{"gene":"HECW2","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2003,"finding":"HECW2 (NEDL2) physically interacts with p73 via its WW domains recognizing PY motifs on p73, ubiquitinates p73, and paradoxically stabilizes rather than degrades it, thereby enhancing p73-dependent transcriptional activation.","method":"Reciprocal co-immunoprecipitation, in vitro pull-down assay, in vitro ubiquitination assay, p73 stability/decay measurements, transcriptional reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus in vitro pull-down plus in vitro ubiquitination assay plus functional transcription assay, single lab","pmids":["12890487"],"is_preprint":false},{"year":2013,"finding":"HECW2 (NEDL2) is a substrate of APC/C-Cdh1 ubiquitin ligase; Cdh1 recognizes a destruction box (R740GSL743) on NEDL2 and targets it for proteasomal degradation during mitotic exit. NEDL2 associates with mitotic spindles, its protein level peaks in mitosis, and its depletion prolongs metaphase while overexpression induces chromosomal lagging.","method":"Co-immunoprecipitation, mass spectrometry, in vitro interaction assay, overexpression and knockdown experiments with mitotic phenotype readouts, destruction-box mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (MS-identified interaction, reciprocal Co-IP in vivo and in vitro, destruction-box mutant, KD/OE phenotypes) in a single rigorous study","pmids":["24163370"],"is_preprint":false},{"year":2014,"finding":"HECW2 (NEDL2) positively regulates enteric neural precursor proliferation through the GDNF/Ret/Akt signaling pathway; NEDL2-deficient mice display intestinal aganglionosis and postnatal lethality, establishing a required in vivo role.","method":"NEDL2 knockout mouse model with histological and functional readout (intestinal aganglionosis, bowel motility), signaling pathway analysis (Akt phosphorylation)","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout mouse with defined cellular phenotype (aganglionosis) and pathway placement (GDNF/Akt), replicated in follow-up study (PMID:27119228)","pmids":["25555806"],"is_preprint":false},{"year":2016,"finding":"HECW2 (NEDL2) acts as a scaffold to recruit SHC, Grb2, PI3K (p110/p85), PDK1, and Akt, promoting GDNF-stimulated Akt signaling. Additionally, NEDL2 harbors intrinsic Nedd8 ligase activity with cysteine 1341 as the catalytic site, and Nedd8 ligase—but not ubiquitin ligase—activity is required for GDNF-stimulated Akt activation and ENS/kidney development.","method":"Biochemical co-immunoprecipitation/scaffold assembly assay, Nedd8 ligase activity assay, site-directed mutagenesis (C1341), double knockout mouse phenotype, kidney development analysis","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis identifying catalytic cysteine, in vivo genetic rescue/phenotype, biochemical complex assembly, single lab with multiple orthogonal methods","pmids":["27119228"],"is_preprint":false},{"year":2016,"finding":"HECW2 physically interacts with AMOTL1 and stabilizes it via K63-linked ubiquitination. HECW2 depletion in human endothelial cells decreases AMOTL1 stability, loosens cell-to-cell junctions, relocalizes YAP from cytoplasm to nucleus, and increases angiogenic sprouting—an effect blocked by ANG-2 depletion.","method":"Co-immunoprecipitation, ubiquitination assay specifying K63-linkage, siRNA knockdown, YAP subcellular localization (imaging), sprouting angiogenesis assay, epistasis (ANG-2 co-depletion)","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, K63 linkage-specific ubiquitination assay, functional KD with defined pathway (YAP/ANG-2) and cellular phenotype, single lab multiple orthogonal methods","pmids":["27498087"],"is_preprint":false},{"year":2018,"finding":"HECW2 interacts with PCNA via a canonical PIP motif and with lamin B1, ubiquitinates both, and targets them for proteasomal degradation. HECW2 also interacts with wild-type lamin A and ubiquitinates it, but this interaction is reduced with laminopathy-causing lamin A mutants (G232E, Q294P).","method":"Co-immunoprecipitation, PIP-motif-dependent interaction assay, ubiquitination assay, proteasome inhibitor rescue, lamin A mutant cell lines","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, proteasome inhibitor rescue, and mutant cell-line comparison in single lab","pmids":["29753763"],"is_preprint":false},{"year":2018,"finding":"HECW2 interacts with HP1α and HP1β (but not HP1γ), ubiquitinates them, and targets them for proteasomal degradation; HECW2 downregulation increases steady-state levels of HP1α and HP1β.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown with western blot quantification of HP1 isoforms","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and KD rescue, single lab with two orthogonal approaches","pmids":["30208514"],"is_preprint":false},{"year":2021,"finding":"HECW2 (NEDL2) localizes to nuclear foci in germinal vesicles of immature oocytes, to pronuclei of zygotes, and to nuclei of blastomeres. Multiple protein isoforms exist with distinct tissue distributions (52 kDa in germ cells/embryos; 91/136/155 kDa in somatic cells). Microinjection of anti-NEDL2 antibody into porcine oocytes inhibited sperm DNA decondensation and male pronuclear formation without affecting fertilization rate or blastocyst formation.","method":"Western blot isoform analysis, immunofluorescence localization across developmental stages, antibody microinjection functional assay in porcine IVF","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence (antibody injection phenotype), single lab, two orthogonal methods","pmids":["33030211"],"is_preprint":false},{"year":2023,"finding":"HECW2 promotes colorectal cancer progression and chemoresistance by mediating ubiquitin-proteasome degradation of lamin B1, which activates the AKT/mTOR signaling pathway.","method":"HECW2 knockdown and overexpression in CRC cells, ubiquitination assay of lamin B1, AKT/mTOR pathway analysis by western blot, functional assays (proliferation, chemoresistance)","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — KD/OE with defined pathway placement (AKT/mTOR via lamin B1 degradation) and ubiquitination assay, single lab","pmids":["37781079"],"is_preprint":false},{"year":2025,"finding":"HECW2 regulates ubiquitination of ALKBH5 (an m6A RNA demethylase), which subsequently enhances LDHA expression through ALKBH5-mediated m6A demethylation of LDHA mRNA, promoting glycolysis in hemangioma endothelial cells.","method":"HECW2 overexpression/knockdown, ubiquitination assay of ALKBH5, m6A demethylation readout on LDHA mRNA, functional glycolysis assays (glucose uptake, lactate, ATP), in vivo xenograft model","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ubiquitination assay, pathway epistasis (LDHA OE rescue), in vivo validation; single lab","pmids":["40520873"],"is_preprint":false}],"current_model":"HECW2 (NEDL2) is a HECT-type E3 ubiquitin ligase that also possesses intrinsic Nedd8 ligase activity (catalytic cysteine C1341); it stabilizes p73 via non-degradative ubiquitination to enhance p73-dependent transcription, mediates K63-linked ubiquitination of AMOTL1 to maintain endothelial junctions and restrain YAP nuclear activity, ubiquitinates and degrades PCNA, lamin B1, HP1α, HP1β, and ALKBH5, acts as a scaffold to assemble and activate the GDNF/Ret/Akt signaling complex in enteric neural development, and is itself degraded by APC/C-Cdh1 via a destruction box during mitotic exit to regulate the metaphase-to-anaphase transition."},"narrative":{"mechanistic_narrative":"HECW2 (NEDL2) is a HECT-domain E3 ligase that uses its WW domains to engage PY- and PIP-motif-bearing substrates and exerts dual catalytic outputs—classical ubiquitin transfer and an intrinsic Nedd8 ligase activity dependent on catalytic cysteine 1341 [PMID:27119228]. Its ubiquitination outcomes are substrate-dependent: it ubiquitinates p73 yet stabilizes it to enhance p73-driven transcription [PMID:12890487], deposits non-degradative K63-linked chains on AMOTL1 to reinforce endothelial cell-cell junctions and retain YAP in the cytoplasm, restraining angiogenic sprouting [PMID:27498087], and conversely directs proteasomal degradation of PCNA, lamin B1, lamin A, and the heterochromatin proteins HP1α and HP1β [PMID:29753763, PMID:30208514]. Beyond catalysis, HECW2 functions as a scaffold that assembles a SHC/Grb2/PI3K/PDK1/Akt complex downstream of GDNF/Ret, and its Nedd8 ligase activity—not its ubiquitin ligase activity—is required for GDNF-stimulated Akt signaling and for enteric nervous system and kidney development in vivo [PMID:25555806, PMID:27119228]. HECW2 is itself cell-cycle-regulated: APC/C-Cdh1 recognizes a destruction box and targets it for degradation at mitotic exit, and HECW2 associates with mitotic spindles and influences the metaphase-to-anaphase transition [PMID:24163370]. In disease contexts, HECW2-mediated lamin B1 degradation activates AKT/mTOR to promote colorectal cancer progression and chemoresistance [PMID:37781079], and HECW2 ubiquitination of the m6A demethylase ALKBH5 drives LDHA-dependent glycolysis in hemangioma endothelial cells [PMID:40520873].","teleology":[{"year":2003,"claim":"Established the founding paradigm that HECW2 is an E3 ligase whose ubiquitination of a substrate can stabilize rather than degrade it, coupling HECW2 to p73 transcriptional output.","evidence":"Reciprocal Co-IP, in vitro pull-down and ubiquitination assays, stability/decay measurements, and transcriptional reporter assays for the HECW2–p73 interaction","pmids":["12890487"],"confidence":"Medium","gaps":["Chain linkage type and the mechanism by which ubiquitination stabilizes p73 not resolved","No in vivo confirmation of the HECW2–p73 axis"]},{"year":2013,"claim":"Placed HECW2 in the cell cycle by showing it is a degradation substrate of APC/C-Cdh1 via a destruction box and that its levels and spindle association couple it to mitotic progression.","evidence":"MS-identified interaction, reciprocal Co-IP in vivo and in vitro, destruction-box mutant analysis, and knockdown/overexpression mitotic phenotypes","pmids":["24163370"],"confidence":"High","gaps":["Mitotic substrates of HECW2 not identified","Mechanism linking HECW2 to metaphase delay and chromosomal lagging unresolved"]},{"year":2014,"claim":"Demonstrated a required physiological role for HECW2 by showing knockout mice develop intestinal aganglionosis, tying HECW2 to GDNF/Ret/Akt-driven enteric neural precursor proliferation.","evidence":"HECW2 knockout mouse with histological/functional intestinal readouts and Akt phosphorylation analysis","pmids":["25555806"],"confidence":"High","gaps":["Molecular mechanism of pathway engagement not defined in this study","Catalytic activity requirement not yet dissected"]},{"year":2016,"claim":"Resolved the GDNF mechanism as scaffold assembly and uncovered an intrinsic Nedd8 ligase activity (Cys1341), showing neddylation—not ubiquitination—drives Akt activation and development.","evidence":"Scaffold co-IP assembly assays, Nedd8 ligase assay, C1341 site-directed mutagenesis, and double-knockout mouse ENS/kidney phenotypes","pmids":["27119228"],"confidence":"High","gaps":["Neddylation target(s) within the GDNF complex not identified","How a single protein toggles between ubiquitin and Nedd8 ligase outputs unknown"]},{"year":2016,"claim":"Extended the stabilizing-ubiquitination paradigm to endothelial biology, showing K63-linked ubiquitination of AMOTL1 maintains junctions and restrains YAP nuclear translocation and angiogenesis.","evidence":"Reciprocal Co-IP, K63-linkage-specific ubiquitination assay, siRNA knockdown, YAP localization imaging, sprouting assay, and ANG-2 epistasis","pmids":["27498087"],"confidence":"High","gaps":["Direct mechanistic link between AMOTL1 K63 chains and YAP sequestration not fully mapped","Single-lab evidence"]},{"year":2018,"claim":"Identified degradative substrates of HECW2—PCNA (via a PIP motif), lamin B1, lamin A, HP1α and HP1β—broadening its role to nuclear architecture and heterochromatin protein turnover.","evidence":"Co-IP, PIP-motif-dependent interaction, ubiquitination assays, proteasome inhibitor rescue, lamin A mutant comparison, and HP1-isoform knockdown westerns","pmids":["29753763","30208514"],"confidence":"Medium","gaps":["Cellular consequences of substrate degradation not established","Reduced binding to laminopathy mutants not linked to disease phenotype"]},{"year":2021,"claim":"Localized HECW2 to germinal-vesicle and pronuclear nuclei and implicated it functionally in sperm DNA decondensation and male pronuclear formation, with distinct tissue-specific isoforms.","evidence":"Western-blot isoform analysis, immunofluorescence across developmental stages, and anti-NEDL2 antibody microinjection in porcine IVF","pmids":["33030211"],"confidence":"Medium","gaps":["Substrate mediating pronuclear formation unknown","Functional roles of the distinct isoforms not characterized"]},{"year":2023,"claim":"Connected HECW2 substrate degradation to oncogenic signaling, showing lamin B1 degradation activates AKT/mTOR to drive colorectal cancer progression and chemoresistance.","evidence":"HECW2 knockdown/overexpression in CRC cells, lamin B1 ubiquitination assay, AKT/mTOR western blots, and proliferation/chemoresistance assays","pmids":["37781079"],"confidence":"Medium","gaps":["Mechanistic link between lamin B1 loss and AKT/mTOR activation not detailed","Single-lab evidence"]},{"year":2025,"claim":"Linked HECW2 to RNA metabolism and tumor metabolism by showing it ubiquitinates the m6A demethylase ALKBH5, which enhances LDHA expression and glycolysis in hemangioma endothelium.","evidence":"HECW2 overexpression/knockdown, ALKBH5 ubiquitination assay, m6A demethylation readout on LDHA mRNA, glycolysis assays, and xenograft model","pmids":["40520873"],"confidence":"Medium","gaps":["Whether ALKBH5 ubiquitination is degradative or activating not clarified","Single-lab evidence"]},{"year":null,"claim":"How HECW2 selects between stabilizing (non-degradative/K63) and degradative ubiquitination, and between ubiquitin and Nedd8 ligase outputs, across its diverse substrates remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of HECW2 catalytic switching","Substrate-specific cofactors or regulatory inputs unknown","Neddylation substrates largely unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,5,6,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,5,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3]}],"complexes":["GDNF/Ret/PI3K/Akt signaling complex"],"partners":["TP73","AMOTL1","PCNA","LMNB1","LMNA","CBX5","ALKBH5","FZR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P2P5","full_name":"E3 ubiquitin-protein ligase HECW2","aliases":["HECT, C2 and WW domain-containing protein 2","HECT-type E3 ubiquitin transferase HECW2","NEDD4-like E3 ubiquitin-protein ligase 2"],"length_aa":1572,"mass_kda":175.8,"function":"E3 ubiquitin-protein ligase that mediates ubiquitination of TP73. Acts to stabilize TP73 and enhance activation of transcription by TP73 (PubMed:12890487). Involved in the regulation of mitotic metaphase/anaphase transition (PubMed:24163370)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton, spindle","url":"https://www.uniprot.org/uniprotkb/Q9P2P5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HECW2","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/HECW2","total_profiled":1310},"omim":[{"mim_id":"617268","title":"NEURODEVELOPMENTAL DISORDER WITH HYPOTONIA, SEIZURES, AND ABSENT LANGUAGE; NDHSAL","url":"https://www.omim.org/entry/617268"},{"mim_id":"617245","title":"HECT, C2, AND WW DOMAINS-CONTAINING E3 UBIQUITIN-PROTEIN LIGASE 2; HECW2","url":"https://www.omim.org/entry/617245"},{"mim_id":"606234","title":"IKAROS FAMILY ZINC FINGER 2; IKZF2","url":"https://www.omim.org/entry/606234"},{"mim_id":"601990","title":"TUMOR PROTEIN p73; TP73","url":"https://www.omim.org/entry/601990"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HECW2"},"hgnc":{"alias_symbol":["KIAA1301","NEDL2"],"prev_symbol":[]},"alphafold":{"accession":"Q9P2P5","domains":[{"cath_id":"2.60.40.2840","chopping":"61-166","consensus_level":"high","plddt":79.8105,"start":61,"end":166},{"cath_id":"2.60.40.150","chopping":"186-324","consensus_level":"high","plddt":78.9084,"start":186,"end":324},{"cath_id":"2.20.70.10","chopping":"927-1013","consensus_level":"medium","plddt":90.0939,"start":927,"end":1013},{"cath_id":"-","chopping":"1082-1149","consensus_level":"high","plddt":88.9221,"start":1082,"end":1149},{"cath_id":"3.90.1750.10","chopping":"1191-1365_1400-1453","consensus_level":"medium","plddt":86.5341,"start":1191,"end":1453},{"cath_id":"3.30.2410.10","chopping":"1454-1572","consensus_level":"medium","plddt":82.5696,"start":1454,"end":1572}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2P5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2P5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2P5-F1-predicted_aligned_error_v6.png","plddt_mean":60.41},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HECW2","jax_strain_url":"https://www.jax.org/strain/search?query=HECW2"},"sequence":{"accession":"Q9P2P5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P2P5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P2P5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2P5"}},"corpus_meta":[{"pmid":"12890487","id":"PMC_12890487","title":"A novel HECT-type E3 ubiquitin ligase, NEDL2, stabilizes p73 and enhances its transcriptional activity.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12890487","citation_count":86,"is_preprint":false},{"pmid":"29260931","id":"PMC_29260931","title":"Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition.","date":"2018","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/29260931","citation_count":83,"is_preprint":false},{"pmid":"27334371","id":"PMC_27334371","title":"Mutations in HECW2 are associated with intellectual disability and epilepsy.","date":"2016","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27334371","citation_count":59,"is_preprint":false},{"pmid":"27498087","id":"PMC_27498087","title":"The endothelial E3 ligase HECW2 promotes endothelial cell junctions by increasing AMOTL1 protein stability via K63-linked ubiquitination.","date":"2016","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/27498087","citation_count":44,"is_preprint":false},{"pmid":"24163370","id":"PMC_24163370","title":"The HECT type ubiquitin ligase NEDL2 is degraded by anaphase-promoting complex/cyclosome (APC/C)-Cdh1, and its tight regulation maintains the metaphase to anaphase transition.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24163370","citation_count":40,"is_preprint":false},{"pmid":"29753763","id":"PMC_29753763","title":"E3 ubiquitin ligase HECW2 targets PCNA and lamin B1.","date":"2018","source":"Biochimica et biophysica acta. 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Gly1452Ser) in a Chinese Patient with Developmental Delay, Neurodevelopmental Delay, and Hypotonia.","date":"2025","source":"Molecular syndromology","url":"https://pubmed.ncbi.nlm.nih.gov/40469502","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16787,"output_tokens":2828,"usd":0.046391,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10036,"output_tokens":3599,"usd":0.070078,"stage2_stop_reason":"end_turn"},"total_usd":0.116469,"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\": 2003,\n      \"finding\": \"HECW2 (NEDL2) physically interacts with p73 via its WW domains recognizing PY motifs on p73, ubiquitinates p73, and paradoxically stabilizes rather than degrades it, thereby enhancing p73-dependent transcriptional activation.\",\n      \"method\": \"Reciprocal co-immunoprecipitation, in vitro pull-down assay, in vitro ubiquitination assay, p73 stability/decay measurements, transcriptional reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus in vitro pull-down plus in vitro ubiquitination assay plus functional transcription assay, single lab\",\n      \"pmids\": [\"12890487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HECW2 (NEDL2) is a substrate of APC/C-Cdh1 ubiquitin ligase; Cdh1 recognizes a destruction box (R740GSL743) on NEDL2 and targets it for proteasomal degradation during mitotic exit. NEDL2 associates with mitotic spindles, its protein level peaks in mitosis, and its depletion prolongs metaphase while overexpression induces chromosomal lagging.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, in vitro interaction assay, overexpression and knockdown experiments with mitotic phenotype readouts, destruction-box mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (MS-identified interaction, reciprocal Co-IP in vivo and in vitro, destruction-box mutant, KD/OE phenotypes) in a single rigorous study\",\n      \"pmids\": [\"24163370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HECW2 (NEDL2) positively regulates enteric neural precursor proliferation through the GDNF/Ret/Akt signaling pathway; NEDL2-deficient mice display intestinal aganglionosis and postnatal lethality, establishing a required in vivo role.\",\n      \"method\": \"NEDL2 knockout mouse model with histological and functional readout (intestinal aganglionosis, bowel motility), signaling pathway analysis (Akt phosphorylation)\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout mouse with defined cellular phenotype (aganglionosis) and pathway placement (GDNF/Akt), replicated in follow-up study (PMID:27119228)\",\n      \"pmids\": [\"25555806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HECW2 (NEDL2) acts as a scaffold to recruit SHC, Grb2, PI3K (p110/p85), PDK1, and Akt, promoting GDNF-stimulated Akt signaling. Additionally, NEDL2 harbors intrinsic Nedd8 ligase activity with cysteine 1341 as the catalytic site, and Nedd8 ligase—but not ubiquitin ligase—activity is required for GDNF-stimulated Akt activation and ENS/kidney development.\",\n      \"method\": \"Biochemical co-immunoprecipitation/scaffold assembly assay, Nedd8 ligase activity assay, site-directed mutagenesis (C1341), double knockout mouse phenotype, kidney development analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis identifying catalytic cysteine, in vivo genetic rescue/phenotype, biochemical complex assembly, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27119228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HECW2 physically interacts with AMOTL1 and stabilizes it via K63-linked ubiquitination. HECW2 depletion in human endothelial cells decreases AMOTL1 stability, loosens cell-to-cell junctions, relocalizes YAP from cytoplasm to nucleus, and increases angiogenic sprouting—an effect blocked by ANG-2 depletion.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay specifying K63-linkage, siRNA knockdown, YAP subcellular localization (imaging), sprouting angiogenesis assay, epistasis (ANG-2 co-depletion)\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, K63 linkage-specific ubiquitination assay, functional KD with defined pathway (YAP/ANG-2) and cellular phenotype, single lab multiple orthogonal methods\",\n      \"pmids\": [\"27498087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HECW2 interacts with PCNA via a canonical PIP motif and with lamin B1, ubiquitinates both, and targets them for proteasomal degradation. HECW2 also interacts with wild-type lamin A and ubiquitinates it, but this interaction is reduced with laminopathy-causing lamin A mutants (G232E, Q294P).\",\n      \"method\": \"Co-immunoprecipitation, PIP-motif-dependent interaction assay, ubiquitination assay, proteasome inhibitor rescue, lamin A mutant cell lines\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, proteasome inhibitor rescue, and mutant cell-line comparison in single lab\",\n      \"pmids\": [\"29753763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HECW2 interacts with HP1α and HP1β (but not HP1γ), ubiquitinates them, and targets them for proteasomal degradation; HECW2 downregulation increases steady-state levels of HP1α and HP1β.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown with western blot quantification of HP1 isoforms\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and KD rescue, single lab with two orthogonal approaches\",\n      \"pmids\": [\"30208514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HECW2 (NEDL2) localizes to nuclear foci in germinal vesicles of immature oocytes, to pronuclei of zygotes, and to nuclei of blastomeres. Multiple protein isoforms exist with distinct tissue distributions (52 kDa in germ cells/embryos; 91/136/155 kDa in somatic cells). Microinjection of anti-NEDL2 antibody into porcine oocytes inhibited sperm DNA decondensation and male pronuclear formation without affecting fertilization rate or blastocyst formation.\",\n      \"method\": \"Western blot isoform analysis, immunofluorescence localization across developmental stages, antibody microinjection functional assay in porcine IVF\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence (antibody injection phenotype), single lab, two orthogonal methods\",\n      \"pmids\": [\"33030211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HECW2 promotes colorectal cancer progression and chemoresistance by mediating ubiquitin-proteasome degradation of lamin B1, which activates the AKT/mTOR signaling pathway.\",\n      \"method\": \"HECW2 knockdown and overexpression in CRC cells, ubiquitination assay of lamin B1, AKT/mTOR pathway analysis by western blot, functional assays (proliferation, chemoresistance)\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — KD/OE with defined pathway placement (AKT/mTOR via lamin B1 degradation) and ubiquitination assay, single lab\",\n      \"pmids\": [\"37781079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HECW2 regulates ubiquitination of ALKBH5 (an m6A RNA demethylase), which subsequently enhances LDHA expression through ALKBH5-mediated m6A demethylation of LDHA mRNA, promoting glycolysis in hemangioma endothelial cells.\",\n      \"method\": \"HECW2 overexpression/knockdown, ubiquitination assay of ALKBH5, m6A demethylation readout on LDHA mRNA, functional glycolysis assays (glucose uptake, lactate, ATP), in vivo xenograft model\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ubiquitination assay, pathway epistasis (LDHA OE rescue), in vivo validation; single lab\",\n      \"pmids\": [\"40520873\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HECW2 (NEDL2) is a HECT-type E3 ubiquitin ligase that also possesses intrinsic Nedd8 ligase activity (catalytic cysteine C1341); it stabilizes p73 via non-degradative ubiquitination to enhance p73-dependent transcription, mediates K63-linked ubiquitination of AMOTL1 to maintain endothelial junctions and restrain YAP nuclear activity, ubiquitinates and degrades PCNA, lamin B1, HP1α, HP1β, and ALKBH5, acts as a scaffold to assemble and activate the GDNF/Ret/Akt signaling complex in enteric neural development, and is itself degraded by APC/C-Cdh1 via a destruction box during mitotic exit to regulate the metaphase-to-anaphase transition.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HECW2 (NEDL2) is a HECT-domain E3 ligase that uses its WW domains to engage PY- and PIP-motif-bearing substrates and exerts dual catalytic outputs—classical ubiquitin transfer and an intrinsic Nedd8 ligase activity dependent on catalytic cysteine 1341 [#3]. Its ubiquitination outcomes are substrate-dependent: it ubiquitinates p73 yet stabilizes it to enhance p73-driven transcription [#0], deposits non-degradative K63-linked chains on AMOTL1 to reinforce endothelial cell-cell junctions and retain YAP in the cytoplasm, restraining angiogenic sprouting [#4], and conversely directs proteasomal degradation of PCNA, lamin B1, lamin A, and the heterochromatin proteins HP1α and HP1β [#5, #6]. Beyond catalysis, HECW2 functions as a scaffold that assembles a SHC/Grb2/PI3K/PDK1/Akt complex downstream of GDNF/Ret, and its Nedd8 ligase activity—not its ubiquitin ligase activity—is required for GDNF-stimulated Akt signaling and for enteric nervous system and kidney development in vivo [#2, #3]. HECW2 is itself cell-cycle-regulated: APC/C-Cdh1 recognizes a destruction box and targets it for degradation at mitotic exit, and HECW2 associates with mitotic spindles and influences the metaphase-to-anaphase transition [#1]. In disease contexts, HECW2-mediated lamin B1 degradation activates AKT/mTOR to promote colorectal cancer progression and chemoresistance [#8], and HECW2 ubiquitination of the m6A demethylase ALKBH5 drives LDHA-dependent glycolysis in hemangioma endothelial cells [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the founding paradigm that HECW2 is an E3 ligase whose ubiquitination of a substrate can stabilize rather than degrade it, coupling HECW2 to p73 transcriptional output.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro pull-down and ubiquitination assays, stability/decay measurements, and transcriptional reporter assays for the HECW2–p73 interaction\",\n      \"pmids\": [\"12890487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Chain linkage type and the mechanism by which ubiquitination stabilizes p73 not resolved\", \"No in vivo confirmation of the HECW2–p73 axis\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed HECW2 in the cell cycle by showing it is a degradation substrate of APC/C-Cdh1 via a destruction box and that its levels and spindle association couple it to mitotic progression.\",\n      \"evidence\": \"MS-identified interaction, reciprocal Co-IP in vivo and in vitro, destruction-box mutant analysis, and knockdown/overexpression mitotic phenotypes\",\n      \"pmids\": [\"24163370\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mitotic substrates of HECW2 not identified\", \"Mechanism linking HECW2 to metaphase delay and chromosomal lagging unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated a required physiological role for HECW2 by showing knockout mice develop intestinal aganglionosis, tying HECW2 to GDNF/Ret/Akt-driven enteric neural precursor proliferation.\",\n      \"evidence\": \"HECW2 knockout mouse with histological/functional intestinal readouts and Akt phosphorylation analysis\",\n      \"pmids\": [\"25555806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of pathway engagement not defined in this study\", \"Catalytic activity requirement not yet dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the GDNF mechanism as scaffold assembly and uncovered an intrinsic Nedd8 ligase activity (Cys1341), showing neddylation—not ubiquitination—drives Akt activation and development.\",\n      \"evidence\": \"Scaffold co-IP assembly assays, Nedd8 ligase assay, C1341 site-directed mutagenesis, and double-knockout mouse ENS/kidney phenotypes\",\n      \"pmids\": [\"27119228\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Neddylation target(s) within the GDNF complex not identified\", \"How a single protein toggles between ubiquitin and Nedd8 ligase outputs unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended the stabilizing-ubiquitination paradigm to endothelial biology, showing K63-linked ubiquitination of AMOTL1 maintains junctions and restrains YAP nuclear translocation and angiogenesis.\",\n      \"evidence\": \"Reciprocal Co-IP, K63-linkage-specific ubiquitination assay, siRNA knockdown, YAP localization imaging, sprouting assay, and ANG-2 epistasis\",\n      \"pmids\": [\"27498087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanistic link between AMOTL1 K63 chains and YAP sequestration not fully mapped\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified degradative substrates of HECW2—PCNA (via a PIP motif), lamin B1, lamin A, HP1α and HP1β—broadening its role to nuclear architecture and heterochromatin protein turnover.\",\n      \"evidence\": \"Co-IP, PIP-motif-dependent interaction, ubiquitination assays, proteasome inhibitor rescue, lamin A mutant comparison, and HP1-isoform knockdown westerns\",\n      \"pmids\": [\"29753763\", \"30208514\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular consequences of substrate degradation not established\", \"Reduced binding to laminopathy mutants not linked to disease phenotype\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Localized HECW2 to germinal-vesicle and pronuclear nuclei and implicated it functionally in sperm DNA decondensation and male pronuclear formation, with distinct tissue-specific isoforms.\",\n      \"evidence\": \"Western-blot isoform analysis, immunofluorescence across developmental stages, and anti-NEDL2 antibody microinjection in porcine IVF\",\n      \"pmids\": [\"33030211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate mediating pronuclear formation unknown\", \"Functional roles of the distinct isoforms not characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected HECW2 substrate degradation to oncogenic signaling, showing lamin B1 degradation activates AKT/mTOR to drive colorectal cancer progression and chemoresistance.\",\n      \"evidence\": \"HECW2 knockdown/overexpression in CRC cells, lamin B1 ubiquitination assay, AKT/mTOR western blots, and proliferation/chemoresistance assays\",\n      \"pmids\": [\"37781079\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between lamin B1 loss and AKT/mTOR activation not detailed\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked HECW2 to RNA metabolism and tumor metabolism by showing it ubiquitinates the m6A demethylase ALKBH5, which enhances LDHA expression and glycolysis in hemangioma endothelium.\",\n      \"evidence\": \"HECW2 overexpression/knockdown, ALKBH5 ubiquitination assay, m6A demethylation readout on LDHA mRNA, glycolysis assays, and xenograft model\",\n      \"pmids\": [\"40520873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ALKBH5 ubiquitination is degradative or activating not clarified\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HECW2 selects between stabilizing (non-degradative/K63) and degradative ubiquitination, and between ubiquitin and Nedd8 ligase outputs, across its diverse substrates remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of HECW2 catalytic switching\", \"Substrate-specific cofactors or regulatory inputs unknown\", \"Neddylation substrates largely unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 5, 6, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"GDNF/Ret/PI3K/Akt signaling complex\"],\n    \"partners\": [\"TP73\", \"AMOTL1\", \"PCNA\", \"LMNB1\", \"LMNA\", \"CBX5\", \"ALKBH5\", \"FZR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}