{"gene":"HECTD3","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2008,"finding":"HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo; overexpression of HECTD3 enhances ubiquitination of Tara and promotes its proteasomal turnover, while HECTD3 depletion decreases Tara degradation and leads to multipolar spindle formation.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, siRNA knockdown, ubiquitination assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus in vitro binding plus functional phenotype (multipolar spindles), single lab","pmids":["18194665"],"is_preprint":false},{"year":2008,"finding":"HECTD3 interacts with Syntaxin 8 (identified by yeast two-hybrid) and co-immunoprecipitation confirms direct interaction; overexpression of HECTD3 promotes ubiquitination of Syntaxin 8; HECTD3 and Syntaxin 8 share similar subcellular localization by immunofluorescence.","method":"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence, ubiquitination assay","journal":"Cellular and molecular neurobiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and overexpression ubiquitination assay, single lab, no mutagenesis or functional consequence established","pmids":["18821010"],"is_preprint":false},{"year":2013,"finding":"HECTD3 interacts with MALT1 via its N-terminal DOC domain (identified by yeast two-hybrid), promotes non-degradative polyubiquitination of MALT1, stabilizes MALT1 protein levels, and HECTD3 depletion decreases MALT1 and increases cisplatin-induced apoptosis; MALT1 overexpression partially rescues HECTD3 depletion-induced apoptosis.","method":"Yeast two-hybrid, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression rescue, domain mapping","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Y2H, Co-IP, ubiquitination, rescue), single lab","pmids":["23358872"],"is_preprint":false},{"year":2013,"finding":"HECTD3 interacts with caspase-8 death effector domains and ubiquitinates caspase-8 with K63-linked polyubiquitin chains at K215, which does not target caspase-8 for degradation but decreases caspase-8 activation; mutation of K215 abolishes HECTD3-mediated protection from TRAIL-induced caspase-8 cleavage; HECTD3 inhibits TRAIL-induced apoptosis in an E3 ligase activity-dependent manner.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K215 caspase-8), E3 ligase-dead mutant, TRAIL apoptosis assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination assay with mutagenesis of ubiquitination site, E3 ligase activity-dependent effect, multiple orthogonal methods, single lab","pmids":["24287696"],"is_preprint":false},{"year":2017,"finding":"HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain; HECTD3 is required for proteasomal degradation of the HSP90 client kinase CRAF (and also MASTL and LKB1) upon HSP90 ATPase inhibition; this DOC domain is mutationally disrupted in tumor cells with activated MAP kinase signaling.","method":"siRNA screen, co-immunoprecipitation, domain mapping (DOC domain), proteasomal degradation assay, tumor cell mutation analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA screen plus Co-IP plus domain mapping, single lab, multiple client kinases tested","pmids":["28636940"],"is_preprint":false},{"year":2017,"finding":"HECTD3 binds and ubiquitinates caspase-9, inhibiting caspase-9 oligomerization and its association with Apaf-1, thereby suppressing caspase-9 activation; this antiapoptotic function requires phosphorylation of HECTD3 at Thr-157 by ERK, as the T157A mutant is non-functional.","method":"Co-immunoprecipitation, ubiquitination assay, caspase-9 oligomerization assay, Apaf-1 interaction assay, site-directed mutagenesis (T157A), ERK kinase assay, xenograft model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including mutagenesis and in vivo xenograft, single lab","pmids":["28716524"],"is_preprint":false},{"year":2018,"finding":"HECTD3 mediates K63-linked polyubiquitination of TRAF3 at residue K138 via its catalytic HECT domain; this ubiquitination enables TRAF3-TBK1 complex formation and type I IFN induction during intracellular bacterial infection; Hectd3-deficient mice have impaired type I IFN response and enhanced bacterial clearance.","method":"Knockout mouse model, in vitro ubiquitination assay, site-directed mutagenesis (K138 TRAF3), co-immunoprecipitation (TRAF3-TBK1 complex), infection models (Francisella, Mycobacterium, Listeria)","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted ubiquitination with site-specific mutagenesis, knockout mouse phenotype, multiple bacterial models, complex formation demonstrated","pmids":["29920190"],"is_preprint":false},{"year":2019,"finding":"HECTD3 promotes K27-linked and K29-linked polyubiquitination of MALT1 (at K648) and K27-linked polyubiquitination of STAT3 (at K180); these non-degradative modifications promote STAT3 tyrosine-705 activating phosphorylation, NF-κB activation via MALT1, and pathogenic Th17 cell differentiation in EAE.","method":"Knockout mouse model (EAE), ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K648 MALT1, K180 STAT3), phosphorylation assay, Th17 differentiation assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — knockout mouse with disease model, site-specific mutagenesis on two substrates, linkage-specific ubiquitination, multiple orthogonal methods","pmids":["30741923"],"is_preprint":false},{"year":2021,"finding":"Both the DOC and HECT domains of HECTD3 directly interact with TRAF3; the catalytic Cys832 in the HECT domain promotes K63-linked polyubiquitination of TRAF3 at Lys138, increasing oxidative stress and activating NF-κB to induce ischemia-reperfusion injury.","method":"Domain mapping (DOC and HECT domains), co-immunoprecipitation, ubiquitination assay, Cys832 mutagenesis, hypoxia/reoxygenation cell model, rat DCD liver model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping plus catalytic site mutagenesis plus in vivo model, single lab, replicates TRAF3 K138 finding from earlier paper","pmids":["33627626"],"is_preprint":false},{"year":2022,"finding":"HECTD3 promotes K29-linked polyubiquitination of c-MYC through interaction between its DOC domain and the CP/bHLHZ domains of c-MYC; mutation of the catalytic Cys823 of HECTD3 reduces c-MYC polyubiquitination; this modification promotes gastric cancer cell proliferation.","method":"Co-immunoprecipitation, domain mapping, ubiquitination assay, site-directed mutagenesis (C823A HECTD3), cell proliferation assay","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping, mutagenesis, ubiquitination assay, single lab","pmids":["35397617"],"is_preprint":false},{"year":2022,"finding":"HECTD3 promotes K63-linked polyubiquitination of PARP1 (at K209 and K221) via interaction between its DOC domain and the DNA-binding domain of PARP1, stabilizing PARP1 expression; Cys823 mutation of HECTD3 reduces PARP1 polyubiquitination; EGFR-mediated signaling activates this process in glioblastoma.","method":"Co-immunoprecipitation, domain mapping, ubiquitination assay, site-directed mutagenesis (C823 HECTD3, K209/K221 PARP1), xenograft mouse model","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping, mutagenesis of both enzyme and substrate, in vivo model, single lab","pmids":["36088509"],"is_preprint":false},{"year":2022,"finding":"HECTD3 promotes stabilization of MALT1 to regulate JNK pathway (c-JUN/p-JNK upregulation) in high-glucose conditions; MALT1 overexpression attenuates neuroprotective effects of HECTD3 silencing, placing MALT1 downstream of HECTD3 in neuronal glucose toxicity.","method":"siRNA knockdown, HECTD3 knockout rat model, western blot, overexpression rescue (MALT1)","journal":"Archives of physiology and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — epistasis by rescue experiment, single lab, indirect mechanistic characterization of MALT1 stabilization","pmids":["35913790"],"is_preprint":false},{"year":2022,"finding":"HECTD3 promotes K27- and K63-linked polyubiquitination of IKKα at K296, stabilizing IKKα, promoting its nuclear localization and kinase activity, increasing H3 phosphorylation and NF-κB target gene transcription; endothelial HECTD3 knockout reduces tumor cell adhesion and lung colonization.","method":"Co-immunoprecipitation, ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K296 IKKα), nuclear fractionation, kinase activity assay, conditional knockout/knockin mouse models, metastasis assays","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific mutagenesis, linkage-specific ubiquitination, nuclear localization by fractionation, kinase activity, conditional mouse models, multiple orthogonal methods","pmids":["35918322"],"is_preprint":false},{"year":2022,"finding":"HECTD3 ubiquitinates LKB1 and positively regulates ZEB1 expression through LKB1 ubiquitination; ZEB1 overexpression abolishes the effects of HECTD3 knockdown on radiation resistance and migration in glioma cells, placing ZEB1 downstream of HECTD3-mediated LKB1 ubiquitination.","method":"siRNA knockdown, ubiquitination assay, overexpression rescue (ZEB1), xenograft mouse model","journal":"The European journal of neuroscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited mechanistic detail on ubiquitination linkage/site, rescue experiment for epistasis","pmids":["35768187"],"is_preprint":false},{"year":2023,"finding":"HECTD3 interacts with PKR and mediates K33-linked polyubiquitination of PKR (first identified non-proteolytic ubiquitin modification for PKR); this disrupts PKR dimerization and phosphorylation, preventing EIF2α activation (accelerating viral replication) while promoting PKR-IKK complex formation and inflammatory response.","method":"Co-immunoprecipitation, ubiquitination assay with K33-linkage-specific analysis, PKR dimerization assay, phosphorylation assay, EIF2α activation assay, PKR-IKK complex co-IP, HECTD3-deficient mouse model, viral infection model","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1 / Strong — K33-linkage-specific ubiquitination with functional consequences on dimerization, phosphorylation, and complex formation demonstrated, knockout mouse validation, single lab but multiple orthogonal methods","pmids":["37402711"],"is_preprint":false},{"year":2023,"finding":"HECTD3 promotes polyubiquitination and proteasomal degradation of SLC7A11; mutation of the catalytic Cys823 impairs SLC7A11 polyubiquitination; HECTD3 suppresses SLC7A11-mediated cystine uptake, enhancing ferroptosis in colon cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, Cys823 mutagenesis, protein half-life assay, ferroptosis assay, xenograft model","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of catalytic site, protein stability assay, functional ferroptosis readout, single lab","pmids":["37422058"],"is_preprint":false},{"year":2024,"finding":"HECTD3 collaborates with UbcH5b (E2 enzyme) to promote p62 ubiquitination and autophagy; HECTD3 deletion leads to p62 accumulation in the nucleus after irradiation, inhibiting RNF168-mediated DNA damage repair; the HECTD3/UbcH5b inhibitor PC3-15 blocks DNA damage repair and increases radiosensitivity.","method":"Co-immunoprecipitation, ubiquitination assay (with UbcH5b), subcellular fractionation (nuclear p62 accumulation), DNA damage repair assay, small molecule inhibitor (PC3-15), HECTD3 knockout cell lines","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — E2-E3 collaboration defined, nuclear localization by fractionation with functional consequence, pharmacological inhibitor validation, single lab","pmids":["39487119"],"is_preprint":false},{"year":2025,"finding":"HECTD3 ubiquitinates AKT-phosphorylated CMTM3 targeting it for proteasomal degradation; AKT1 directly phosphorylates CMTM3 at Ser181, and this phosphorylation is required for HECTD3 recognition; knockdown of HECTD3 or PI3K/AKT inhibition stabilizes CMTM3; non-phosphorylatable CMTM3-S181A resists HECTD3-mediated degradation.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (S181A CMTM3), kinase assay (AKT1), pharmacological PI3K/AKT inhibition, xenograft model","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-dependent ubiquitination with site-specific mutagenesis of both phosphorylation and recognition site, in vivo model, single lab","pmids":["40836897"],"is_preprint":false}],"current_model":"HECTD3 is a HECT-domain E3 ubiquitin ligase that uses its N-terminal DOC domain for substrate/HSP90 docking and its catalytic Cys (C823/C832) for ubiquitin transfer; it assembles non-degradative K27-, K29-, K33-, and K63-linked polyubiquitin chains on diverse substrates—including MALT1 (K648), STAT3 (K180), TRAF3 (K138), caspase-8 (K215), IKKα (K296), PARP1 (K209/K221), PKR (K33-linked), p62, and c-MYC—to modulate NF-κB and interferon signaling, Th17 differentiation, apoptosis resistance, viral replication, and metastasis; it also targets selected proteins (Tara, LKB1, CRAF/MASTL as HSP90 clients, SLC7A11, CMTM3) for proteasomal degradation, with its own activity regulated by ERK-mediated phosphorylation at Thr-157."},"narrative":{"mechanistic_narrative":"HECTD3 is a HECT-domain E3 ubiquitin ligase that controls inflammatory signaling, apoptosis resistance, and tumor cell behavior by assembling predominantly non-degradative polyubiquitin chains on a range of signaling proteins [PMID:24287696, PMID:29920190, PMID:30741923]. It engages substrates through an N-terminal DOC domain and transfers ubiquitin via a catalytic cysteine in its HECT domain (C823/C832), as demonstrated by catalytic-site mutagenesis that abolishes substrate ubiquitination [PMID:33627626, PMID:35397617, PMID:36088509]. A recurring theme is the building of atypical, signal-modifying chains: K63-linked ubiquitination of TRAF3 at K138 to drive TRAF3-TBK1 complex formation and type I interferon induction during bacterial infection [PMID:29920190], K27/K29-linked modification of MALT1 (K648) and K27-linked modification of STAT3 (K180) to promote NF-κB activation, STAT3 Tyr705 phosphorylation, and pathogenic Th17 differentiation [PMID:30741923], K27/K63-linked ubiquitination of IKKα at K296 to enhance its nuclear kinase activity and tumor metastasis [PMID:35918322], and K33-linked ubiquitination of PKR that blocks PKR dimerization and eIF2α activation while redirecting PKR into an IKK-associated inflammatory complex [PMID:37402711]. HECTD3 also restrains apoptosis: it K63-ubiquitinates caspase-8 at K215 to limit its activation and TRAIL-induced death in an E3-activity-dependent manner [PMID:24287696], and ubiquitinates caspase-9 to block its oligomerization and Apaf-1 association, a function gated by ERK-mediated phosphorylation of HECTD3 at Thr-157 [PMID:28716524]. In addition to stabilizing or activating substrates, HECTD3 directs selected proteins to proteasomal degradation, including the HSP90 client kinases CRAF, MASTL, and LKB1 [PMID:28636940] and the cystine transporter SLC7A11, the latter sensitizing colon cancer cells to ferroptosis [PMID:37422058]. Through these activities HECTD3 promotes c-MYC- and PARP1-dependent tumor proliferation and DNA repair [PMID:35397617, PMID:36088509], links to p62 ubiquitination and autophagy [PMID:39487119], and is itself a pharmacological target via the HECTD3/UbcH5b inhibitor PC3-15 [PMID:39487119].","teleology":[{"year":2008,"claim":"Established HECTD3 as a functional E3 ligase by identifying its first substrate, defining a degradative mode of action linked to mitotic spindle integrity.","evidence":"Yeast two-hybrid, reciprocal Co-IP, in vitro binding, siRNA, and ubiquitination assays on Tara (also Syntaxin 8 in a parallel study)","pmids":["18194665","18821010"],"confidence":"Medium","gaps":["Ubiquitin chain linkage on Tara not defined","Catalytic residue dependence not tested","Syntaxin 8 interaction lacked functional readout or mutagenesis"]},{"year":2013,"claim":"Revealed HECTD3's non-degradative ubiquitination mode and its anti-apoptotic role, showing it stabilizes MALT1 and ubiquitinates caspase-8 at K215 to blunt death receptor signaling.","evidence":"Y2H/DOC-domain mapping, Co-IP, linkage and site-specific ubiquitination, K215 and ligase-dead mutagenesis, TRAIL apoptosis and rescue assays","pmids":["23358872","24287696"],"confidence":"High","gaps":["Endogenous chain-type confirmation for MALT1 in this work","How non-degradative chains mechanistically block caspase-8 activation not resolved structurally"]},{"year":2017,"claim":"Defined two opposing branches of HECTD3 function—DOC-domain-dependent degradation of HSP90 client kinases versus ERK-phosphorylation-gated suppression of caspase-9 apoptosis.","evidence":"siRNA screen, Co-IP, DOC-domain mapping, proteasomal degradation assays (CRAF/MASTL/LKB1); caspase-9 oligomerization/Apaf-1 assays, T157A mutant, ERK kinase assay, xenografts","pmids":["28636940","28716524"],"confidence":"Medium","gaps":["Chain linkages on client kinases and caspase-9 not specified","Structural basis of DOC-domain client recognition unresolved","How Thr-157 phosphorylation alters ligase activity not mechanistically defined"]},{"year":2018,"claim":"Demonstrated HECTD3 drives innate antibacterial immunity through K63-linked TRAF3 ubiquitination enabling TRAF3-TBK1 complex assembly and type I IFN.","evidence":"Hectd3 knockout mice, in vitro ubiquitination with K138 mutagenesis, Co-IP of TRAF3-TBK1, Francisella/Mycobacterium/Listeria infection models","pmids":["29920190"],"confidence":"High","gaps":["E2 partner in this context not identified","Structural detail of K63-chain placement on TRAF3 not resolved"]},{"year":2019,"claim":"Showed HECTD3 builds K27/K29 chains on MALT1 and K27 chains on STAT3 to coordinately activate NF-κB and STAT3 and drive pathogenic Th17 autoimmunity.","evidence":"EAE knockout mouse model, linkage-specific ubiquitination, K648 MALT1 and K180 STAT3 mutagenesis, STAT3 phosphorylation and Th17 differentiation assays","pmids":["30741923"],"confidence":"High","gaps":["How K27/K29 chains are read by downstream effectors not defined","Whether MALT1 and STAT3 modification are coupled events unclear"]},{"year":2021,"claim":"Confirmed and extended the TRAF3 mechanism, mapping dual DOC/HECT engagement of TRAF3 and catalytic Cys832 dependence in oxidative stress-driven ischemia-reperfusion injury.","evidence":"Domain mapping, Co-IP, ubiquitination assays, Cys832 mutagenesis, hypoxia/reoxygenation and rat DCD liver models","pmids":["33627626"],"confidence":"Medium","gaps":["Tissue-specific regulation of HECTD3 in this context not defined","Single-lab in vivo model"]},{"year":2022,"claim":"Expanded the substrate repertoire across cancer contexts, establishing catalytic-cysteine-dependent ubiquitination of c-MYC (K29), PARP1 (K63, K209/K221), and IKKα (K27/K63, K296) to promote proliferation, DNA repair, and metastasis.","evidence":"Co-IP, DOC-domain mapping, linkage/site-specific ubiquitination, C823 and substrate-lysine mutagenesis, kinase activity and nuclear fractionation, conditional knockout/knockin mice and xenografts","pmids":["35397617","36088509","35918322"],"confidence":"High","gaps":["Upstream signals selecting among these substrates not fully defined","Whether one E2 supports all linkage types unknown"]},{"year":2022,"claim":"Linked HECTD3-MALT1 and HECTD3-LKB1 axes to disease phenotypes (neuronal glucose toxicity, glioma radioresistance) via epistasis, implicating JNK and ZEB1 as downstream effectors.","evidence":"siRNA, knockout rat/xenograft models, western blot, MALT1 and ZEB1 overexpression rescue","pmids":["35913790","35768187"],"confidence":"Low","gaps":["Ubiquitin linkage and site on LKB1 in this context not defined","Mechanism of MALT1 stabilization indirect","Epistasis inferred from rescue rather than direct biochemistry"]},{"year":2023,"claim":"Identified a novel K33-linked ubiquitination of PKR and a degradative role on SLC7A11, broadening HECTD3 control to antiviral/inflammatory signaling and ferroptosis.","evidence":"K33-linkage-specific ubiquitination, PKR dimerization/phosphorylation/eIF2α and PKR-IKK complex assays, knockout mouse and viral model; SLC7A11 C823 mutagenesis, half-life and ferroptosis assays, xenografts","pmids":["37402711","37422058"],"confidence":"High","gaps":["Structural basis distinguishing K33 vs degradative chains on different substrates unknown","Determinants of degradative versus non-degradative outcome unresolved"]},{"year":2024,"claim":"Defined a specific E2-E3 pairing (UbcH5b) and a druggable axis, linking HECTD3 to p62 ubiquitination, autophagy, and DNA damage repair through nuclear p62/RNF168 control.","evidence":"Co-IP, ubiquitination with UbcH5b, subcellular fractionation, DNA repair assays, PC3-15 inhibitor, HECTD3 knockout cells","pmids":["39487119"],"confidence":"Medium","gaps":["p62 chain linkage not specified","Generality of UbcH5b across other substrates not tested"]},{"year":2025,"claim":"Established phosphodegron-based substrate recognition, showing AKT1 phosphorylation of CMTM3 at Ser181 licenses HECTD3-mediated degradation.","evidence":"Co-IP, ubiquitination, S181A CMTM3 mutagenesis, AKT1 kinase assay, PI3K/AKT inhibition, xenografts","pmids":["40836897"],"confidence":"Medium","gaps":["Whether DOC domain directly reads phospho-Ser181 not structurally shown","Chain linkage on CMTM3 not defined"]},{"year":null,"claim":"The structural determinants that allow HECTD3 to choose between non-degradative (K27/K29/K33/K63) and degradative chain assembly on different substrates, and how DOC-domain engagement and Thr-157 phosphorylation specify these outcomes, remain unresolved.","evidence":"No structural or biochemical study in the corpus reconstitutes linkage-selection determinants","pmids":[],"confidence":"Medium","gaps":["No structure of HECTD3 DOC or HECT domain with substrate","Linkage-specificity mechanism undefined","Full E2 enzyme repertoire across substrates unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3,6,7,8,9,10,12,14]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3,6,8,9,10,12,15]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6,7,12,14,15,17]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[12,16]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,7,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,5,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,12,14]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,4,6,15,17]}],"complexes":[],"partners":["MALT1","TRAF3","STAT3","IKKΑ","CASPASE-8","PKR","C-MYC","UBCH5B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5T447","full_name":"E3 ubiquitin-protein ligase HECTD3","aliases":["HECT domain-containing protein 3","HECT-type E3 ubiquitin transferase HECTD3"],"length_aa":861,"mass_kda":97.1,"function":"E3 ubiquitin ligases accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. Mediates ubiquitination of TRIOBP and its subsequent proteasomal degradation, thus facilitating cell cycle progression by regulating the turn-over of TRIOBP. Mediates also ubiquitination of STX8 (By similarity)","subcellular_location":"Cytoplasm, perinuclear region","url":"https://www.uniprot.org/uniprotkb/Q5T447/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HECTD3","classification":"Not Classified","n_dependent_lines":15,"n_total_lines":1208,"dependency_fraction":0.012417218543046357},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HECTD3","total_profiled":1310},"omim":[{"mim_id":"618638","title":"HECT DOMAIN E3 UBIQUITIN PROTEIN LIGASE 3; HECTD3","url":"https://www.omim.org/entry/618638"}],"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/HECTD3"},"hgnc":{"alias_symbol":["FLJ21156"],"prev_symbol":[]},"alphafold":{"accession":"Q5T447","domains":[{"cath_id":"-","chopping":"12-41_175-230_381-449","consensus_level":"medium","plddt":86.8916,"start":12,"end":449},{"cath_id":"-","chopping":"47-74_92-154","consensus_level":"medium","plddt":80.9331,"start":47,"end":154},{"cath_id":"2.60.120.260","chopping":"239-372","consensus_level":"high","plddt":87.3247,"start":239,"end":372},{"cath_id":"3.30.2410.10","chopping":"750-847","consensus_level":"high","plddt":84.2421,"start":750,"end":847}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T447","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T447-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T447-F1-predicted_aligned_error_v6.png","plddt_mean":83.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HECTD3","jax_strain_url":"https://www.jax.org/strain/search?query=HECTD3"},"sequence":{"accession":"Q5T447","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5T447.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5T447/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T447"}},"corpus_meta":[{"pmid":"30741923","id":"PMC_30741923","title":"Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30741923","citation_count":71,"is_preprint":false},{"pmid":"24287696","id":"PMC_24287696","title":"The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8.","date":"2013","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/24287696","citation_count":56,"is_preprint":false},{"pmid":"29920190","id":"PMC_29920190","title":"HECTD3 mediates TRAF3 polyubiquitination and type I interferon induction during bacterial infection.","date":"2018","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/29920190","citation_count":53,"is_preprint":false},{"pmid":"27508098","id":"PMC_27508098","title":"MiR-153 promotes breast cancer cell apoptosis by targeting HECTD3.","date":"2016","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/27508098","citation_count":41,"is_preprint":false},{"pmid":"23358872","id":"PMC_23358872","title":"The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1.","date":"2013","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/23358872","citation_count":37,"is_preprint":false},{"pmid":"31637449","id":"PMC_31637449","title":"The role of E3 ubiquitin ligase HECTD3 in cancer and beyond.","date":"2019","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/31637449","citation_count":30,"is_preprint":false},{"pmid":"33627626","id":"PMC_33627626","title":"Hypothermic oxygenated perfusion inhibits HECTD3-mediated TRAF3 polyubiquitination to alleviate DCD liver ischemia-reperfusion injury.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33627626","citation_count":30,"is_preprint":false},{"pmid":"35918322","id":"PMC_35918322","title":"Targeting HECTD3-IKKα axis inhibits inflammation-related metastasis.","date":"2022","source":"Signal transduction and targeted therapy","url":"https://pubmed.ncbi.nlm.nih.gov/35918322","citation_count":29,"is_preprint":false},{"pmid":"18194665","id":"PMC_18194665","title":"The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18194665","citation_count":28,"is_preprint":false},{"pmid":"28636940","id":"PMC_28636940","title":"HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients.","date":"2017","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28636940","citation_count":22,"is_preprint":false},{"pmid":"28989055","id":"PMC_28989055","title":"Down-regulation of HECTD3 by HER2 inhibition makes serous ovarian cancer cells sensitive to platinum treatment.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/28989055","citation_count":22,"is_preprint":false},{"pmid":"28716524","id":"PMC_28716524","title":"The E3 ligase HECTD3 promotes esophageal squamous cell carcinoma (ESCC) growth and cell survival through targeting and inhibiting caspase-9 activation.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/28716524","citation_count":21,"is_preprint":false},{"pmid":"35397617","id":"PMC_35397617","title":"HECTD3 promotes gastric cancer progression by mediating the polyubiquitination of c-MYC.","date":"2022","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/35397617","citation_count":17,"is_preprint":false},{"pmid":"37422058","id":"PMC_37422058","title":"E3 ubiquitin ligase HECTD3 is a tumor suppressor and mediates the polyubiquitination of SLC7A11 to promote ferroptosis in colon cancer.","date":"2023","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/37422058","citation_count":17,"is_preprint":false},{"pmid":"36088509","id":"PMC_36088509","title":"HECTD3 regulates the tumourigenesis of glioblastoma by polyubiquitinating PARP1 and activating EGFR signalling pathway.","date":"2022","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/36088509","citation_count":16,"is_preprint":false},{"pmid":"18821010","id":"PMC_18821010","title":"Interaction between syntaxin 8 and HECTd3, a HECT domain ligase.","date":"2008","source":"Cellular and molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/18821010","citation_count":12,"is_preprint":false},{"pmid":"37402711","id":"PMC_37402711","title":"E3 ligase HECTD3 promotes RNA virus replication and virus-induced inflammation via K33-linked polyubiquitination of PKR.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/37402711","citation_count":11,"is_preprint":false},{"pmid":"35913790","id":"PMC_35913790","title":"HECTD3 promotes NLRP3 inflammasome and pyroptosis to exacerbate diabetes-related cognitive impairment by stabilising MALT1 to regulate JNK pathway.","date":"2022","source":"Archives of physiology and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35913790","citation_count":10,"is_preprint":false},{"pmid":"39487119","id":"PMC_39487119","title":"Targeting the HECTD3-p62 axis increases the radiosensitivity of triple negative breast cancer cells.","date":"2024","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/39487119","citation_count":7,"is_preprint":false},{"pmid":"35768187","id":"PMC_35768187","title":"HECTD3 enhances cell radiation resistance and migration by regulating LKB1 mediated ZEB1 in glioma.","date":"2022","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/35768187","citation_count":3,"is_preprint":false},{"pmid":"40836897","id":"PMC_40836897","title":"HECTD3 E3 ligase mediates ubiquitination of AKT-phosphorylated CMTM3 in HER2-overexpressed breast cancer cells.","date":"2025","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/40836897","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11950,"output_tokens":4889,"usd":0.054593,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12953,"output_tokens":4554,"usd":0.089307,"stage2_stop_reason":"end_turn"},"total_usd":0.1439,"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\": 2008,\n      \"finding\": \"HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo; overexpression of HECTD3 enhances ubiquitination of Tara and promotes its proteasomal turnover, while HECTD3 depletion decreases Tara degradation and leads to multipolar spindle formation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding, siRNA knockdown, ubiquitination assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus in vitro binding plus functional phenotype (multipolar spindles), single lab\",\n      \"pmids\": [\"18194665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HECTD3 interacts with Syntaxin 8 (identified by yeast two-hybrid) and co-immunoprecipitation confirms direct interaction; overexpression of HECTD3 promotes ubiquitination of Syntaxin 8; HECTD3 and Syntaxin 8 share similar subcellular localization by immunofluorescence.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence, ubiquitination assay\",\n      \"journal\": \"Cellular and molecular neurobiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and overexpression ubiquitination assay, single lab, no mutagenesis or functional consequence established\",\n      \"pmids\": [\"18821010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HECTD3 interacts with MALT1 via its N-terminal DOC domain (identified by yeast two-hybrid), promotes non-degradative polyubiquitination of MALT1, stabilizes MALT1 protein levels, and HECTD3 depletion decreases MALT1 and increases cisplatin-induced apoptosis; MALT1 overexpression partially rescues HECTD3 depletion-induced apoptosis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression rescue, domain mapping\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Y2H, Co-IP, ubiquitination, rescue), single lab\",\n      \"pmids\": [\"23358872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HECTD3 interacts with caspase-8 death effector domains and ubiquitinates caspase-8 with K63-linked polyubiquitin chains at K215, which does not target caspase-8 for degradation but decreases caspase-8 activation; mutation of K215 abolishes HECTD3-mediated protection from TRAIL-induced caspase-8 cleavage; HECTD3 inhibits TRAIL-induced apoptosis in an E3 ligase activity-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K215 caspase-8), E3 ligase-dead mutant, TRAIL apoptosis assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination assay with mutagenesis of ubiquitination site, E3 ligase activity-dependent effect, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"24287696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain; HECTD3 is required for proteasomal degradation of the HSP90 client kinase CRAF (and also MASTL and LKB1) upon HSP90 ATPase inhibition; this DOC domain is mutationally disrupted in tumor cells with activated MAP kinase signaling.\",\n      \"method\": \"siRNA screen, co-immunoprecipitation, domain mapping (DOC domain), proteasomal degradation assay, tumor cell mutation analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA screen plus Co-IP plus domain mapping, single lab, multiple client kinases tested\",\n      \"pmids\": [\"28636940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HECTD3 binds and ubiquitinates caspase-9, inhibiting caspase-9 oligomerization and its association with Apaf-1, thereby suppressing caspase-9 activation; this antiapoptotic function requires phosphorylation of HECTD3 at Thr-157 by ERK, as the T157A mutant is non-functional.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, caspase-9 oligomerization assay, Apaf-1 interaction assay, site-directed mutagenesis (T157A), ERK kinase assay, xenograft model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including mutagenesis and in vivo xenograft, single lab\",\n      \"pmids\": [\"28716524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HECTD3 mediates K63-linked polyubiquitination of TRAF3 at residue K138 via its catalytic HECT domain; this ubiquitination enables TRAF3-TBK1 complex formation and type I IFN induction during intracellular bacterial infection; Hectd3-deficient mice have impaired type I IFN response and enhanced bacterial clearance.\",\n      \"method\": \"Knockout mouse model, in vitro ubiquitination assay, site-directed mutagenesis (K138 TRAF3), co-immunoprecipitation (TRAF3-TBK1 complex), infection models (Francisella, Mycobacterium, Listeria)\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted ubiquitination with site-specific mutagenesis, knockout mouse phenotype, multiple bacterial models, complex formation demonstrated\",\n      \"pmids\": [\"29920190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HECTD3 promotes K27-linked and K29-linked polyubiquitination of MALT1 (at K648) and K27-linked polyubiquitination of STAT3 (at K180); these non-degradative modifications promote STAT3 tyrosine-705 activating phosphorylation, NF-κB activation via MALT1, and pathogenic Th17 cell differentiation in EAE.\",\n      \"method\": \"Knockout mouse model (EAE), ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K648 MALT1, K180 STAT3), phosphorylation assay, Th17 differentiation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — knockout mouse with disease model, site-specific mutagenesis on two substrates, linkage-specific ubiquitination, multiple orthogonal methods\",\n      \"pmids\": [\"30741923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Both the DOC and HECT domains of HECTD3 directly interact with TRAF3; the catalytic Cys832 in the HECT domain promotes K63-linked polyubiquitination of TRAF3 at Lys138, increasing oxidative stress and activating NF-κB to induce ischemia-reperfusion injury.\",\n      \"method\": \"Domain mapping (DOC and HECT domains), co-immunoprecipitation, ubiquitination assay, Cys832 mutagenesis, hypoxia/reoxygenation cell model, rat DCD liver model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping plus catalytic site mutagenesis plus in vivo model, single lab, replicates TRAF3 K138 finding from earlier paper\",\n      \"pmids\": [\"33627626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HECTD3 promotes K29-linked polyubiquitination of c-MYC through interaction between its DOC domain and the CP/bHLHZ domains of c-MYC; mutation of the catalytic Cys823 of HECTD3 reduces c-MYC polyubiquitination; this modification promotes gastric cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ubiquitination assay, site-directed mutagenesis (C823A HECTD3), cell proliferation assay\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping, mutagenesis, ubiquitination assay, single lab\",\n      \"pmids\": [\"35397617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HECTD3 promotes K63-linked polyubiquitination of PARP1 (at K209 and K221) via interaction between its DOC domain and the DNA-binding domain of PARP1, stabilizing PARP1 expression; Cys823 mutation of HECTD3 reduces PARP1 polyubiquitination; EGFR-mediated signaling activates this process in glioblastoma.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ubiquitination assay, site-directed mutagenesis (C823 HECTD3, K209/K221 PARP1), xenograft mouse model\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping, mutagenesis of both enzyme and substrate, in vivo model, single lab\",\n      \"pmids\": [\"36088509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HECTD3 promotes stabilization of MALT1 to regulate JNK pathway (c-JUN/p-JNK upregulation) in high-glucose conditions; MALT1 overexpression attenuates neuroprotective effects of HECTD3 silencing, placing MALT1 downstream of HECTD3 in neuronal glucose toxicity.\",\n      \"method\": \"siRNA knockdown, HECTD3 knockout rat model, western blot, overexpression rescue (MALT1)\",\n      \"journal\": \"Archives of physiology and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — epistasis by rescue experiment, single lab, indirect mechanistic characterization of MALT1 stabilization\",\n      \"pmids\": [\"35913790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HECTD3 promotes K27- and K63-linked polyubiquitination of IKKα at K296, stabilizing IKKα, promoting its nuclear localization and kinase activity, increasing H3 phosphorylation and NF-κB target gene transcription; endothelial HECTD3 knockout reduces tumor cell adhesion and lung colonization.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K296 IKKα), nuclear fractionation, kinase activity assay, conditional knockout/knockin mouse models, metastasis assays\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific mutagenesis, linkage-specific ubiquitination, nuclear localization by fractionation, kinase activity, conditional mouse models, multiple orthogonal methods\",\n      \"pmids\": [\"35918322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HECTD3 ubiquitinates LKB1 and positively regulates ZEB1 expression through LKB1 ubiquitination; ZEB1 overexpression abolishes the effects of HECTD3 knockdown on radiation resistance and migration in glioma cells, placing ZEB1 downstream of HECTD3-mediated LKB1 ubiquitination.\",\n      \"method\": \"siRNA knockdown, ubiquitination assay, overexpression rescue (ZEB1), xenograft mouse model\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited mechanistic detail on ubiquitination linkage/site, rescue experiment for epistasis\",\n      \"pmids\": [\"35768187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HECTD3 interacts with PKR and mediates K33-linked polyubiquitination of PKR (first identified non-proteolytic ubiquitin modification for PKR); this disrupts PKR dimerization and phosphorylation, preventing EIF2α activation (accelerating viral replication) while promoting PKR-IKK complex formation and inflammatory response.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with K33-linkage-specific analysis, PKR dimerization assay, phosphorylation assay, EIF2α activation assay, PKR-IKK complex co-IP, HECTD3-deficient mouse model, viral infection model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — K33-linkage-specific ubiquitination with functional consequences on dimerization, phosphorylation, and complex formation demonstrated, knockout mouse validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37402711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HECTD3 promotes polyubiquitination and proteasomal degradation of SLC7A11; mutation of the catalytic Cys823 impairs SLC7A11 polyubiquitination; HECTD3 suppresses SLC7A11-mediated cystine uptake, enhancing ferroptosis in colon cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Cys823 mutagenesis, protein half-life assay, ferroptosis assay, xenograft model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of catalytic site, protein stability assay, functional ferroptosis readout, single lab\",\n      \"pmids\": [\"37422058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HECTD3 collaborates with UbcH5b (E2 enzyme) to promote p62 ubiquitination and autophagy; HECTD3 deletion leads to p62 accumulation in the nucleus after irradiation, inhibiting RNF168-mediated DNA damage repair; the HECTD3/UbcH5b inhibitor PC3-15 blocks DNA damage repair and increases radiosensitivity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (with UbcH5b), subcellular fractionation (nuclear p62 accumulation), DNA damage repair assay, small molecule inhibitor (PC3-15), HECTD3 knockout cell lines\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — E2-E3 collaboration defined, nuclear localization by fractionation with functional consequence, pharmacological inhibitor validation, single lab\",\n      \"pmids\": [\"39487119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HECTD3 ubiquitinates AKT-phosphorylated CMTM3 targeting it for proteasomal degradation; AKT1 directly phosphorylates CMTM3 at Ser181, and this phosphorylation is required for HECTD3 recognition; knockdown of HECTD3 or PI3K/AKT inhibition stabilizes CMTM3; non-phosphorylatable CMTM3-S181A resists HECTD3-mediated degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (S181A CMTM3), kinase assay (AKT1), pharmacological PI3K/AKT inhibition, xenograft model\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-dependent ubiquitination with site-specific mutagenesis of both phosphorylation and recognition site, in vivo model, single lab\",\n      \"pmids\": [\"40836897\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HECTD3 is a HECT-domain E3 ubiquitin ligase that uses its N-terminal DOC domain for substrate/HSP90 docking and its catalytic Cys (C823/C832) for ubiquitin transfer; it assembles non-degradative K27-, K29-, K33-, and K63-linked polyubiquitin chains on diverse substrates—including MALT1 (K648), STAT3 (K180), TRAF3 (K138), caspase-8 (K215), IKKα (K296), PARP1 (K209/K221), PKR (K33-linked), p62, and c-MYC—to modulate NF-κB and interferon signaling, Th17 differentiation, apoptosis resistance, viral replication, and metastasis; it also targets selected proteins (Tara, LKB1, CRAF/MASTL as HSP90 clients, SLC7A11, CMTM3) for proteasomal degradation, with its own activity regulated by ERK-mediated phosphorylation at Thr-157.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HECTD3 is a HECT-domain E3 ubiquitin ligase that controls inflammatory signaling, apoptosis resistance, and tumor cell behavior by assembling predominantly non-degradative polyubiquitin chains on a range of signaling proteins [#3, #6, #7]. It engages substrates through an N-terminal DOC domain and transfers ubiquitin via a catalytic cysteine in its HECT domain (C823/C832), as demonstrated by catalytic-site mutagenesis that abolishes substrate ubiquitination [#8, #9, #10]. A recurring theme is the building of atypical, signal-modifying chains: K63-linked ubiquitination of TRAF3 at K138 to drive TRAF3-TBK1 complex formation and type I interferon induction during bacterial infection [#6], K27/K29-linked modification of MALT1 (K648) and K27-linked modification of STAT3 (K180) to promote NF-\\u03baB activation, STAT3 Tyr705 phosphorylation, and pathogenic Th17 differentiation [#7], K27/K63-linked ubiquitination of IKK\\u03b1 at K296 to enhance its nuclear kinase activity and tumor metastasis [#12], and K33-linked ubiquitination of PKR that blocks PKR dimerization and eIF2\\u03b1 activation while redirecting PKR into an IKK-associated inflammatory complex [#14]. HECTD3 also restrains apoptosis: it K63-ubiquitinates caspase-8 at K215 to limit its activation and TRAIL-induced death in an E3-activity-dependent manner [#3], and ubiquitinates caspase-9 to block its oligomerization and Apaf-1 association, a function gated by ERK-mediated phosphorylation of HECTD3 at Thr-157 [#5]. In addition to stabilizing or activating substrates, HECTD3 directs selected proteins to proteasomal degradation, including the HSP90 client kinases CRAF, MASTL, and LKB1 [#4] and the cystine transporter SLC7A11, the latter sensitizing colon cancer cells to ferroptosis [#15]. Through these activities HECTD3 promotes c-MYC- and PARP1-dependent tumor proliferation and DNA repair [#9, #10], links to p62 ubiquitination and autophagy [#16], and is itself a pharmacological target via the HECTD3/UbcH5b inhibitor PC3-15 [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established HECTD3 as a functional E3 ligase by identifying its first substrate, defining a degradative mode of action linked to mitotic spindle integrity.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, in vitro binding, siRNA, and ubiquitination assays on Tara (also Syntaxin 8 in a parallel study)\",\n      \"pmids\": [\"18194665\", \"18821010\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain linkage on Tara not defined\", \"Catalytic residue dependence not tested\", \"Syntaxin 8 interaction lacked functional readout or mutagenesis\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed HECTD3's non-degradative ubiquitination mode and its anti-apoptotic role, showing it stabilizes MALT1 and ubiquitinates caspase-8 at K215 to blunt death receptor signaling.\",\n      \"evidence\": \"Y2H/DOC-domain mapping, Co-IP, linkage and site-specific ubiquitination, K215 and ligase-dead mutagenesis, TRAIL apoptosis and rescue assays\",\n      \"pmids\": [\"23358872\", \"24287696\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous chain-type confirmation for MALT1 in this work\", \"How non-degradative chains mechanistically block caspase-8 activation not resolved structurally\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined two opposing branches of HECTD3 function\\u2014DOC-domain-dependent degradation of HSP90 client kinases versus ERK-phosphorylation-gated suppression of caspase-9 apoptosis.\",\n      \"evidence\": \"siRNA screen, Co-IP, DOC-domain mapping, proteasomal degradation assays (CRAF/MASTL/LKB1); caspase-9 oligomerization/Apaf-1 assays, T157A mutant, ERK kinase assay, xenografts\",\n      \"pmids\": [\"28636940\", \"28716524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Chain linkages on client kinases and caspase-9 not specified\", \"Structural basis of DOC-domain client recognition unresolved\", \"How Thr-157 phosphorylation alters ligase activity not mechanistically defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated HECTD3 drives innate antibacterial immunity through K63-linked TRAF3 ubiquitination enabling TRAF3-TBK1 complex assembly and type I IFN.\",\n      \"evidence\": \"Hectd3 knockout mice, in vitro ubiquitination with K138 mutagenesis, Co-IP of TRAF3-TBK1, Francisella/Mycobacterium/Listeria infection models\",\n      \"pmids\": [\"29920190\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E2 partner in this context not identified\", \"Structural detail of K63-chain placement on TRAF3 not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed HECTD3 builds K27/K29 chains on MALT1 and K27 chains on STAT3 to coordinately activate NF-\\u03baB and STAT3 and drive pathogenic Th17 autoimmunity.\",\n      \"evidence\": \"EAE knockout mouse model, linkage-specific ubiquitination, K648 MALT1 and K180 STAT3 mutagenesis, STAT3 phosphorylation and Th17 differentiation assays\",\n      \"pmids\": [\"30741923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How K27/K29 chains are read by downstream effectors not defined\", \"Whether MALT1 and STAT3 modification are coupled events unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed and extended the TRAF3 mechanism, mapping dual DOC/HECT engagement of TRAF3 and catalytic Cys832 dependence in oxidative stress-driven ischemia-reperfusion injury.\",\n      \"evidence\": \"Domain mapping, Co-IP, ubiquitination assays, Cys832 mutagenesis, hypoxia/reoxygenation and rat DCD liver models\",\n      \"pmids\": [\"33627626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific regulation of HECTD3 in this context not defined\", \"Single-lab in vivo model\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded the substrate repertoire across cancer contexts, establishing catalytic-cysteine-dependent ubiquitination of c-MYC (K29), PARP1 (K63, K209/K221), and IKK\\u03b1 (K27/K63, K296) to promote proliferation, DNA repair, and metastasis.\",\n      \"evidence\": \"Co-IP, DOC-domain mapping, linkage/site-specific ubiquitination, C823 and substrate-lysine mutagenesis, kinase activity and nuclear fractionation, conditional knockout/knockin mice and xenografts\",\n      \"pmids\": [\"35397617\", \"36088509\", \"35918322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals selecting among these substrates not fully defined\", \"Whether one E2 supports all linkage types unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked HECTD3-MALT1 and HECTD3-LKB1 axes to disease phenotypes (neuronal glucose toxicity, glioma radioresistance) via epistasis, implicating JNK and ZEB1 as downstream effectors.\",\n      \"evidence\": \"siRNA, knockout rat/xenograft models, western blot, MALT1 and ZEB1 overexpression rescue\",\n      \"pmids\": [\"35913790\", \"35768187\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Ubiquitin linkage and site on LKB1 in this context not defined\", \"Mechanism of MALT1 stabilization indirect\", \"Epistasis inferred from rescue rather than direct biochemistry\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a novel K33-linked ubiquitination of PKR and a degradative role on SLC7A11, broadening HECTD3 control to antiviral/inflammatory signaling and ferroptosis.\",\n      \"evidence\": \"K33-linkage-specific ubiquitination, PKR dimerization/phosphorylation/eIF2\\u03b1 and PKR-IKK complex assays, knockout mouse and viral model; SLC7A11 C823 mutagenesis, half-life and ferroptosis assays, xenografts\",\n      \"pmids\": [\"37402711\", \"37422058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis distinguishing K33 vs degradative chains on different substrates unknown\", \"Determinants of degradative versus non-degradative outcome unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a specific E2-E3 pairing (UbcH5b) and a druggable axis, linking HECTD3 to p62 ubiquitination, autophagy, and DNA damage repair through nuclear p62/RNF168 control.\",\n      \"evidence\": \"Co-IP, ubiquitination with UbcH5b, subcellular fractionation, DNA repair assays, PC3-15 inhibitor, HECTD3 knockout cells\",\n      \"pmids\": [\"39487119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"p62 chain linkage not specified\", \"Generality of UbcH5b across other substrates not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established phosphodegron-based substrate recognition, showing AKT1 phosphorylation of CMTM3 at Ser181 licenses HECTD3-mediated degradation.\",\n      \"evidence\": \"Co-IP, ubiquitination, S181A CMTM3 mutagenesis, AKT1 kinase assay, PI3K/AKT inhibition, xenografts\",\n      \"pmids\": [\"40836897\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DOC domain directly reads phospho-Ser181 not structurally shown\", \"Chain linkage on CMTM3 not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural determinants that allow HECTD3 to choose between non-degradative (K27/K29/K33/K63) and degradative chain assembly on different substrates, and how DOC-domain engagement and Thr-157 phosphorylation specify these outcomes, remain unresolved.\",\n      \"evidence\": \"No structural or biochemical study in the corpus reconstitutes linkage-selection determinants\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of HECTD3 DOC or HECT domain with substrate\", \"Linkage-specificity mechanism undefined\", \"Full E2 enzyme repertoire across substrates unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3, 6, 7, 8, 9, 10, 12, 14]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3, 6, 8, 9, 10, 12, 15]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6, 7, 12, 14, 15, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [12, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 7, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 5, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 12, 14]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 4, 6, 15, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MALT1\", \"TRAF3\", \"STAT3\", \"IKK\\u03b1\", \"caspase-8\", \"PKR\", \"c-MYC\", \"UbcH5b\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}