in sciatic nerve of tambaleante mice","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function mouse model with defined morphological and electrophysiological phenotype; single lab, multiple methods","pmids":["29603094"],"is_preprint":false},{"year":2020,"finding":"HERC1 regulates cell migration through the MKK3/p38 pathway in a RAF-dependent manner: HERC1 ubiquitylates C-RAF for proteasomal degradation; C-RAF stabilization then increases MKK3 mRNA levels, which activates p38 to drive migration. HERC1 knockdown induces C-RAF accumulation, MKK3 upregulation, and p38 phosphorylation.","method":"siRNA knockdown, in vitro ubiquitylation assay, RAF pharmacological inhibitors, qRT-PCR for MKK3 mRNA, migration assays, Western blot for p38 phosphorylation","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitylation confirms direct substrate relationship, epistasis by RAF inhibition places RAF upstream of MKK3, functional migration readout; multiple orthogonal methods, single lab","pmids":["31965002"],"is_preprint":false},{"year":2020,"finding":"HERC1 mutation (gain-of-function, Arg4691Pro in HECT domain) causes mTORC1 hyperactivation and failure to suppress mTORC1 during nutrient starvation (catabolic state), with abnormally high S6K1 phosphorylation and reduced autophagy in patient fibroblasts.","method":"Patient fibroblast functional assay under nutrient starvation, Western blot for pS6K1, autophagy markers (LC3, p62)","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cell functional validation with biochemical pathway readouts; single lab, patient material supports physiological relevance","pmids":["32921582"],"is_preprint":false},{"year":2020,"finding":"HERC1 mutation in tambaleante hippocampal neurons reduces synaptic vesicle number, decreases active zone size, reduces clathrin immunoreactivity, and increases endosomes and autophagosomes at presynaptic endings, demonstrating HERC1's role in presynaptic membrane dynamics.","method":"Transmission electron microscopy, FM1-43 destaining assay, immunocytochemistry in cultured hippocampal neurons from tambaleante mice","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function model with TEM and functional dye assay for vesicle recycling; single lab, multiple morphological and functional methods","pmids":["32694577"],"is_preprint":false},{"year":2021,"finding":"HERC1 physically interacts with BCR-ABL1 fusion protein in CML cells and is directly tyrosine-phosphorylated by ABL kinase.","method":"Co-immunoprecipitation, phosphorylation assay in CML cell lines","journal":"Cancers","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP experiment, single lab, no reconstitution or mutagenesis to map sites","pmids":["33477751"],"is_preprint":false},{"year":2023,"finding":"HERC1 regulates osteoblastogenesis and osteoclastogenesis; its depletion increases C-RAF levels and phosphorylated ERK and p38 during osteogenic differentiation of mesenchymal stem cells, and Herc1-knockout mice develop osteopenia with increased osteoclast numbers in young females and elevated Rankl/Opg ratio in osteocytes.","method":"Herc1-knockout mouse model, osteogenic differentiation of MSCs, Western blot for C-RAF/pERK/pp38, bone histomorphometry, RANKL/OPG gene expression","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined cellular and in vivo phenotype, mechanistic link to C-RAF/ERK/p38 confirmed biochemically; single lab","pmids":["36635269"],"is_preprint":false},{"year":2024,"finding":"HERC1 ubiquitinates and degrades NCOA4 (a cargo receptor for ferritinophagy); targeting the HERC1-NCOA4 axis during photodynamic therapy activates ferroptosis in osteosarcoma cells. NRF2 is identified as a potential upstream regulator of this axis.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell death assays in osteosarcoma cell lines","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay supporting substrate relationship; single lab, functional ferroptosis readout","pmids":["39216271"],"is_preprint":false},{"year":2024,"finding":"HERC1 ubiquitinates deoxycytidine kinase (dCK) for degradation in AML cells; loss of HERC1 enhances Ara-C (cytarabine)-induced cell death by compromising cell cycle progression. This was identified by genome-wide CRISPR pharmacogenomic screening and validated by proteomic analysis.","method":"Genome-wide CRISPR screen, in vitro and in vivo AML models, quantitative proteomics, validation of dCK as HERC1 substrate","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen validated in multiple models with proteomic substrate identification; single lab, substrate relationship inferred rather than directly reconstituted in vitro","pmids":["39093953"],"is_preprint":false},{"year":2025,"finding":"HERC1 mutation in tambaleante mice does not alter proteasomal activity or specific UPS gene expression in brain or muscle tissue; proteasome inhibition with MG-132 does not affect evoked neurotransmitter release, whereas autophagy inhibition with wortmannin impairs it. HERC1 mutation increases p62 expression in muscle and alters clathrin and synaptophysin levels, indicating HERC1 regulates autophagy and vesicular recycling but not proteasomal function at the NMJ.","method":"Proteasome activity assay, MG-132 and wortmannin pharmacology at the NMJ, Western blot for p62/clathrin/synaptophysin in tambaleante mice","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — negative result on proteasome (multiple methods) and positive result on autophagy pathway both experimentally established; single lab","pmids":["39859507"],"is_preprint":false},{"year":2025,"finding":"HERC1 promotes ubiquitination and proteasomal degradation of KAT2A (a lysine acetyltransferase); KAT2A depletion inhibits lysine acetylation of PIK3CB, inactivating the PI3K/AKT axis and suppressing autophagy, thereby enhancing gemcitabine sensitivity in lung cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown in gemcitabine-resistant lung cancer cells, xenograft model, Western blot for PI3K/AKT pathway","journal":"Neoplasma","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay establish substrate relationship; rescue experiments with KAT2A connect pathway; single lab","pmids":["41567019"],"is_preprint":false},{"year":2025,"finding":"HERC1 interacts with C-RAF, promotes its polyubiquitination, accelerates its proteasomal degradation, and promotes ferroptosis in lung adenocarcinoma cells; C-RAF overexpression partially rescues the ferroptosis phenotype induced by HERC1 overexpression.","method":"Co-immunoprecipitation, ubiquitination assay, protein stability assay, ferroptosis markers (ROS, MDA, GSH, Fe2+), CCK-8/LDH assays, rescue by C-RAF overexpression","journal":"Cancer genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods confirm C-RAF as substrate; functional rescue establishes epistasis; single lab, consistent with prior HERC1–C-RAF literature","pmids":["39983667"],"is_preprint":false}],"current_model":"HERC1 is a giant multidomain E3 ubiquitin ligase (HECT domain) and guanine nucleotide exchange factor (RCC1-like domains) that (1) targets C-RAF for K48-linked polyubiquitination and proteasomal degradation, thereby suppressing ERK, p38/MKK3, and PI3K/AKT signaling and regulating cell proliferation, migration, and ferroptosis; (2) destabilizes TSC2 (counteracted by TSC1 binding), thereby modulating mTORC1 activity and autophagy; (3) ubiquitinates additional substrates including BAK (in HPV5 E6-expressing cells), NCOA4, KAT2A, and deoxycytidine kinase; (4) stimulates GDP release from ARF1, ARF6, and Rab GTPases in a PI(4,5)P2-dependent manner at the Golgi; and (5) is essential in neurons for presynaptic vesicle recycling, neuromuscular junction function, axonal myelination, and autophagy homeostasis, with loss-of-function causing Purkinje cell degeneration and intellectual disability."},"narrative":{"mechanistic_narrative":"HERC1 is a giant multidomain protein that couples guanine-nucleotide exchange activity to HECT-domain E3 ubiquitin ligase function, governing membrane trafficking, growth signaling, and neuronal homeostasis [PMID:8861955, PMID:30140388]. Through its N-terminal RCC1-like domain it acts as a guanine-nucleotide exchange factor that stimulates GDP release from ARF1, ARF6, and Rab GTPases at the cytosol and Golgi, an activity that strictly requires PI(4,5)P2 bound to the RLD [PMID:8861955, PMID:15642342]. As an E3 ligase, HERC1 targets multiple substrates for K48-linked polyubiquitination and proteasomal degradation: it destabilizes C-RAF to restrain ERK and MKK3/p38 signaling, thereby limiting cell proliferation, migration, and promoting ferroptosis [PMID:30140388, PMID:31965002, PMID:39983667]; it destabilizes TSC2 in a manner blocked by TSC1 binding, linking HERC1 to mTORC1 regulation and autophagy [PMID:16464865, PMID:32921582]; and it degrades additional substrates including NCOA4, KAT2A, and deoxycytidine kinase to modulate ferritinophagy, PI3K/AKT signaling, and chemosensitivity [PMID:39216271, PMID:41567019, PMID:39093953]. In neurons, HERC1 is essential for presynaptic vesicle recycling, neuromuscular junction structure and neurotransmitter release, and axonal myelination, acting through autophagy rather than proteasomal control at the synapse [PMID:25746226, PMID:32694577, PMID:39859507]. A missense mutation in the RLD causes Purkinje cell degeneration via excessive autophagy in tambaleante mice, and human HERC1 HECT-domain mutation causes mTORC1 hyperactivation with intellectual disability [PMID:20041218, PMID:32921582].","teleology":[{"year":1996,"claim":"Established HERC1 as a Golgi/cytosolic guanine-nucleotide exchange factor, defining its first molecular activity through its RCC1-like domains acting on ARF and Rab GTPases.","evidence":"Subcellular fractionation, immunofluorescence with Brefeldin A, and in vitro GEF assays with recombinant domains","pmids":["8861955"],"confidence":"High","gaps":["Did not connect GEF activity to a downstream trafficking pathway","E3 ligase function not yet known"]},{"year":2003,"claim":"Tested whether the HECT domain has substrate roles beyond ubiquitination by identifying an M2-PK interaction, but found no functional consequence.","evidence":"Co-IP, recombinant HECT pulldown, and colocalization","pmids":["12650930"],"confidence":"Medium","gaps":["No ubiquitination or activity change observed","Functional significance of the interaction unresolved"]},{"year":2005,"claim":"Defined the biochemical regulation of HERC1 GEF activity, showing it requires PI(4,5)P2 and modulates ARF6 nucleotide exchange.","evidence":"In vitro nucleotide exchange and lipid co-purification assays with recombinant RLD1","pmids":["15642342"],"confidence":"High","gaps":["Cellular context for PI(4,5)P2-dependent GEF activity not established","Relationship to E3 ligase function unclear"]},{"year":2006,"claim":"Revealed HERC1 as an E3 ligase that destabilizes TSC2, with TSC1 binding protecting TSC2, linking HERC1 to mTOR pathway control.","evidence":"Reciprocal Co-IP, pulldown, and protein stability assays with WT and mutant TSC2","pmids":["16464865"],"confidence":"High","gaps":["Direct in vitro ubiquitylation of TSC2 not reconstituted","Physiological consequence on mTORC1 output not measured here"]},{"year":2009,"claim":"Demonstrated HERC1 is causally required for Purkinje cell survival, linking an RLD mutation to autophagy-driven neurodegeneration in vivo.","evidence":"Positional cloning and transgenic rescue in tambaleante mice with pathway Western blots","pmids":["20041218"],"confidence":"High","gaps":["Mechanism linking RLD mutation to excess autophagy not resolved","Whether the mutation is gain- or loss-of-function ambiguous"]},{"year":2014,"claim":"Identified activated BAK as a HERC1 substrate co-opted by HPV5 E6, extending HERC1 ligase activity into apoptotic regulation.","evidence":"siRNA screen, Co-IP in E6/UV-treated cells, BAK lysine and HERC1 BH3-domain mutagenesis","pmids":["25408501"],"confidence":"High","gaps":["Whether HERC1 targets BAK outside HPV E6 context unknown","Direct in vitro ubiquitylation not shown"]},{"year":2018,"claim":"Established C-RAF as a direct HERC1 substrate degraded via K48 ubiquitination, defining a node controlling ERK signaling and proliferation.","evidence":"Reciprocal Co-IP, in vitro ubiquitylation, linkage-specific ubiquitin detection, RAF inhibitor and siRNA epistasis","pmids":["30140388"],"confidence":"High","gaps":["Recruitment mechanism for C-RAF not defined","Regulation of HERC1-C-RAF interaction unknown"]},{"year":2018,"claim":"Extended HERC1 neuronal function to myelin integrity and axon conduction, implicating Akt2 dysregulation in peripheral nerve pathology.","evidence":"Nerve conduction, electron microscopy, and pAkt2 Western blot in tambaleante sciatic nerve","pmids":["29603094"],"confidence":"Medium","gaps":["Substrate driving myelin/Akt2 changes not identified","Schwann-cell-autonomous vs neuronal contribution unresolved"]},{"year":2020,"claim":"Connected HERC1-mediated C-RAF degradation to cell migration through a RAF-dependent MKK3/p38 transcriptional axis.","evidence":"siRNA, in vitro ubiquitylation, RAF inhibition epistasis, MKK3 qRT-PCR, and migration assays","pmids":["31965002"],"confidence":"High","gaps":["Mechanism by which C-RAF raises MKK3 mRNA not defined","In vivo relevance of migration phenotype untested here"]},{"year":2020,"claim":"Linked a human HECT-domain mutation to mTORC1 hyperactivation and impaired starvation-induced autophagy, establishing disease relevance.","evidence":"Patient fibroblast assays under starvation with pS6K1 and autophagy marker Western blots","pmids":["32921582"],"confidence":"Medium","gaps":["Whether mutation acts through TSC2 not directly tested","Single patient-derived material"]},{"year":2020,"claim":"Defined HERC1's presynaptic role in vesicle recycling and active zone structure at central synapses.","evidence":"TEM, FM1-43 destaining, and immunocytochemistry in tambaleante hippocampal neurons","pmids":["32694577"],"confidence":"Medium","gaps":["Molecular substrate at the presynapse not identified","Loss-of-function vs mutant gain-of-function not distinguished"]},{"year":2021,"claim":"Reported HERC1 association with and tyrosine phosphorylation by BCR-ABL1, hinting at regulation in CML.","evidence":"Co-IP and phosphorylation assay in CML cell lines","pmids":["33477751"],"confidence":"Low","gaps":["Single Co-IP without phosphosite mapping or reconstitution","Functional consequence of phosphorylation unknown"]},{"year":2023,"claim":"Showed HERC1 controls bone remodeling via C-RAF/ERK/p38 in mesenchymal cells, extending the C-RAF axis to skeletal biology.","evidence":"Herc1-knockout mice, MSC osteogenic differentiation, bone histomorphometry, and RANKL/OPG expression","pmids":["36635269"],"confidence":"Medium","gaps":["Cell-autonomous osteoclast vs osteoblast contributions unresolved","Direct ubiquitylation in bone cells not reconstituted"]},{"year":2024,"claim":"Identified NCOA4 as a HERC1 substrate, coupling HERC1 to ferritinophagy and ferroptosis regulation.","evidence":"Co-IP, ubiquitination assay, and ferroptosis readouts in osteosarcoma cells","pmids":["39216271"],"confidence":"Medium","gaps":["NRF2 upstream link only correlative","Direct in vitro ubiquitylation of NCOA4 not shown"]},{"year":2024,"claim":"Discovered HERC1 degrades deoxycytidine kinase, defining a pharmacogenomic determinant of cytarabine response in AML.","evidence":"Genome-wide CRISPR screen, in vivo AML models, and quantitative proteomics","pmids":["39093953"],"confidence":"Medium","gaps":["Substrate relationship inferred from proteomics, not reconstituted in vitro","Mechanism of dCK recognition unknown"]},{"year":2025,"claim":"Distinguished HERC1's neuromuscular role as autophagy-dependent rather than proteasome-dependent, refining its synaptic mechanism.","evidence":"Proteasome activity assays, MG-132 and wortmannin pharmacology, and autophagy/vesicle marker Western blots at the NMJ","pmids":["39859507"],"confidence":"Medium","gaps":["Substrate driving autophagy regulation at synapse not identified","Tissue specificity of autophagy dependence unclear"]},{"year":2025,"claim":"Identified KAT2A as a HERC1 substrate, connecting HERC1 to PI3K/AKT signaling and chemoresistance via PIK3CB acetylation.","evidence":"Co-IP, ubiquitination assay, knockdown/overexpression rescue, and xenografts in lung cancer cells","pmids":["41567019"],"confidence":"Medium","gaps":["Direct in vitro ubiquitylation of KAT2A not reconstituted","Generality beyond gemcitabine resistance untested"]},{"year":2025,"claim":"Confirmed the HERC1-C-RAF axis drives ferroptosis in lung adenocarcinoma, reinforcing C-RAF as a central effector.","evidence":"Co-IP, ubiquitination and stability assays, ferroptosis markers, and C-RAF rescue","pmids":["39983667"],"confidence":"Medium","gaps":["Link between C-RAF stability and ferroptosis effectors not detailed","Single lab"]},{"year":null,"claim":"How HERC1's GEF activity, multiple ubiquitination substrates, and giant scaffold architecture are coordinated into a unified regulatory logic, and how distinct mutations produce opposing mTORC1 outcomes, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating RLD and HECT domains","Substrate selection rules across contexts undefined","Gain- vs loss-of-function basis of different disease mutations unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,8,10,15,16,18,19]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[8,10,19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,10,18,19]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,8,19]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[5,11,17]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[7,12,17]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6,15,19]}],"complexes":[],"partners":["RAF1","TSC2","NCOA4","KAT2A","BAK1","ARF1","ARF6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15751","full_name":"Probable E3 ubiquitin-protein ligase HERC1","aliases":["HECT domain and RCC1-like domain-containing protein 1","HECT-type E3 ubiquitin transferase HERC1","p532","p619"],"length_aa":4861,"mass_kda":532.2,"function":"Involved in membrane trafficking via some guanine nucleotide exchange factor (GEF) activity and its ability to bind clathrin. Acts as a GEF for Arf and Rab, by exchanging bound GDP for free GTP. Binds phosphatidylinositol 4,5-bisphosphate, which is required for GEF activity. May also act as a 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":"Membrane; Cytoplasm, cytosol; Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/Q15751/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HERC1","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HERC1","total_profiled":1310},"omim":[{"mim_id":"617011","title":"MACROCEPHALY, DYSMORPHIC FACIES, AND PSYCHOMOTOR RETARDATION; MDFPMR","url":"https://www.omim.org/entry/617011"},{"mim_id":"609587","title":"REGULATOR OF CHROMOSOME CONDENSATION 2; RCC2","url":"https://www.omim.org/entry/609587"},{"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"},{"mim_id":"605109","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 1; HERC1","url":"https://www.omim.org/entry/605109"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HERC1"},"hgnc":{"alias_symbol":["p532","p619"],"prev_symbol":[]},"alphafold":{"accession":"Q15751","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15751","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HERC1","jax_strain_url":"https://www.jax.org/strain/search?query=HERC1"},"sequence":{"accession":"Q15751","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15751.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15751/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15751"}},"corpus_meta":[{"pmid":"16464865","id":"PMC_16464865","title":"TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16464865","citation_count":186,"is_preprint":false},{"pmid":"8861955","id":"PMC_8861955","title":"p619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.","date":"1996","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/8861955","citation_count":117,"is_preprint":false},{"pmid":"20041218","id":"PMC_20041218","title":"Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.","date":"2009","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20041218","citation_count":55,"is_preprint":false},{"pmid":"15592436","id":"PMC_15592436","title":"Apoptosis-stimulating protein of p53-2 (ASPP2/53BP2L) is an E2F target gene.","date":"2005","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/15592436","citation_count":50,"is_preprint":false},{"pmid":"26153217","id":"PMC_26153217","title":"A nonsense variant in HERC1 is associated with intellectual disability, megalencephaly, thick corpus callosum and cerebellar atrophy.","date":"2015","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/26153217","citation_count":44,"is_preprint":false},{"pmid":"28323226","id":"PMC_28323226","title":"HERC1 mutations in idiopathic intellectual disability.","date":"2017","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28323226","citation_count":40,"is_preprint":false},{"pmid":"26138117","id":"PMC_26138117","title":"Biallelic HERC1 mutations in a syndromic form of overgrowth and intellectual disability.","date":"2015","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26138117","citation_count":38,"is_preprint":false},{"pmid":"25746226","id":"PMC_25746226","title":"The HERC1 E3 Ubiquitin Ligase is essential for normal development and for neurotransmission at the mouse neuromuscular junction.","date":"2015","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/25746226","citation_count":37,"is_preprint":false},{"pmid":"12650930","id":"PMC_12650930","title":"Interaction between HERC1 and M2-type pyruvate kinase.","date":"2003","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12650930","citation_count":35,"is_preprint":false},{"pmid":"25408501","id":"PMC_25408501","title":"Resistance to UV-induced apoptosis by β-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.","date":"2014","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25408501","citation_count":35,"is_preprint":false},{"pmid":"27108999","id":"PMC_27108999","title":"A splice site mutation in HERC1 leads to syndromic intellectual disability with macrocephaly and facial dysmorphism: Further delineation of the phenotypic spectrum.","date":"2016","source":"American journal of medical genetics. 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\"Subcellular fractionation, immunofluorescence, Brefeldin A treatment, in vitro GEF assay with purified recombinant domains\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro GEF activity assay with domain mapping, subcellular localization with pharmacological perturbation, multiple orthogonal methods in a single foundational study\",\n      \"pmids\": [\"8861955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The HECT domain of HERC1 physically interacts with M2-type pyruvate kinase (M2-PK); this interaction does not induce M2-PK ubiquitination nor affect its enzymatic activity.\",\n      \"method\": \"Co-immunoprecipitation, pulldown with recombinant HECT domain, immunofluorescence colocalization\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal pulldown and colocalization in a single lab; negative result on ubiquitination also experimentally established\",\n      \"pmids\": [\"12650930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"HERC1 is recruited to ARF6-induced, aluminum fluoride-stimulated actin-rich surface protrusions in HeLa cells; HERC1 overexpression alone does not stimulate protrusion formation, indicating its recruitment is downstream of ARF6 activation rather than HERC1 acting as an ARF6-GEF in this context. A phosphoinositide-binding mechanism was proposed for translocation.\",\n      \"method\": \"Immunofluorescence of transfected HeLa cells treated with AlF4⁻, overexpression experiments\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (immunofluorescence), mechanistic conclusion is partially negative/inferred\",\n      \"pmids\": [\"14960311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HERC1 RLD1 stimulates GDP release from ARF6 but inhibits GDP/GTP exchange under conditions where ARNO promotes it; HERC1's GEF activity requires phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) bound to RLD1, and purified HERC1 contains PI(4,5)P2 co-purifying with RLD1.\",\n      \"method\": \"In vitro guanine nucleotide exchange assay with purified recombinant RLD1, lipid co-purification/binding assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution of GEF activity with defined lipid requirement, single lab but multiple orthogonal biochemical methods\",\n      \"pmids\": [\"15642342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"HERC1 interacts with TSC2 and functions as an E3 ubiquitin ligase that destabilizes TSC2; TSC1 binding to TSC2 sterically excludes HERC1 from the TSC2 complex, thereby stabilizing TSC2. Disease-causing TSC2 mutations allow HERC1 binding even in the presence of TSC1.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assays, protein stability assays with wild-type and mutant TSC2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mutant analysis, protein stability experiments; replicated in multiple subsequent studies confirming the HERC1-TSC2 axis\",\n      \"pmids\": [\"16464865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A missense mutation Gly483Glu in the N-terminal RCC1-like domain of HERC1 causes progressive Purkinje cell degeneration in tambaleante mice via extensive autophagy, accompanied by increased mutant HERC1 protein levels and decreased mTOR activity; transgenic rescue with wild-type Herc1 BAC or human HERC1 cDNA validated the causal role.\",\n      \"method\": \"Positional cloning, transgenic rescue, histology, Western blot for mTOR pathway components, autophagy markers\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — transgenic rescue with two constructs, positional cloning, biochemical pathway analysis; independently foundational for HERC1 neurological function\",\n      \"pmids\": [\"20041218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HPV5 E6 recruits HERC1 to ubiquitinate and degrade activated BAK (after UV-induced conformational change and phosphorylation); HERC1 physically interacts with activated BAK via a putative BH3-like domain in HERC1, and a specific lysine on BAK is required for E6-mediated proteolysis.\",\n      \"method\": \"siRNA functional screen, co-immunoprecipitation in E6-expressing UV-treated cells, site-directed mutagenesis of BAK lysine residue, domain analysis\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional siRNA screen validated by Co-IP, mutagenesis of substrate, BH3 domain mapping; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"25408501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HERC1 E3 ubiquitin ligase is required for normal neuromuscular junction morphology and evoked neurotransmitter release; the tambaleante HERC1 mutation reduces motor end-plate area and impairs presynaptic vesicle release before cerebellar degeneration occurs.\",\n      \"method\": \"Electrophysiology (evoked neurotransmitter release), morphometry of motor end-plates, in vivo motor function assays in tambaleante mice\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular phenotype with loss-of-function model, electrophysiological readout, single lab\",\n      \"pmids\": [\"25746226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HERC1 controls C-RAF protein stability by promoting its K48-linked polyubiquitination and proteasomal degradation; HERC1 directly interacts with C-RAF as shown by Co-IP and pulldown; in vitro ubiquitylation assay confirms C-RAF is a direct substrate of HERC1. HERC1 knockdown increases ERK phosphorylation and cell proliferation via C-RAF stabilization.\",\n      \"method\": \"Co-immunoprecipitation, pulldown, confocal microscopy, in vitro ubiquitylation assay, pharmacological RAF inhibitors, siRNA, K48-linkage-specific ubiquitin detection\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitylation assay establishing direct substrate relationship, reciprocal Co-IP, linkage-specific ubiquitin analysis, and functional pathway rescue; multiple orthogonal methods\",\n      \"pmids\": [\"30140388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HERC1 mutation in tambaleante mice causes compact myelin damage (tomacula, hypermyelination foci) in motor axons, delays action potential propagation, and alters terminal Schwann cells at the NMJ, associated with increased phospho-Akt2 in sciatic nerve.\",\n      \"method\": \"Electrophysiology (nerve conduction velocity), electron microscopy, immunohistochemistry, Western blot for pAkt2 in sciatic nerve of tambaleante mice\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function mouse model with defined morphological and electrophysiological phenotype; single lab, multiple methods\",\n      \"pmids\": [\"29603094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HERC1 regulates cell migration through the MKK3/p38 pathway in a RAF-dependent manner: HERC1 ubiquitylates C-RAF for proteasomal degradation; C-RAF stabilization then increases MKK3 mRNA levels, which activates p38 to drive migration. HERC1 knockdown induces C-RAF accumulation, MKK3 upregulation, and p38 phosphorylation.\",\n      \"method\": \"siRNA knockdown, in vitro ubiquitylation assay, RAF pharmacological inhibitors, qRT-PCR for MKK3 mRNA, migration assays, Western blot for p38 phosphorylation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitylation confirms direct substrate relationship, epistasis by RAF inhibition places RAF upstream of MKK3, functional migration readout; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"31965002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HERC1 mutation (gain-of-function, Arg4691Pro in HECT domain) causes mTORC1 hyperactivation and failure to suppress mTORC1 during nutrient starvation (catabolic state), with abnormally high S6K1 phosphorylation and reduced autophagy in patient fibroblasts.\",\n      \"method\": \"Patient fibroblast functional assay under nutrient starvation, Western blot for pS6K1, autophagy markers (LC3, p62)\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cell functional validation with biochemical pathway readouts; single lab, patient material supports physiological relevance\",\n      \"pmids\": [\"32921582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HERC1 mutation in tambaleante hippocampal neurons reduces synaptic vesicle number, decreases active zone size, reduces clathrin immunoreactivity, and increases endosomes and autophagosomes at presynaptic endings, demonstrating HERC1's role in presynaptic membrane dynamics.\",\n      \"method\": \"Transmission electron microscopy, FM1-43 destaining assay, immunocytochemistry in cultured hippocampal neurons from tambaleante mice\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function model with TEM and functional dye assay for vesicle recycling; single lab, multiple morphological and functional methods\",\n      \"pmids\": [\"32694577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HERC1 physically interacts with BCR-ABL1 fusion protein in CML cells and is directly tyrosine-phosphorylated by ABL kinase.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay in CML cell lines\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP experiment, single lab, no reconstitution or mutagenesis to map sites\",\n      \"pmids\": [\"33477751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC1 regulates osteoblastogenesis and osteoclastogenesis; its depletion increases C-RAF levels and phosphorylated ERK and p38 during osteogenic differentiation of mesenchymal stem cells, and Herc1-knockout mice develop osteopenia with increased osteoclast numbers in young females and elevated Rankl/Opg ratio in osteocytes.\",\n      \"method\": \"Herc1-knockout mouse model, osteogenic differentiation of MSCs, Western blot for C-RAF/pERK/pp38, bone histomorphometry, RANKL/OPG gene expression\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined cellular and in vivo phenotype, mechanistic link to C-RAF/ERK/p38 confirmed biochemically; single lab\",\n      \"pmids\": [\"36635269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC1 ubiquitinates and degrades NCOA4 (a cargo receptor for ferritinophagy); targeting the HERC1-NCOA4 axis during photodynamic therapy activates ferroptosis in osteosarcoma cells. NRF2 is identified as a potential upstream regulator of this axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell death assays in osteosarcoma cell lines\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay supporting substrate relationship; single lab, functional ferroptosis readout\",\n      \"pmids\": [\"39216271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC1 ubiquitinates deoxycytidine kinase (dCK) for degradation in AML cells; loss of HERC1 enhances Ara-C (cytarabine)-induced cell death by compromising cell cycle progression. This was identified by genome-wide CRISPR pharmacogenomic screening and validated by proteomic analysis.\",\n      \"method\": \"Genome-wide CRISPR screen, in vitro and in vivo AML models, quantitative proteomics, validation of dCK as HERC1 substrate\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen validated in multiple models with proteomic substrate identification; single lab, substrate relationship inferred rather than directly reconstituted in vitro\",\n      \"pmids\": [\"39093953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC1 mutation in tambaleante mice does not alter proteasomal activity or specific UPS gene expression in brain or muscle tissue; proteasome inhibition with MG-132 does not affect evoked neurotransmitter release, whereas autophagy inhibition with wortmannin impairs it. HERC1 mutation increases p62 expression in muscle and alters clathrin and synaptophysin levels, indicating HERC1 regulates autophagy and vesicular recycling but not proteasomal function at the NMJ.\",\n      \"method\": \"Proteasome activity assay, MG-132 and wortmannin pharmacology at the NMJ, Western blot for p62/clathrin/synaptophysin in tambaleante mice\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — negative result on proteasome (multiple methods) and positive result on autophagy pathway both experimentally established; single lab\",\n      \"pmids\": [\"39859507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC1 promotes ubiquitination and proteasomal degradation of KAT2A (a lysine acetyltransferase); KAT2A depletion inhibits lysine acetylation of PIK3CB, inactivating the PI3K/AKT axis and suppressing autophagy, thereby enhancing gemcitabine sensitivity in lung cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown in gemcitabine-resistant lung cancer cells, xenograft model, Western blot for PI3K/AKT pathway\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay establish substrate relationship; rescue experiments with KAT2A connect pathway; single lab\",\n      \"pmids\": [\"41567019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC1 interacts with C-RAF, promotes its polyubiquitination, accelerates its proteasomal degradation, and promotes ferroptosis in lung adenocarcinoma cells; C-RAF overexpression partially rescues the ferroptosis phenotype induced by HERC1 overexpression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein stability assay, ferroptosis markers (ROS, MDA, GSH, Fe2+), CCK-8/LDH assays, rescue by C-RAF overexpression\",\n      \"journal\": \"Cancer genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods confirm C-RAF as substrate; functional rescue establishes epistasis; single lab, consistent with prior HERC1–C-RAF literature\",\n      \"pmids\": [\"39983667\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HERC1 is a giant multidomain E3 ubiquitin ligase (HECT domain) and guanine nucleotide exchange factor (RCC1-like domains) that (1) targets C-RAF for K48-linked polyubiquitination and proteasomal degradation, thereby suppressing ERK, p38/MKK3, and PI3K/AKT signaling and regulating cell proliferation, migration, and ferroptosis; (2) destabilizes TSC2 (counteracted by TSC1 binding), thereby modulating mTORC1 activity and autophagy; (3) ubiquitinates additional substrates including BAK (in HPV5 E6-expressing cells), NCOA4, KAT2A, and deoxycytidine kinase; (4) stimulates GDP release from ARF1, ARF6, and Rab GTPases in a PI(4,5)P2-dependent manner at the Golgi; and (5) is essential in neurons for presynaptic vesicle recycling, neuromuscular junction function, axonal myelination, and autophagy homeostasis, with loss-of-function causing Purkinje cell degeneration and intellectual disability.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HERC1 is a giant multidomain protein that couples guanine-nucleotide exchange activity to HECT-domain E3 ubiquitin ligase function, governing membrane trafficking, growth signaling, and neuronal homeostasis [#0, #8]. Through its N-terminal RCC1-like domain it acts as a guanine-nucleotide exchange factor that stimulates GDP release from ARF1, ARF6, and Rab GTPases at the cytosol and Golgi, an activity that strictly requires PI(4,5)P2 bound to the RLD [#0, #3]. As an E3 ligase, HERC1 targets multiple substrates for K48-linked polyubiquitination and proteasomal degradation: it destabilizes C-RAF to restrain ERK and MKK3/p38 signaling, thereby limiting cell proliferation, migration, and promoting ferroptosis [#8, #10, #19]; it destabilizes TSC2 in a manner blocked by TSC1 binding, linking HERC1 to mTORC1 regulation and autophagy [#4, #11]; and it degrades additional substrates including NCOA4, KAT2A, and deoxycytidine kinase to modulate ferritinophagy, PI3K/AKT signaling, and chemosensitivity [#15, #18, #16]. In neurons, HERC1 is essential for presynaptic vesicle recycling, neuromuscular junction structure and neurotransmitter release, and axonal myelination, acting through autophagy rather than proteasomal control at the synapse [#7, #12, #17]. A missense mutation in the RLD causes Purkinje cell degeneration via excessive autophagy in tambaleante mice, and human HERC1 HECT-domain mutation causes mTORC1 hyperactivation with intellectual disability [#5, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established HERC1 as a Golgi/cytosolic guanine-nucleotide exchange factor, defining its first molecular activity through its RCC1-like domains acting on ARF and Rab GTPases.\",\n      \"evidence\": \"Subcellular fractionation, immunofluorescence with Brefeldin A, and in vitro GEF assays with recombinant domains\",\n      \"pmids\": [\"8861955\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect GEF activity to a downstream trafficking pathway\", \"E3 ligase function not yet known\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Tested whether the HECT domain has substrate roles beyond ubiquitination by identifying an M2-PK interaction, but found no functional consequence.\",\n      \"evidence\": \"Co-IP, recombinant HECT pulldown, and colocalization\",\n      \"pmids\": [\"12650930\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ubiquitination or activity change observed\", \"Functional significance of the interaction unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the biochemical regulation of HERC1 GEF activity, showing it requires PI(4,5)P2 and modulates ARF6 nucleotide exchange.\",\n      \"evidence\": \"In vitro nucleotide exchange and lipid co-purification assays with recombinant RLD1\",\n      \"pmids\": [\"15642342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular context for PI(4,5)P2-dependent GEF activity not established\", \"Relationship to E3 ligase function unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed HERC1 as an E3 ligase that destabilizes TSC2, with TSC1 binding protecting TSC2, linking HERC1 to mTOR pathway control.\",\n      \"evidence\": \"Reciprocal Co-IP, pulldown, and protein stability assays with WT and mutant TSC2\",\n      \"pmids\": [\"16464865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct in vitro ubiquitylation of TSC2 not reconstituted\", \"Physiological consequence on mTORC1 output not measured here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated HERC1 is causally required for Purkinje cell survival, linking an RLD mutation to autophagy-driven neurodegeneration in vivo.\",\n      \"evidence\": \"Positional cloning and transgenic rescue in tambaleante mice with pathway Western blots\",\n      \"pmids\": [\"20041218\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking RLD mutation to excess autophagy not resolved\", \"Whether the mutation is gain- or loss-of-function ambiguous\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified activated BAK as a HERC1 substrate co-opted by HPV5 E6, extending HERC1 ligase activity into apoptotic regulation.\",\n      \"evidence\": \"siRNA screen, Co-IP in E6/UV-treated cells, BAK lysine and HERC1 BH3-domain mutagenesis\",\n      \"pmids\": [\"25408501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HERC1 targets BAK outside HPV E6 context unknown\", \"Direct in vitro ubiquitylation not shown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established C-RAF as a direct HERC1 substrate degraded via K48 ubiquitination, defining a node controlling ERK signaling and proliferation.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro ubiquitylation, linkage-specific ubiquitin detection, RAF inhibitor and siRNA epistasis\",\n      \"pmids\": [\"30140388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recruitment mechanism for C-RAF not defined\", \"Regulation of HERC1-C-RAF interaction unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended HERC1 neuronal function to myelin integrity and axon conduction, implicating Akt2 dysregulation in peripheral nerve pathology.\",\n      \"evidence\": \"Nerve conduction, electron microscopy, and pAkt2 Western blot in tambaleante sciatic nerve\",\n      \"pmids\": [\"29603094\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate driving myelin/Akt2 changes not identified\", \"Schwann-cell-autonomous vs neuronal contribution unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected HERC1-mediated C-RAF degradation to cell migration through a RAF-dependent MKK3/p38 transcriptional axis.\",\n      \"evidence\": \"siRNA, in vitro ubiquitylation, RAF inhibition epistasis, MKK3 qRT-PCR, and migration assays\",\n      \"pmids\": [\"31965002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which C-RAF raises MKK3 mRNA not defined\", \"In vivo relevance of migration phenotype untested here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked a human HECT-domain mutation to mTORC1 hyperactivation and impaired starvation-induced autophagy, establishing disease relevance.\",\n      \"evidence\": \"Patient fibroblast assays under starvation with pS6K1 and autophagy marker Western blots\",\n      \"pmids\": [\"32921582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether mutation acts through TSC2 not directly tested\", \"Single patient-derived material\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined HERC1's presynaptic role in vesicle recycling and active zone structure at central synapses.\",\n      \"evidence\": \"TEM, FM1-43 destaining, and immunocytochemistry in tambaleante hippocampal neurons\",\n      \"pmids\": [\"32694577\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate at the presynapse not identified\", \"Loss-of-function vs mutant gain-of-function not distinguished\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reported HERC1 association with and tyrosine phosphorylation by BCR-ABL1, hinting at regulation in CML.\",\n      \"evidence\": \"Co-IP and phosphorylation assay in CML cell lines\",\n      \"pmids\": [\"33477751\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without phosphosite mapping or reconstitution\", \"Functional consequence of phosphorylation unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed HERC1 controls bone remodeling via C-RAF/ERK/p38 in mesenchymal cells, extending the C-RAF axis to skeletal biology.\",\n      \"evidence\": \"Herc1-knockout mice, MSC osteogenic differentiation, bone histomorphometry, and RANKL/OPG expression\",\n      \"pmids\": [\"36635269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-autonomous osteoclast vs osteoblast contributions unresolved\", \"Direct ubiquitylation in bone cells not reconstituted\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified NCOA4 as a HERC1 substrate, coupling HERC1 to ferritinophagy and ferroptosis regulation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, and ferroptosis readouts in osteosarcoma cells\",\n      \"pmids\": [\"39216271\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NRF2 upstream link only correlative\", \"Direct in vitro ubiquitylation of NCOA4 not shown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovered HERC1 degrades deoxycytidine kinase, defining a pharmacogenomic determinant of cytarabine response in AML.\",\n      \"evidence\": \"Genome-wide CRISPR screen, in vivo AML models, and quantitative proteomics\",\n      \"pmids\": [\"39093953\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate relationship inferred from proteomics, not reconstituted in vitro\", \"Mechanism of dCK recognition unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Distinguished HERC1's neuromuscular role as autophagy-dependent rather than proteasome-dependent, refining its synaptic mechanism.\",\n      \"evidence\": \"Proteasome activity assays, MG-132 and wortmannin pharmacology, and autophagy/vesicle marker Western blots at the NMJ\",\n      \"pmids\": [\"39859507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate driving autophagy regulation at synapse not identified\", \"Tissue specificity of autophagy dependence unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified KAT2A as a HERC1 substrate, connecting HERC1 to PI3K/AKT signaling and chemoresistance via PIK3CB acetylation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, knockdown/overexpression rescue, and xenografts in lung cancer cells\",\n      \"pmids\": [\"41567019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro ubiquitylation of KAT2A not reconstituted\", \"Generality beyond gemcitabine resistance untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Confirmed the HERC1-C-RAF axis drives ferroptosis in lung adenocarcinoma, reinforcing C-RAF as a central effector.\",\n      \"evidence\": \"Co-IP, ubiquitination and stability assays, ferroptosis markers, and C-RAF rescue\",\n      \"pmids\": [\"39983667\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link between C-RAF stability and ferroptosis effectors not detailed\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HERC1's GEF activity, multiple ubiquitination substrates, and giant scaffold architecture are coordinated into a unified regulatory logic, and how distinct mutations produce opposing mTORC1 outcomes, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating RLD and HECT domains\", \"Substrate selection rules across contexts undefined\", \"Gain- vs loss-of-function basis of different disease mutations unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 8, 10, 15, 16, 18, 19]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [8, 10, 19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 10, 18, 19]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 8, 19]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [5, 11, 17]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [7, 12, 17]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6, 15, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RAF1\", \"TSC2\", \"NCOA4\", \"KAT2A\", \"BAK1\", \"ARF1\", \"ARF6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}