{"gene":"HUWE1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2008,"finding":"HUWE1 ubiquitinates N-Myc via K48-linked polyubiquitin chains, targeting it for proteasomal degradation; genetic and RNAi-mediated inactivation of Huwe1 stabilizes N-Myc, prevents cell-cycle exit, and blocks neuronal differentiation in ES cells and mouse brain cortex.","method":"Co-IP, in vitro ubiquitination assay, genetic knockout, RNAi knockdown in ES cells and mouse brain with phenotypic rescue by N-myc silencing","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination assay showing K48 linkage, genetic epistasis confirmed by N-myc rescue, replicated across ES cells and in vivo mouse cortex","pmids":["18488021"],"is_preprint":false},{"year":2005,"finding":"The HECT domain of HUWE1 (hUREB1) ubiquitinates p53 and promotes its proteasomal degradation, suppressing p53 transcriptional activity; point mutations abolishing HECT catalytic activity block this effect.","method":"Transient transfection overexpression, p53 reporter assay, MG132 proteasome inhibition, HECT domain deletion/point mutation analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic domain mutagenesis plus functional reporter and degradation assay, single lab","pmids":["15567145"],"is_preprint":false},{"year":2007,"finding":"HUWE1 directly binds Cdc6 and polyubiquitinates it in vitro; Huwe1 is required for Cdc6 degradation after UV or MMS-induced DNA damage, and this function is conserved (yeast ortholog Tom1 performs the same role).","method":"Co-IP, in vitro polyubiquitination assay, siRNA knockdown, yeast genetic analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination reconstitution plus genetic conservation demonstrated in yeast, two orthogonal methods","pmids":["17567951"],"is_preprint":false},{"year":2009,"finding":"The N-terminal helix of the HUWE1 HECT domain structurally stabilizes the domain and modulates activity: its absence increases self-ubiquitination while having distinct effects on substrate (Mcl-1) ubiquitination, revealing a structural element that differentially controls self- vs. substrate-ubiquitination.","method":"Crystal structure determination, deletion mutagenesis, in vitro self-ubiquitination and substrate ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro activity assays in single rigorous study","pmids":["20007713"],"is_preprint":false},{"year":2009,"finding":"Huwe1 operates upstream of the N-Myc–DLL3–Notch pathway in the developing brain; conditional Huwe1 knockout causes N-Myc accumulation, DLL3 elevation, and Notch suppression, resulting in neural stem cell over-expansion and failure of neurogenesis.","method":"Conditional knockout mouse, gain- and loss-of-function experiments in mouse cortex, epistasis analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with pathway epistasis confirmed in vivo, multiple phenotypic readouts","pmids":["19686682"],"is_preprint":false},{"year":2010,"finding":"Huwe1 loss in cerebellar granule neuron precursors causes N-Myc accumulation and uncontrolled proliferation; additional loss in Bergmann glia disrupts glial differentiation and granule neuron migration, demonstrating Huwe1 coordinates neuronal and glial programming.","method":"Conditional knockout mouse (cerebellar-specific), immunohistochemistry, N-Myc protein level assessment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with cell-type-specific phenotypes in vivo, substrate (N-Myc) accumulation confirmed","pmids":["20231446"],"is_preprint":false},{"year":2010,"finding":"The C. elegans HUWE1 ortholog EEL-1 fine-tunes myotactin (hemidesmosome ECM receptor) abundance ~2-fold; EEL-1 activity is essential for hemidesmosomes to mature into a tension-resistant form during embryonic development.","method":"Genome-wide RNAi screen in C. elegans, quantitative protein level analysis, genetic interaction with plakin mutant","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi phenotype in vivo plus quantitative protein measurement, single lab","pmids":["20153198"],"is_preprint":false},{"year":2012,"finding":"HUWE1 ubiquitinates MyoD at its N-terminal residue and targets it for proteasomal degradation, acting as an E3 ligase for this muscle-regulatory transcription factor.","method":"In vitro ubiquitination assay, proteasome inhibitor treatment, co-IP","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro ubiquitination assay plus co-IP, single lab, single study","pmids":["22277673"],"is_preprint":false},{"year":2012,"finding":"HUWE1 is associated with the UFD substrate Ub(G76V)-YFP and the 26S proteasome; HUWE1 knockdown stabilizes ubiquitin-fusion degradation (UFD) substrates and the pathological UFD substrate UBB+1; HUWE1 and TRIP12 function in parallel to degrade UFD substrates.","method":"High-throughput siRNA imaging screen, co-IP with proteasome, substrate half-life analysis, double knockdown epistasis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (screen, co-IP, half-life), epistasis with TRIP12, single lab","pmids":["23209776"],"is_preprint":false},{"year":2012,"finding":"ARF-BP1/HUWE1 forms a multiprotein complex with ARF, p53, MYC, and CTCF; HUWE1 ubiquitylates CTCF leading to its proteasomal degradation, linking the MYC proliferative and p53-ARF apoptotic pathways.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown with apoptosis/growth arrest readout","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP identifying multiprotein complex plus ubiquitination assay, single lab","pmids":["22754359"],"is_preprint":false},{"year":2013,"finding":"HUWE1 ubiquitylates the antiapoptotic factor Mcl-1; MDM2 ubiquitylates HUWE1 itself; in HER2+ breast cancer cells treated with lapatinib, MDM2 degradation stabilizes HUWE1 enabling Mcl-1 degradation and apoptosis, while lapatinib-resistant cells maintain MDM2 to degrade HUWE1.","method":"Co-IP, ubiquitination assay, siRNA knockdown, xenograft models, half-life analysis","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell-based assays plus in vivo xenograft, single lab","pmids":["23652204"],"is_preprint":false},{"year":2013,"finding":"In Ras-driven skin tumorigenesis, Mule/HUWE1 suppresses tumor formation by preventing accumulation of c-Myc/Miz1 transcriptional complexes that otherwise downregulate p21 and p15; genetic knockout of c-Myc (but not p53 or p19Arf) reverses the Mule-KO tumorigenic phenotype.","method":"Conditional knockout mouse (K14Cre), chemical carcinogenesis model, genetic epistasis (c-Myc, p53, p19Arf co-knockouts), Miz1 knockdown rescue","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with genetic epistasis resolved by multiple co-KO experiments in one study","pmids":["23699408"],"is_preprint":false},{"year":2014,"finding":"Huwe1 ubiquitylates Dishevelled (Dvl) with K63-linked polyubiquitin chains in a Wnt3a- and CK1ε-dependent manner; this inhibits Dvl DIX-domain multimerization required for Wnt signaling, creating a negative feedback loop upstream of β-catenin and downstream of CK1/LRP6.","method":"RNAi screen in C. elegans, reporter assay in HEK293T cells, co-IP, mass spectrometry for K63-linkage determination, Dvl multimerization assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — evolutionary conservation (worm + human), mass spectrometry linkage determination, genetic epistasis, and functional Dvl multimerization assay in one study","pmids":["24643799"],"is_preprint":false},{"year":2014,"finding":"HUWE1 ubiquitylates and degrades TIAM1 preferentially at cell-cell adhesions in response to HGF, facilitating junction disassembly, cell scattering, migration, and invasion; simultaneous TIAM1 depletion rescues migration/invasion in HUWE1-depleted cells.","method":"Co-IP, ubiquitination assay, siRNA knockdown, HGF stimulation assays, invasion assay, epistasis (TIAM1 double depletion), human lung cancer cell validation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, ubiquitination assay, epistasis, spatial localization at cell-cell adhesions, validated in cancer cells","pmids":["25543140"],"is_preprint":false},{"year":2014,"finding":"HUWE1 binds Shoc2 and mediates its ubiquitination and degradation; HUWE1 also controls RAF-1 ubiquitination and its kinase levels through the Shoc2 scaffold; HUWE1 depletion abolishes RAF-1 ubiquitination, altering ERK1/2 pathway activity.","method":"Co-IP, ubiquitination assay, siRNA knockdown, ERK1/2 activity measurement","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and ubiquitination assay with functional ERK pathway readout, single lab","pmids":["25022756"],"is_preprint":false},{"year":2014,"finding":"HUWE1 is required for growth of colorectal cancer cells; HUWE1 associates with both MYC and MIZ1; HUWE1 inhibition stabilizes MIZ1, which accumulates on MYC target genes and represses MYC-activated transcription, revealing that continuous MIZ1 degradation is required for MYC transactivation.","method":"Small-molecule HUWE1 inhibitors (high-throughput screen), genetic knockdown, gene expression analysis, orthotopic xenograft models","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — chemical and genetic inhibition converging on same mechanism, orthogonal in vitro and in vivo validation","pmids":["25253726"],"is_preprint":false},{"year":2014,"finding":"HUWE1 degrades DDIT4 (REDD1) through ubiquitination; diGly proteomics coupled with inducible HUWE1 RNAi identified DDIT4 as a HUWE1 substrate; HUWE1 interacts with and regulates DDIT4 stability in cell-based assays.","method":"Inducible RNAi, quantitative diGly (ubiquitin remnant) proteomics, co-IP, protein half-life assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased proteomics plus co-IP and stability assay, single lab","pmids":["25147182"],"is_preprint":false},{"year":2014,"finding":"Huwe1 phosphodependently degrades the transcription factor Atoh1; SHH signaling prevents this phosphodependent degradation, stabilizing Atoh1 to promote neuronal precursor expansion; Huwe1 loss in mouse SHH medulloblastoma disrupts this developmental mechanism.","method":"In vivo mouse cerebellar studies, genetic Huwe1 loss-of-function, phosphorylation analysis, protein stability assay","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic and biochemical analysis, single lab, mechanism partially inferred","pmids":["24960692"],"is_preprint":false},{"year":2015,"finding":"HUWE1 mediates K48-linked polyubiquitination of Atoh1 following CK1-mediated phosphorylation at serine 334; this targets Atoh1 for proteasomal degradation; Huwe1 knockout in cochlear-supporting cells causes extra inner hair cells and synaptic defects.","method":"Co-IP, proximity ligation assay, mass spectrometry, cell-free ubiquitination system, CK1 inhibition, Huwe1 conditional knockout in cochlea","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — cell-free ubiquitination reconstitution, mass spectrometry phosphosite identification, CK1 epistasis, in vivo knockout phenotype","pmids":["27542412"],"is_preprint":false},{"year":2015,"finding":"ATM kinase, SIRT6, and SNF2H cooperate to block HUWE1-mediated poly-ubiquitination of H2AX upon DSB formation, transiently stabilizing H2AX to allow efficient γH2AX foci formation and chromatin incorporation; ATM phosphorylation of H2AX Ser139 is essential for this stabilization.","method":"Proteasome inhibition, ubiquitination assay, siRNA knockdown of HUWE1/ATM/SIRT6/SNF2H, γH2AX foci analysis, Ser139 mutation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic perturbations converging on HUWE1 as the ubiquitin ligase, Ser139 mutagenesis, orthogonal methods","pmids":["26711340"],"is_preprint":false},{"year":2015,"finding":"DNA damage activates CUL4B, which ubiquitinates HUWE1 in a NEDD8-dependent manner, targeting HUWE1 for proteasomal degradation; CUL4B depletion stabilizes HUWE1, accelerating MCL-1 degradation and increasing apoptosis, which is rescued by simultaneous HUWE1 depletion.","method":"Co-IP, in vitro ubiquitination assay, NEDD8 inhibition, CUL4B siRNA knockdown, epistasis with HUWE1","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination reconstitution, in vivo epistasis, NEDD8 dependency tested","pmids":["25883150"],"is_preprint":false},{"year":2015,"finding":"HUWE1 and NEDD4-1 are E3 ligases for ABCG1 and ABCG4; silencing both ligases increases ABCG1/ABCG4 protein levels and cholesterol export; overexpression of either ligase reduces transporter levels.","method":"LC-MS identification of ligases, siRNA silencing, overexpression, cholesterol export assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification plus functional validation, single lab","pmids":["26296893"],"is_preprint":false},{"year":2016,"finding":"HUWE1 ubiquitinates unassembled soluble proteins bearing hydrophobic segments in the cytoplasm; this requires p97/NPL4; inhibition of nuclear entry enhances HUWE1-mediated ubiquitination, placing USPD primarily in the cytoplasm; SILAC proteomics identified nuclear complex subunits as endogenous HUWE1 substrates.","method":"Stable isotope labeling (SILAC) proteomics, in vitro ubiquitination, p97 inhibition, nuclear import inhibition","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution plus unbiased proteomics plus mechanistic epistasis with p97, single comprehensive study","pmids":["27867533"],"is_preprint":false},{"year":2016,"finding":"HUWE1 is essential for replication of damaged DNA; HUWE1-knockout cells fail to mitigate replication stress; HUWE1 interacts with PCNA at stalled replication forks and mono-ubiquitinates H2AX to promote signaling at these sites.","method":"HUWE1 knockout cells, replication stress assays, co-IP with PCNA, H2AX ubiquitination assay, DNA fiber analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout with defined phenotype, co-IP demonstrating PCNA interaction at forks, mono-ubiquitination activity demonstrated, multiple orthogonal methods","pmids":["27146073"],"is_preprint":false},{"year":2016,"finding":"Huwe1 is essential for HSC self-renewal, quiescence, and lymphoid-fate specification by post-translationally controlling N-myc; N-myc levels are upregulated after Huwe1 loss or stress, leading to HSC exhaustion; Mycn depletion reverses most Huwe1-KO phenotypes in vivo.","method":"Conditional knockout mouse, fluorescent Mycn fusion allele (live imaging), genetic rescue by Mycn deletion","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with in vivo fluorescent substrate tracking and genetic epistasis rescue","pmids":["27668798"],"is_preprint":false},{"year":2017,"finding":"HUWE1 is a bona fide tumor suppressor in intestinal epithelium; identified mutations in colorectal cancer inactivate HUWE1 E3 ligase activity; Huwe1 deletion accelerates APC-loss-driven tumorigenesis through increased MYC levels and rapid DNA damage accumulation, leading to loss of the second APC allele.","method":"Modeling of clinical mutations in cells, conditional knockout in Apc-mutant mice, MYC protein level analysis, comet assay, epistasis with MCL1","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO in disease model, mutation functional validation, mechanistic epistasis","pmids":["28003334"],"is_preprint":false},{"year":2017,"finding":"HUWE1 mediates UV-induced polyubiquitination of histone H1 at multiple lysines; HUWE1-depleted cells show reduced RNF168 and 53BP1 recruitment to DNA damage sites (downstream of RNF8-mediated H1 ubiquitination) but normal MDC1 recruitment (upstream); HUWE1 primes H1 to allow K63-chain elongation by RNF8.","method":"Quantitative diGly proteomics, siRNA knockdown, recruitment assays (RNF168, 53BP1, MDC1) at DNA damage sites","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased proteomics plus functional knockdown with ordered pathway epistasis, single lab","pmids":["29127375"],"is_preprint":false},{"year":2017,"finding":"HUWE1 forms an auto-inhibited asymmetric dimer in solution and in cells through a crystallographic dimer interface; disruption of the dimer activates HUWE1; a conserved intramolecular segment counteracts dimerization; tumor suppressor p14ARF binds this segment and may shift HUWE1 toward the inactive state.","method":"Crystal structure determination, solution dimerization assays (biochemical), cellular self-association assays, HUWE1 activation upon dimer disruption, ARF binding experiment","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus multiple biochemical validation experiments demonstrating functional relevance of dimer interface","pmids":["28193319"],"is_preprint":false},{"year":2017,"finding":"HUWE1 ubiquitinates histone H1.3, targeting it for proteasomal degradation; Huwe1-deficient cells accumulate H1.3, which silences H19 expression; H1.3 silencing partially rescues H19 expression and the Huwe1-null phenotype in ovarian epithelial transformation.","method":"Co-IP, ubiquitination assay, Huwe1 conditional knockout in mice, H1.3/H19 epistasis analysis","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and ubiquitination assay in vivo confirmed with epistasis, single lab","pmids":["28687618"],"is_preprint":false},{"year":2018,"finding":"HUWE1 promotes PINK1/PARKIN-independent mitophagy by enabling IKKα-mediated phosphorylation of AMBRA1 at serine 1014, which induces structural changes in AMBRA1 promoting its interaction with LC3/GABARAP proteins and mitophagic activity.","method":"Co-IP, siRNA knockdown, phosphorylation mutant analysis, mitophagy assays, IKKα kinase identification","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, mutagenesis, structural change, functional mitophagy assay), mechanistic pathway established","pmids":["30217973"],"is_preprint":false},{"year":2018,"finding":"mTORC1 phosphorylates WIPI2 at Ser395, directing it to interact specifically with HUWE1 for ubiquitination and proteasomal degradation; HUWE1 loss stabilizes WIPI2, enhances autophagosome formation, and improves hepatic lipid clearance in fasting mice.","method":"In vitro mTORC1 phosphorylation assay, co-IP, ubiquitination assay, HUWE1 siRNA in mouse liver, WIPI2 stabilization and autophagy flux measurement","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay identifying phosphodegron, co-IP, in vivo liver silencing, multiple orthogonal methods","pmids":["30340022"],"is_preprint":false},{"year":2018,"finding":"HUWE1 promotes ubiquitination and proteasomal degradation of PPARα in coordination with the scaffold protein PAQR3; PAQR3 directly interacts with PPARα and enhances the PPARα–HUWE1 interaction; PAQR3 deficiency in liver increases PPARα-target gene expression during fasting.","method":"Co-IP, polyubiquitination assay, adenoviral knockdown/liver-specific KO of PAQR3, PPARα target gene expression, fasting metabolic assays","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vivo genetic model, ubiquitination assay, single lab","pmids":["29331071"],"is_preprint":false},{"year":2018,"finding":"HUWE1 controls lung cancer cell malignancy by promoting proteasomal degradation of p53; HUWE1 inactivation increases p53, upregulates p21 and downregulates HIF1α; Huwe1 conditional deletion abolishes EGFRVIII-induced lung cancer in mice.","method":"siRNA/shRNA knockdown, soft agar and xenograft assays, western blot for p53/p21/HIF1α, genetically engineered Huwe1 conditional KO mouse","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO in vivo plus molecular mechanism via p53 pathway, single lab","pmids":["30026863"],"is_preprint":false},{"year":2019,"finding":"HUWE1 controls AMBRA1-mediated mitophagy through MCL1 stability: MCL1 overexpression inhibits HUWE1 recruitment to mitochondria during AMBRA1-induced mitophagy; GSK-3β phosphorylates MCL1 at S159, which triggers HUWE1-dependent MCL1 degradation, enabling mitophagy to proceed.","method":"Co-IP, overexpression/knockdown, GSK-3β inhibitor treatment, phosphorylation site mutagenesis, mitophagy assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, phosphosite mutagenesis, and functional mitophagy readout, single lab","pmids":["31434979"],"is_preprint":false},{"year":2019,"finding":"HUWE1 directly ubiquitinates Chk1 at multiple lysines within the kinase domain, controlling Chk1 protein stability; HUWE1 knockdown prolongs Chk1 half-life and elevates Chk1 levels independently of ATM/ATR and p53; replication stress reduces Chk1 levels in a HUWE1-dependent manner.","method":"HUWE1 siRNA knockdown, in vitro ubiquitination assay with lysine mapping, Chk1 half-life measurement, replication stress (HU, camptothecin)","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro ubiquitination with residue mapping plus cellular stability assay, single lab","pmids":["31713291"],"is_preprint":false},{"year":2020,"finding":"HUWE1 interacts with AIM2, NLRP3, and NLRC4 through specific domain interactions (BH3 domain of HUWE1; HIN domain of AIM2; NACHT domains of NLRP3/NLRC4); HUWE1 mediates K27-linked polyubiquitination of these inflammasome sensors, promoting inflammasome assembly, ASC speck formation, and caspase-1 activation.","method":"Co-IP, domain-mapping experiments, ubiquitination assay (K27 linkage), HUWE1-KO macrophages, HUWE1 inhibitor (BI8622), in vivo bacterial infection models","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-mapping co-IP, ubiquitin linkage determination, KO cells and in vivo models all converge on same mechanism","pmids":["33104527"],"is_preprint":false},{"year":2020,"finding":"HUWE1-dependent neddylation of DNA-PKcs at its kinase domain, catalyzed by E2 UBE2M, is required for DNA-PKcs autophosphorylation at Ser2056 and efficient NHEJ repair.","method":"Co-IP, neddylation assay, HUWE1 siRNA, DNA-PKcs autophosphorylation assay, NHEJ efficiency measurement","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional autophosphorylation and NHEJ assays, single lab","pmids":["32457294"],"is_preprint":false},{"year":2020,"finding":"HAPSTR1 (C16orf72) is required for HUWE1 nuclear localization; nuclear HUWE1 is needed for cell proliferation and modulation of p53 and NF-κB stress signaling pathways; unbiased quantitative proteomics shows HUWE1 substrate targeting is largely cell-type-specific.","method":"Quantitative proteomics, HAPSTR1 KO, subcellular fractionation, p53/NF-κB reporter assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased proteomics, genetic KO demonstrating localization-function link, multiple pathway readouts","pmids":["37167062"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structures of full-length HUWE1 reveal an alpha-solenoid assembly with a central pore decorated with protein interaction modules; the N-terminal ~3900 amino acids are indispensable for proper ligase function; neurodevelopmental disorder-associated variants and substrate binding were mapped onto the three-dimensional organization.","method":"Cryo-EM structure determination, functional analysis of deletion constructs, structure of disease-variant HUWE1 and substrate-bound complex","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — comprehensive cryo-EM with functional validation, disease variant structures, and substrate binding in single study","pmids":["34314700"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of Nematocida HUWE1 reveals a snake-like alpha-solenoid ring with the C-terminal HECT domain at the head; the ring structure is highly dynamic, allowing the flexible HECT domain to access substrate-presenting acceptor sites distributed around the ring; this explains how HUWE1 ubiquitinates diverse unrelated substrates.","method":"Crystal structure determination, integrative structural analysis, functional validation of ring architecture","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus integrative structural analysis, functional model validated","pmids":["34294896"],"is_preprint":false},{"year":2021,"finding":"HUWE1 ubiquitinates ATG101 for proteasomal degradation; HUWE1-depleted cells have stable ATG101 and elevated autophagy; HUWE1 thus suppresses autophagy flux by targeting both WIPI2 and ATG101.","method":"CRISPR KO of ATG101, co-IP, siRNA knockdown, autophagy assays, ATG101 domain mapping of ubiquitination site","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, domain mapping, functional autophagy readout, single lab","pmids":["34502089"],"is_preprint":false},{"year":2022,"finding":"HUWE1 directly ubiquitinates transferrin receptor 1 (TfR1) for proteasomal degradation, thereby regulating cellular iron metabolism; Huwe1 hepatocyte-specific knockout leads to TfR1 accumulation, excess iron uptake, lipid peroxidation, and ferroptosis in acute liver injury models.","method":"Hepatocyte-specific Huwe1 KO mice, ubiquitination assay, TfR1 protein level analysis, ferroptosis markers, chemical/genetic TfR1 inhibition rescue","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo hepatocyte KO, ubiquitination assay, epistasis rescue by TfR1 inhibition, multiple orthogonal methods","pmids":["35260822"],"is_preprint":false},{"year":2022,"finding":"HUWE1 promotes ubiquitination and degradation of EGFR; HUWE1 physically interacts with EGFR; HUWE1 overexpression decreases EGFR levels and protects against TGF-β-induced renal tubular injury.","method":"Co-IP, ubiquitination assay, overexpression/knockdown in HK-2 cells, UUO mouse model","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ubiquitination assay plus in vivo model, single lab","pmids":["32017279"],"is_preprint":false},{"year":2023,"finding":"HUWE1 controls TTP (tristetraprolin) stability indirectly by regulating its phosphorylation; HUWE1 function requires phosphatase and E3 ligase binding sites in the TTP C-terminus; HUWE1-regulated phosphorylation is independent of MAPK-mediated S52/S178 phosphorylation and controls TTP stability at late stages of pro-inflammatory stimulation.","method":"Genetic screen, pharmacological kinase inhibition, TTP phosphorylation analysis, stability assays, domain-mapping","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen, pharmacological dissection, domain mapping, single lab","pmids":["36961408"],"is_preprint":false},{"year":2024,"finding":"HUWE1 acts as a centrosomal E3 ligase targeting TTBK2 for degradation; TTBK2 stabilizes primary cilia and promotes GNP proliferation in response to SHH; HUWE1-mediated TTBK2 degradation triggers primary cilia disassembly, thereby driving GNP differentiation and migration in cerebellar development.","method":"Conditional knockout mouse (cerebellar GNPs), co-IP, ubiquitination assay, TTBK2 overexpression/depletion, primary cilia dynamics assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO plus ubiquitination assay plus primary cilia functional assays, mechanistic pathway ordered by genetic epistasis","pmids":["38879724"],"is_preprint":false},{"year":2024,"finding":"CDK9 recruits HUWE1 to ubiquitinate and degrade RARα; pharmacological CDK9 inhibition stabilizes RARα and synergizes with ATRA in suppressing CTCL tumor growth.","method":"Co-IP (CDK9–HUWE1 interaction), RARα ubiquitination assay, CDK9 PROTAC, in vitro and xenograft combination treatment","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating CDK9–HUWE1 interaction, ubiquitination assay, functional in vivo validation, single lab","pmids":["39632829"],"is_preprint":false},{"year":2023,"finding":"HUWE1 possesses a Ubiquitin-Directed ubiquitin Ligase (UDL) activity: it recognizes substrates and aggregates/condensates bearing high-density pre-existing ubiquitin chains and amplifies ubiquitin modifications on them; this recruits p97/VCP for processing and downstream proteasomal or autophagic clearance.","method":"Biochemical reconstitution, aggregate clearance assays, p97 co-recruitment assay, cell-cycle transition analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — reconstitution with novel UDL activity, but preprint not yet peer-reviewed","pmids":["37398461"],"is_preprint":true},{"year":2014,"finding":"HUWE1 interacts with and promotes proteasomal degradation of BRCA1 via the ubiquitin-proteasome pathway; HUWE1 binds BRCA1 through an N-terminal degron domain; HUWE1 depletion increases BRCA1 levels and resistance to ionizing radiation and mitomycin.","method":"Co-IP, siRNA knockdown, BRCA1 half-life assay, clonogenic survival after IR/mitomycin","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus functional radiation sensitivity assay, single lab, two publications reporting same finding","pmids":["24472556","24342616"],"is_preprint":false},{"year":2018,"finding":"CRL4B E3 ligase with the DCAF adaptor RBBP7 polyubiquitinates HUWE1 and targets it for proteasomal degradation; RBBP7 depletion stabilizes HUWE1 and accelerates MCL-1 and BRCA1 degradation.","method":"Co-IP identifying RBBP7 as DCAF, in vitro ubiquitination assay, RBBP7 overexpression/depletion, MCL-1/BRCA1 levels","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vitro ubiquitination identifying specific adaptor, functional substrate effects measured, single lab","pmids":["29738775"],"is_preprint":false},{"year":2019,"finding":"HUWE1 ubiquitinates the GlyR-α1 subunit of glycine receptors in the spinal cord dorsal horn, reducing surface expression of GlyRs via endocytosis; HUWE1 knockdown blunts GlyR-α1 ubiquitination, potentiates glycinergic transmission, and attenuates inflammatory pain in mice.","method":"Co-IP (HUWE1–GlyR-α1 interaction), ubiquitination assay, shRNA knockdown in dorsal horn, surface expression assay, electrophysiology, CFA pain model","journal":"Neuropharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, ubiquitination assay, in vivo knockdown with defined electrophysiological and behavioral phenotypes","pmids":["30721695"],"is_preprint":false},{"year":2020,"finding":"HUWE1 also functions as an E3 ligase for Ezh1α (PRC2 complex component): reduced HUWE1 association is required for Ezh1α stabilization under oxidative stress in skeletal muscle cells.","method":"Co-IP, ubiquitination assay, kinase inhibition of Ser560 phosphorylation on Ezh1β","journal":"Epigenetics & chromatin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP observation with limited mechanistic follow-up, single lab","pmids":["31856907"],"is_preprint":false},{"year":2021,"finding":"HUWE1 ubiquitinates Ets-1 transcription factor for proteasomal degradation; this interaction is regulated by Ets-1 Thr38 phosphorylation status; HUWE1-mediated Ets-1 degradation reduces Treg cell differentiation and function, causing immune imbalance in ITP.","method":"Co-IP, cycloheximide-chase assay, ubiquitination assay, Thr38 phosphorylation analysis, HUWE1 overexpression/knockdown, in vivo ITP mouse model with HUWE1 inhibitor","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, phosphosite analysis, in vivo pharmacological rescue, single lab","pmids":["34900980"],"is_preprint":false},{"year":2013,"finding":"Increased HUWE1 dosage in Drosophila (modeling human duplication) decreases Dishevelled (dsh) protein levels ~50% and causes axon branching defects in dorsal cluster neurons; fz2 and dsh mutants phenocopy this; constitutively active β-catenin (armadillo) partially rescues, placing HUWE1 upstream of Wnt/β-catenin in axon development.","method":"Drosophila overexpression model, protein level quantification, genetic epistasis with dsh/fz2/armadillo mutants","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo Drosophila genetic epistasis with substrate level measurement, consistent with Wnt pathway finding in vertebrates","pmids":["24303071"],"is_preprint":false},{"year":2022,"finding":"HUWE1 ubiquitinates TOMM20 directly and also regulates TOMM20 degradation via a PARKIN-mediated pathway; HUWE1 overexpression impairs mitochondrial ATP production and membrane potential, increasing ROS and apoptosis, sensitizing CRC cells to oxaliplatin.","method":"Co-IP, ubiquitination assay, mitochondrial function assays (ATP, membrane potential), HUWE1 overexpression, oxaliplatin sensitivity assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ubiquitination assay plus functional mitochondrial readouts, single lab","pmids":["38184713"],"is_preprint":false},{"year":2025,"finding":"HUWE1 ubiquitinates and degrades MUTYH (DNA repair glycosylase) in renal tubular epithelial cells; upregulation of HUWE1 during AKI causes MUTYH depletion, leading to 8-oxoG accumulation, mitochondrial dysfunction, and tubular injury; HUWE1 KO or BI8622 treatment protects against cisplatin-induced AKI.","method":"Co-IP, ubiquitination assay, HUWE1 KO mice, BI8622 pharmacological inhibition, cisplatin/FA AKI models, 8-oxoG quantification, mitochondrial function assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo KO and pharmacological rescue, multiple functional readouts, single lab","pmids":["39921445"],"is_preprint":false},{"year":1995,"finding":"UREB1 (HUWE1) inhibits p53 transcriptional activity when co-expressed; this inhibitory effect requires tyrosine phosphorylation of UREB1, as a tyrosine-to-alanine mutation in the phosphorylation site significantly attenuates suppression.","method":"Transient co-transfection in p53-null H1299 cells, p53 reporter assay, tyrosine phosphorylation site mutagenesis","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single reporter assay with mutagenesis, no direct ubiquitination demonstrated, early study predating molecular characterization","pmids":["7478539"],"is_preprint":false}],"current_model":"HUWE1 is a giant HECT-domain E3 ubiquitin ligase that adopts an alpha-solenoid ring structure presenting multiple substrate-binding modules to a flexible catalytic HECT domain; its activity is regulated by auto-inhibitory dimerization (relieved by ARF) and by phosphorylation/neddylation-controlled substrate recruitment. HUWE1 ubiquitinates a wide array of substrates—including N-Myc/c-Myc (K48-linked, for proteasomal degradation controlling neural stem cell differentiation and HSC quiescence), Mcl-1 (pro-apoptotic), p53 (degradation), Cdc6 (DNA-damage-responsive degradation), WIPI2 and ATG101 (mTORC1-directed autophagy suppression), Dishevelled (K63-linked, Wnt negative feedback), Atoh1/Shh-dependent phosphodegron (neural progenitor differentiation), TfR1 (iron metabolism/ferroptosis suppression), TTBK2 (primary cilia disassembly), GlyR-α1 (synaptic inhibition), H2AX (mono-ubiquitination at stalled forks), histone H1 (UV-damage DDR priming), DNA-PKcs (neddylation for NHEJ), and many unassembled soluble nuclear proteins (protein quality control); HUWE1 is itself regulated by CRL4B(RBBP7)-mediated ubiquitination after DNA damage, and its nuclear functions are gated by the adaptor HAPSTR1."},"narrative":{"mechanistic_narrative":"HUWE1 is a giant HECT-domain E3 ubiquitin ligase that uses a dynamic alpha-solenoid ring architecture to present a flexible catalytic HECT domain to a structurally diverse repertoire of substrates, thereby acting as a master regulator of protein turnover across development, the DNA-damage response, cell death, and metabolism [PMID:34314700, PMID:34294896]. Cryo-EM and crystallographic studies show that the N-terminal ~3900 residues form a ring decorated with substrate-interaction modules whose mobility relative to the HECT head explains how a single ligase ubiquitinates unrelated targets [PMID:34314700, PMID:34294896], and its catalytic output is tuned by an N-terminal HECT helix that differentially controls self- versus substrate-ubiquitination [PMID:20007713] and by an auto-inhibited asymmetric dimer that is relieved upon dimer disruption and modulated by p14ARF binding [PMID:28193319]. A dominant theme is control of MYC-family oncoproteins: HUWE1 catalyzes K48-linked polyubiquitination and proteasomal degradation of N-Myc to drive cell-cycle exit and neuronal differentiation, acting upstream of the N-Myc–DLL3–Notch axis in neural stem cells and of N-myc-dependent HSC quiescence [PMID:18488021, PMID:19686682, PMID:27668798], while in intestinal and skin epithelia it suppresses c-Myc/Miz1 transcriptional output and functions as a bona fide tumor suppressor whose cancer-associated mutations inactivate its ligase activity [PMID:23699408, PMID:25253726, PMID:28003334]. HUWE1 broadly governs apoptosis and the DNA-damage response by degrading Mcl-1 [PMID:23652204], p53 [PMID:15567145], and Cdc6 [PMID:17567951], by mono-ubiquitinating H2AX at stalled forks via PCNA [PMID:26711340, PMID:27146073], and by priming histone H1 for RNF8-dependent chain elongation to recruit RNF168/53BP1 [PMID:29127375]. It additionally suppresses autophagy by degrading WIPI2 and ATG101 downstream of mTORC1 [PMID:30340022, PMID:34502089], drives AMBRA1/MCL1-dependent mitophagy [PMID:30217973, PMID:31434979], imposes negative feedback on Wnt signaling through K63-linked ubiquitination of Dishevelled [PMID:24643799, PMID:24303071], controls iron homeostasis and ferroptosis via TfR1 degradation [PMID:35260822], promotes inflammasome assembly through K27-linked ubiquitination of AIM2/NLRP3/NLRC4 [PMID:33104527], and performs cytoplasmic protein quality control of unassembled hydrophobic-segment-bearing proteins in a p97/NPL4-dependent manner [PMID:27867533]. HUWE1 itself is a regulated node: it is targeted for degradation by CRL4B/CUL4B(RBBP7) in a NEDD8-dependent, DNA-damage-responsive manner [PMID:25883150, PMID:29738775], and its nuclear localization and stress-pathway functions are gated by the adaptor HAPSTR1 [PMID:37167062].","teleology":[{"year":1995,"claim":"Established the earliest functional link between HUWE1 (UREB1) and p53, indicating it negatively regulates p53 transcriptional activity before any ligase mechanism was known.","evidence":"Co-transfection p53 reporter assay with tyrosine phosphosite mutagenesis in p53-null cells","pmids":["7478539"],"confidence":"Low","gaps":["No ubiquitination demonstrated","Mechanism of phosphorylation-dependent suppression not defined","Single reporter readout, not independently confirmed at the time"]},{"year":2005,"claim":"Resolved that HUWE1 acts as an E3 ligase for p53, showing the HECT catalytic domain drives p53 proteasomal degradation.","evidence":"Overexpression, p53 reporter, MG132 rescue, and HECT catalytic point mutants","pmids":["15567145"],"confidence":"Medium","gaps":["Ubiquitin linkage type on p53 not defined","Degron on p53 unmapped","Overexpression-based, physiological context limited"]},{"year":2007,"claim":"Demonstrated a DNA-damage-responsive substrate role by showing HUWE1 directly polyubiquitinates Cdc6 for degradation, with evolutionary conservation in the yeast ortholog Tom1.","evidence":"In vitro polyubiquitination, co-IP, siRNA, and yeast genetics","pmids":["17567951"],"confidence":"High","gaps":["Chain linkage on Cdc6 not specified","Signal coupling damage to Cdc6 recognition unresolved"]},{"year":2008,"claim":"Defined the canonical developmental role of HUWE1: K48-linked ubiquitination and degradation of N-Myc enforces cell-cycle exit and neuronal differentiation, validated by genetic rescue.","evidence":"In vitro ubiquitination with K48 linkage, knockout/RNAi, and N-myc-silencing rescue in ES cells and mouse cortex","pmids":["18488021"],"confidence":"High","gaps":["Degron/phosphorylation requirements for N-Myc recognition not defined here"]},{"year":2009,"claim":"Placed HUWE1 in a defined developmental signaling circuit (N-Myc–DLL3–Notch) and provided structural insight into how the HECT domain self-regulates activity.","evidence":"Conditional knockout mouse with pathway epistasis; crystal structure plus deletion mutagenesis and in vitro activity assays","pmids":["19686682","20007713"],"confidence":"High","gaps":["Full-length regulatory architecture not yet resolved","How HECT helix discriminates substrates mechanistically unclear"]},{"year":2013,"claim":"Established HUWE1 as a context-dependent tumor suppressor that restrains c-Myc/Miz1 oncogenic transcription, and revealed reciprocal regulation by MDM2.","evidence":"Conditional knockout chemical carcinogenesis with c-Myc/p53/p19Arf co-knockout epistasis; co-IP, ubiquitination, and HER2+ xenograft analyses","pmids":["23699408","23652204"],"confidence":"High","gaps":["Determinants of tumor-suppressor versus oncogenic outcome across tissues unresolved","MDM2-HUWE1 hierarchy in normal physiology unclear"]},{"year":2014,"claim":"Broadly expanded the substrate landscape into signaling and adhesion control, including K63-linked Dishevelled ubiquitination as Wnt negative feedback, TIAM1 degradation at junctions, MIZ1 turnover required for MYC transactivation, and metabolic/transcriptional targets.","evidence":"Cross-species RNAi, mass-spectrometry linkage determination, co-IP, ubiquitination assays, HUWE1 small-molecule inhibitors, and diGly proteomics","pmids":["24643799","25543140","25253726","25147182","25022756","22277673","24472556"],"confidence":"High","gaps":["How distinct chain linkages are selected per substrate unresolved","Cell-type specificity of substrate choice not explained"]},{"year":2016,"claim":"Defined HUWE1's protein quality-control function for unassembled cytoplasmic hydrophobic-segment proteins and its role in replication stress via PCNA-coupled H2AX mono-ubiquitination.","evidence":"SILAC proteomics with p97/nuclear-import inhibition; knockout cells, DNA fiber analysis, and PCNA co-IP","pmids":["27867533","27146073","26711340"],"confidence":"High","gaps":["Recognition code for orphan hydrophobic segments not defined","Coordination of fork-localized activity with bulk QC unclear"]},{"year":2017,"claim":"Resolved the autoregulatory mechanism: HUWE1 forms an auto-inhibited asymmetric dimer relieved by an intramolecular segment and modulated by p14ARF, and extended H1-based DDR priming and tissue-specific Atoh1 control.","evidence":"Crystal structure with dimerization and activation assays; diGly proteomics with ordered RNF168/53BP1 recruitment; in vivo cochlear knockout","pmids":["28193319","29127375","27542412"],"confidence":"High","gaps":["In-cell triggers of dimer disruption not identified","Quantitative contribution of ARF in vivo unresolved"]},{"year":2018,"claim":"Connected HUWE1 to autophagy/mitophagy and metabolic regulation, showing mTORC1-phosphorylated WIPI2 degradation suppresses autophagy and IKKα/AMBRA1 phosphorylation drives PINK1/PARKIN-independent mitophagy.","evidence":"In vitro kinase and ubiquitination assays, co-IP, phospho-mutant analysis, and in vivo liver silencing","pmids":["30340022","30217973","29331071","30026863"],"confidence":"High","gaps":["Switch between autophagy suppression and mitophagy promotion not reconciled","Substrate selection under nutrient stress incompletely mapped"]},{"year":2020,"claim":"Identified non-degradative and atypical-linkage activities (K27 ubiquitination of inflammasome sensors, neddylation of DNA-PKcs) and the adaptor HAPSTR1 gating nuclear HUWE1 function.","evidence":"Domain-mapping co-IP, K27 linkage determination, KO macrophages and infection models; neddylation assays; quantitative proteomics with HAPSTR1 KO and fractionation","pmids":["33104527","32457294","37167062"],"confidence":"High","gaps":["Structural basis for switching between ubiquitin and NEDD8 transfer unknown","How HAPSTR1 directs nuclear substrate selection unresolved"]},{"year":2021,"claim":"Delivered the full-length architectural model: alpha-solenoid ring with a mobile HECT head presenting substrates around the ring, explaining promiscuous substrate engagement and mapping neurodevelopmental disorder variants.","evidence":"Cryo-EM and crystal structures with deletion-construct functional validation and substrate-bound complexes","pmids":["34314700","34294896"],"confidence":"High","gaps":["How specific substrate modules are licensed in cells unresolved","Conformational dynamics during catalysis not directly visualized"]},{"year":2022,"claim":"Extended HUWE1's physiological reach to iron homeostasis and ferroptosis through TfR1 degradation, with hepatocyte-specific genetic and epistatic validation.","evidence":"Hepatocyte-specific knockout mice, ubiquitination assays, and TfR1 inhibition rescue","pmids":["35260822"],"confidence":"High","gaps":["Signal coupling HUWE1 activity to iron status not defined"]},{"year":2024,"claim":"Identified a centrosomal role controlling ciliary dynamics and SHH-driven proliferation by degrading TTBK2 to trigger primary cilia disassembly during cerebellar development.","evidence":"Conditional knockout in cerebellar granule precursors, co-IP, ubiquitination, and cilia dynamics assays","pmids":["38879724"],"confidence":"High","gaps":["How HUWE1 is recruited to the centrosome unresolved","Spatial control of TTBK2 access not defined"]},{"year":null,"claim":"How HUWE1 achieves substrate, linkage, and tissue specificity from a single promiscuous ring-shaped ligase—and how its proposed ubiquitin-directed ligase activity integrates with adaptor- and phosphorylation-gated recruitment—remains the central open question.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking conformational state to substrate choice","UDL activity reported only in preprint, awaiting peer review","Adaptor inventory beyond HAPSTR1/PAQR3 incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,35,36]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,19,23,35]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,12,35]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,2,30]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[27,37]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[22,23,26,37]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[22]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[29,33,53]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[44]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[13,49]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,22]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,19,23,26,36]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[10,20,33]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[29,30,40]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,4,5,17,44]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,14,52]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[35,51]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,15,25]}],"complexes":[],"partners":["MYCN","MYC","MIZ1","MCL1","TP53","DVL","PCNA","HAPSTR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z6Z7","full_name":"E3 ubiquitin-protein ligase HUWE1","aliases":["ARF-binding protein 1","ARF-BP1","HECT, UBA and WWE domain-containing protein 1","HECT-type E3 ubiquitin transferase HUWE1","Homologous to E6AP carboxyl terminus homologous protein 9","HectH9","Large structure of UREB1","LASU1","Mcl-1 ubiquitin ligase E3","Mule","Upstream regulatory element-binding protein 1","URE-B1","URE-binding protein 1"],"length_aa":4374,"mass_kda":481.9,"function":"E3 ubiquitin-protein ligase which mediates ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:15567145, PubMed:15767685, PubMed:15989957, PubMed:17567951, PubMed:18488021, PubMed:19037095, PubMed:19713937, PubMed:20534529, PubMed:30217973). Regulates apoptosis by catalyzing the polyubiquitination and degradation of MCL1 (PubMed:15989957). Mediates monoubiquitination of DNA polymerase beta (POLB) at 'Lys-41', 'Lys-61' and 'Lys-81', thereby playing a role in base-excision repair (PubMed:19713937). Also ubiquitinates the p53/TP53 tumor suppressor and core histones including H1, H2A, H2B, H3 and H4 (PubMed:15567145, PubMed:15767685, PubMed:15989956). Ubiquitinates MFN2 to negatively regulate mitochondrial fusion in response to decreased stearoylation of TFRC (PubMed:26214738). Ubiquitination of MFN2 also takes place following induction of mitophagy; AMBRA1 acts as a cofactor for HUWE1-mediated ubiquitination (PubMed:30217973). Regulates neural differentiation and proliferation by catalyzing the polyubiquitination and degradation of MYCN (PubMed:18488021). May regulate abundance of CDC6 after DNA damage by polyubiquitinating and targeting CDC6 to degradation (PubMed:17567951). Mediates polyubiquitination of isoform 2 of PA2G4 (PubMed:19037095). Acts in concert with MYCBP2 to regulate the circadian clock gene expression by promoting the lithium-induced ubiquitination and degradation of NR1D1 (PubMed:20534529). Binds to an upstream initiator-like sequence in the preprodynorphin gene (By similarity). Mediates HAPSTR1 degradation, but is also a required cofactor in the pathway by which HAPSTR1 governs stress signaling (PubMed:35776542). Acts as a regulator of the JNK and NF-kappa-B signaling pathways by mediating assembly of heterotypic 'Lys-63'-/'Lys-48'-linked branched ubiquitin chains that are then recognized by TAB2: HUWE1 mediates branching of 'Lys-48'-linked chains of substrates initially modified with 'Lys-63'-linked conjugates by TRAF6 (PubMed:27746020). 'Lys-63'-/'Lys-48'-linked branched ubiquitin chains protect 'Lys-63'-linkages from CYLD deubiquitination (PubMed:27746020). Ubiquitinates PPARA in hepatocytes (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q7Z6Z7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/HUWE1","classification":"Common Essential","n_dependent_lines":1086,"n_total_lines":1208,"dependency_fraction":0.8990066225165563},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SNRPA","stoichiometry":0.2},{"gene":"UBA52","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HUWE1","total_profiled":1310},"omim":[{"mim_id":"602952","title":"NUCLEAR RECEPTOR-BINDING SET DOMAIN PROTEIN 2; NSD2","url":"https://www.omim.org/entry/602952"},{"mim_id":"602101","title":"CLAUDIN 5; CLDN5","url":"https://www.omim.org/entry/602101"},{"mim_id":"400036","title":"TESTIS-SPECIFIC TRANSCRIPT, Y-LINKED, 3; TTTY3","url":"https://www.omim.org/entry/400036"},{"mim_id":"314320","title":"TRIGONOCEPHALY WITH SHORT STATURE AND DEVELOPMENTAL DELAY","url":"https://www.omim.org/entry/314320"},{"mim_id":"309590","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, TURNER TYPE; MRXST","url":"https://www.omim.org/entry/309590"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nuclear membrane","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"},{"location":"End piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HUWE1"},"hgnc":{"alias_symbol":["Ib772","KIAA0312","UREB1"],"prev_symbol":[]},"alphafold":{"accession":"Q7Z6Z7","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6Z7","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HUWE1","jax_strain_url":"https://www.jax.org/strain/search?query=HUWE1"},"sequence":{"accession":"Q7Z6Z7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7Z6Z7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7Z6Z7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z6Z7"}},"corpus_meta":[{"pmid":"30217973","id":"PMC_30217973","title":"HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.","date":"2018","source":"Nature 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MCR","url":"https://pubmed.ncbi.nlm.nih.gov/34654719","citation_count":7,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49333,"output_tokens":13020,"usd":0.17165,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24829,"output_tokens":5407,"usd":0.12966,"stage2_stop_reason":"end_turn"},"total_usd":0.30131,"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\": \"HUWE1 ubiquitinates N-Myc via K48-linked polyubiquitin chains, targeting it for proteasomal degradation; genetic and RNAi-mediated inactivation of Huwe1 stabilizes N-Myc, prevents cell-cycle exit, and blocks neuronal differentiation in ES cells and mouse brain cortex.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, genetic knockout, RNAi knockdown in ES cells and mouse brain with phenotypic rescue by N-myc silencing\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination assay showing K48 linkage, genetic epistasis confirmed by N-myc rescue, replicated across ES cells and in vivo mouse cortex\",\n      \"pmids\": [\"18488021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The HECT domain of HUWE1 (hUREB1) ubiquitinates p53 and promotes its proteasomal degradation, suppressing p53 transcriptional activity; point mutations abolishing HECT catalytic activity block this effect.\",\n      \"method\": \"Transient transfection overexpression, p53 reporter assay, MG132 proteasome inhibition, HECT domain deletion/point mutation analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic domain mutagenesis plus functional reporter and degradation assay, single lab\",\n      \"pmids\": [\"15567145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HUWE1 directly binds Cdc6 and polyubiquitinates it in vitro; Huwe1 is required for Cdc6 degradation after UV or MMS-induced DNA damage, and this function is conserved (yeast ortholog Tom1 performs the same role).\",\n      \"method\": \"Co-IP, in vitro polyubiquitination assay, siRNA knockdown, yeast genetic analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination reconstitution plus genetic conservation demonstrated in yeast, two orthogonal methods\",\n      \"pmids\": [\"17567951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The N-terminal helix of the HUWE1 HECT domain structurally stabilizes the domain and modulates activity: its absence increases self-ubiquitination while having distinct effects on substrate (Mcl-1) ubiquitination, revealing a structural element that differentially controls self- vs. substrate-ubiquitination.\",\n      \"method\": \"Crystal structure determination, deletion mutagenesis, in vitro self-ubiquitination and substrate ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro activity assays in single rigorous study\",\n      \"pmids\": [\"20007713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Huwe1 operates upstream of the N-Myc–DLL3–Notch pathway in the developing brain; conditional Huwe1 knockout causes N-Myc accumulation, DLL3 elevation, and Notch suppression, resulting in neural stem cell over-expansion and failure of neurogenesis.\",\n      \"method\": \"Conditional knockout mouse, gain- and loss-of-function experiments in mouse cortex, epistasis analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with pathway epistasis confirmed in vivo, multiple phenotypic readouts\",\n      \"pmids\": [\"19686682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Huwe1 loss in cerebellar granule neuron precursors causes N-Myc accumulation and uncontrolled proliferation; additional loss in Bergmann glia disrupts glial differentiation and granule neuron migration, demonstrating Huwe1 coordinates neuronal and glial programming.\",\n      \"method\": \"Conditional knockout mouse (cerebellar-specific), immunohistochemistry, N-Myc protein level assessment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with cell-type-specific phenotypes in vivo, substrate (N-Myc) accumulation confirmed\",\n      \"pmids\": [\"20231446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The C. elegans HUWE1 ortholog EEL-1 fine-tunes myotactin (hemidesmosome ECM receptor) abundance ~2-fold; EEL-1 activity is essential for hemidesmosomes to mature into a tension-resistant form during embryonic development.\",\n      \"method\": \"Genome-wide RNAi screen in C. elegans, quantitative protein level analysis, genetic interaction with plakin mutant\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi phenotype in vivo plus quantitative protein measurement, single lab\",\n      \"pmids\": [\"20153198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HUWE1 ubiquitinates MyoD at its N-terminal residue and targets it for proteasomal degradation, acting as an E3 ligase for this muscle-regulatory transcription factor.\",\n      \"method\": \"In vitro ubiquitination assay, proteasome inhibitor treatment, co-IP\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro ubiquitination assay plus co-IP, single lab, single study\",\n      \"pmids\": [\"22277673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HUWE1 is associated with the UFD substrate Ub(G76V)-YFP and the 26S proteasome; HUWE1 knockdown stabilizes ubiquitin-fusion degradation (UFD) substrates and the pathological UFD substrate UBB+1; HUWE1 and TRIP12 function in parallel to degrade UFD substrates.\",\n      \"method\": \"High-throughput siRNA imaging screen, co-IP with proteasome, substrate half-life analysis, double knockdown epistasis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (screen, co-IP, half-life), epistasis with TRIP12, single lab\",\n      \"pmids\": [\"23209776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ARF-BP1/HUWE1 forms a multiprotein complex with ARF, p53, MYC, and CTCF; HUWE1 ubiquitylates CTCF leading to its proteasomal degradation, linking the MYC proliferative and p53-ARF apoptotic pathways.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown with apoptosis/growth arrest readout\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP identifying multiprotein complex plus ubiquitination assay, single lab\",\n      \"pmids\": [\"22754359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HUWE1 ubiquitylates the antiapoptotic factor Mcl-1; MDM2 ubiquitylates HUWE1 itself; in HER2+ breast cancer cells treated with lapatinib, MDM2 degradation stabilizes HUWE1 enabling Mcl-1 degradation and apoptosis, while lapatinib-resistant cells maintain MDM2 to degrade HUWE1.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA knockdown, xenograft models, half-life analysis\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell-based assays plus in vivo xenograft, single lab\",\n      \"pmids\": [\"23652204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In Ras-driven skin tumorigenesis, Mule/HUWE1 suppresses tumor formation by preventing accumulation of c-Myc/Miz1 transcriptional complexes that otherwise downregulate p21 and p15; genetic knockout of c-Myc (but not p53 or p19Arf) reverses the Mule-KO tumorigenic phenotype.\",\n      \"method\": \"Conditional knockout mouse (K14Cre), chemical carcinogenesis model, genetic epistasis (c-Myc, p53, p19Arf co-knockouts), Miz1 knockdown rescue\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with genetic epistasis resolved by multiple co-KO experiments in one study\",\n      \"pmids\": [\"23699408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Huwe1 ubiquitylates Dishevelled (Dvl) with K63-linked polyubiquitin chains in a Wnt3a- and CK1ε-dependent manner; this inhibits Dvl DIX-domain multimerization required for Wnt signaling, creating a negative feedback loop upstream of β-catenin and downstream of CK1/LRP6.\",\n      \"method\": \"RNAi screen in C. elegans, reporter assay in HEK293T cells, co-IP, mass spectrometry for K63-linkage determination, Dvl multimerization assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — evolutionary conservation (worm + human), mass spectrometry linkage determination, genetic epistasis, and functional Dvl multimerization assay in one study\",\n      \"pmids\": [\"24643799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HUWE1 ubiquitylates and degrades TIAM1 preferentially at cell-cell adhesions in response to HGF, facilitating junction disassembly, cell scattering, migration, and invasion; simultaneous TIAM1 depletion rescues migration/invasion in HUWE1-depleted cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA knockdown, HGF stimulation assays, invasion assay, epistasis (TIAM1 double depletion), human lung cancer cell validation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, ubiquitination assay, epistasis, spatial localization at cell-cell adhesions, validated in cancer cells\",\n      \"pmids\": [\"25543140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HUWE1 binds Shoc2 and mediates its ubiquitination and degradation; HUWE1 also controls RAF-1 ubiquitination and its kinase levels through the Shoc2 scaffold; HUWE1 depletion abolishes RAF-1 ubiquitination, altering ERK1/2 pathway activity.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA knockdown, ERK1/2 activity measurement\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and ubiquitination assay with functional ERK pathway readout, single lab\",\n      \"pmids\": [\"25022756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HUWE1 is required for growth of colorectal cancer cells; HUWE1 associates with both MYC and MIZ1; HUWE1 inhibition stabilizes MIZ1, which accumulates on MYC target genes and represses MYC-activated transcription, revealing that continuous MIZ1 degradation is required for MYC transactivation.\",\n      \"method\": \"Small-molecule HUWE1 inhibitors (high-throughput screen), genetic knockdown, gene expression analysis, orthotopic xenograft models\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chemical and genetic inhibition converging on same mechanism, orthogonal in vitro and in vivo validation\",\n      \"pmids\": [\"25253726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HUWE1 degrades DDIT4 (REDD1) through ubiquitination; diGly proteomics coupled with inducible HUWE1 RNAi identified DDIT4 as a HUWE1 substrate; HUWE1 interacts with and regulates DDIT4 stability in cell-based assays.\",\n      \"method\": \"Inducible RNAi, quantitative diGly (ubiquitin remnant) proteomics, co-IP, protein half-life assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased proteomics plus co-IP and stability assay, single lab\",\n      \"pmids\": [\"25147182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Huwe1 phosphodependently degrades the transcription factor Atoh1; SHH signaling prevents this phosphodependent degradation, stabilizing Atoh1 to promote neuronal precursor expansion; Huwe1 loss in mouse SHH medulloblastoma disrupts this developmental mechanism.\",\n      \"method\": \"In vivo mouse cerebellar studies, genetic Huwe1 loss-of-function, phosphorylation analysis, protein stability assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic and biochemical analysis, single lab, mechanism partially inferred\",\n      \"pmids\": [\"24960692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HUWE1 mediates K48-linked polyubiquitination of Atoh1 following CK1-mediated phosphorylation at serine 334; this targets Atoh1 for proteasomal degradation; Huwe1 knockout in cochlear-supporting cells causes extra inner hair cells and synaptic defects.\",\n      \"method\": \"Co-IP, proximity ligation assay, mass spectrometry, cell-free ubiquitination system, CK1 inhibition, Huwe1 conditional knockout in cochlea\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — cell-free ubiquitination reconstitution, mass spectrometry phosphosite identification, CK1 epistasis, in vivo knockout phenotype\",\n      \"pmids\": [\"27542412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ATM kinase, SIRT6, and SNF2H cooperate to block HUWE1-mediated poly-ubiquitination of H2AX upon DSB formation, transiently stabilizing H2AX to allow efficient γH2AX foci formation and chromatin incorporation; ATM phosphorylation of H2AX Ser139 is essential for this stabilization.\",\n      \"method\": \"Proteasome inhibition, ubiquitination assay, siRNA knockdown of HUWE1/ATM/SIRT6/SNF2H, γH2AX foci analysis, Ser139 mutation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic perturbations converging on HUWE1 as the ubiquitin ligase, Ser139 mutagenesis, orthogonal methods\",\n      \"pmids\": [\"26711340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DNA damage activates CUL4B, which ubiquitinates HUWE1 in a NEDD8-dependent manner, targeting HUWE1 for proteasomal degradation; CUL4B depletion stabilizes HUWE1, accelerating MCL-1 degradation and increasing apoptosis, which is rescued by simultaneous HUWE1 depletion.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, NEDD8 inhibition, CUL4B siRNA knockdown, epistasis with HUWE1\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination reconstitution, in vivo epistasis, NEDD8 dependency tested\",\n      \"pmids\": [\"25883150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HUWE1 and NEDD4-1 are E3 ligases for ABCG1 and ABCG4; silencing both ligases increases ABCG1/ABCG4 protein levels and cholesterol export; overexpression of either ligase reduces transporter levels.\",\n      \"method\": \"LC-MS identification of ligases, siRNA silencing, overexpression, cholesterol export assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification plus functional validation, single lab\",\n      \"pmids\": [\"26296893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HUWE1 ubiquitinates unassembled soluble proteins bearing hydrophobic segments in the cytoplasm; this requires p97/NPL4; inhibition of nuclear entry enhances HUWE1-mediated ubiquitination, placing USPD primarily in the cytoplasm; SILAC proteomics identified nuclear complex subunits as endogenous HUWE1 substrates.\",\n      \"method\": \"Stable isotope labeling (SILAC) proteomics, in vitro ubiquitination, p97 inhibition, nuclear import inhibition\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution plus unbiased proteomics plus mechanistic epistasis with p97, single comprehensive study\",\n      \"pmids\": [\"27867533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HUWE1 is essential for replication of damaged DNA; HUWE1-knockout cells fail to mitigate replication stress; HUWE1 interacts with PCNA at stalled replication forks and mono-ubiquitinates H2AX to promote signaling at these sites.\",\n      \"method\": \"HUWE1 knockout cells, replication stress assays, co-IP with PCNA, H2AX ubiquitination assay, DNA fiber analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout with defined phenotype, co-IP demonstrating PCNA interaction at forks, mono-ubiquitination activity demonstrated, multiple orthogonal methods\",\n      \"pmids\": [\"27146073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Huwe1 is essential for HSC self-renewal, quiescence, and lymphoid-fate specification by post-translationally controlling N-myc; N-myc levels are upregulated after Huwe1 loss or stress, leading to HSC exhaustion; Mycn depletion reverses most Huwe1-KO phenotypes in vivo.\",\n      \"method\": \"Conditional knockout mouse, fluorescent Mycn fusion allele (live imaging), genetic rescue by Mycn deletion\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with in vivo fluorescent substrate tracking and genetic epistasis rescue\",\n      \"pmids\": [\"27668798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HUWE1 is a bona fide tumor suppressor in intestinal epithelium; identified mutations in colorectal cancer inactivate HUWE1 E3 ligase activity; Huwe1 deletion accelerates APC-loss-driven tumorigenesis through increased MYC levels and rapid DNA damage accumulation, leading to loss of the second APC allele.\",\n      \"method\": \"Modeling of clinical mutations in cells, conditional knockout in Apc-mutant mice, MYC protein level analysis, comet assay, epistasis with MCL1\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO in disease model, mutation functional validation, mechanistic epistasis\",\n      \"pmids\": [\"28003334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HUWE1 mediates UV-induced polyubiquitination of histone H1 at multiple lysines; HUWE1-depleted cells show reduced RNF168 and 53BP1 recruitment to DNA damage sites (downstream of RNF8-mediated H1 ubiquitination) but normal MDC1 recruitment (upstream); HUWE1 primes H1 to allow K63-chain elongation by RNF8.\",\n      \"method\": \"Quantitative diGly proteomics, siRNA knockdown, recruitment assays (RNF168, 53BP1, MDC1) at DNA damage sites\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased proteomics plus functional knockdown with ordered pathway epistasis, single lab\",\n      \"pmids\": [\"29127375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HUWE1 forms an auto-inhibited asymmetric dimer in solution and in cells through a crystallographic dimer interface; disruption of the dimer activates HUWE1; a conserved intramolecular segment counteracts dimerization; tumor suppressor p14ARF binds this segment and may shift HUWE1 toward the inactive state.\",\n      \"method\": \"Crystal structure determination, solution dimerization assays (biochemical), cellular self-association assays, HUWE1 activation upon dimer disruption, ARF binding experiment\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus multiple biochemical validation experiments demonstrating functional relevance of dimer interface\",\n      \"pmids\": [\"28193319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HUWE1 ubiquitinates histone H1.3, targeting it for proteasomal degradation; Huwe1-deficient cells accumulate H1.3, which silences H19 expression; H1.3 silencing partially rescues H19 expression and the Huwe1-null phenotype in ovarian epithelial transformation.\",\n      \"method\": \"Co-IP, ubiquitination assay, Huwe1 conditional knockout in mice, H1.3/H19 epistasis analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and ubiquitination assay in vivo confirmed with epistasis, single lab\",\n      \"pmids\": [\"28687618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HUWE1 promotes PINK1/PARKIN-independent mitophagy by enabling IKKα-mediated phosphorylation of AMBRA1 at serine 1014, which induces structural changes in AMBRA1 promoting its interaction with LC3/GABARAP proteins and mitophagic activity.\",\n      \"method\": \"Co-IP, siRNA knockdown, phosphorylation mutant analysis, mitophagy assays, IKKα kinase identification\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, mutagenesis, structural change, functional mitophagy assay), mechanistic pathway established\",\n      \"pmids\": [\"30217973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"mTORC1 phosphorylates WIPI2 at Ser395, directing it to interact specifically with HUWE1 for ubiquitination and proteasomal degradation; HUWE1 loss stabilizes WIPI2, enhances autophagosome formation, and improves hepatic lipid clearance in fasting mice.\",\n      \"method\": \"In vitro mTORC1 phosphorylation assay, co-IP, ubiquitination assay, HUWE1 siRNA in mouse liver, WIPI2 stabilization and autophagy flux measurement\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay identifying phosphodegron, co-IP, in vivo liver silencing, multiple orthogonal methods\",\n      \"pmids\": [\"30340022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HUWE1 promotes ubiquitination and proteasomal degradation of PPARα in coordination with the scaffold protein PAQR3; PAQR3 directly interacts with PPARα and enhances the PPARα–HUWE1 interaction; PAQR3 deficiency in liver increases PPARα-target gene expression during fasting.\",\n      \"method\": \"Co-IP, polyubiquitination assay, adenoviral knockdown/liver-specific KO of PAQR3, PPARα target gene expression, fasting metabolic assays\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vivo genetic model, ubiquitination assay, single lab\",\n      \"pmids\": [\"29331071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HUWE1 controls lung cancer cell malignancy by promoting proteasomal degradation of p53; HUWE1 inactivation increases p53, upregulates p21 and downregulates HIF1α; Huwe1 conditional deletion abolishes EGFRVIII-induced lung cancer in mice.\",\n      \"method\": \"siRNA/shRNA knockdown, soft agar and xenograft assays, western blot for p53/p21/HIF1α, genetically engineered Huwe1 conditional KO mouse\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO in vivo plus molecular mechanism via p53 pathway, single lab\",\n      \"pmids\": [\"30026863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HUWE1 controls AMBRA1-mediated mitophagy through MCL1 stability: MCL1 overexpression inhibits HUWE1 recruitment to mitochondria during AMBRA1-induced mitophagy; GSK-3β phosphorylates MCL1 at S159, which triggers HUWE1-dependent MCL1 degradation, enabling mitophagy to proceed.\",\n      \"method\": \"Co-IP, overexpression/knockdown, GSK-3β inhibitor treatment, phosphorylation site mutagenesis, mitophagy assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, phosphosite mutagenesis, and functional mitophagy readout, single lab\",\n      \"pmids\": [\"31434979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HUWE1 directly ubiquitinates Chk1 at multiple lysines within the kinase domain, controlling Chk1 protein stability; HUWE1 knockdown prolongs Chk1 half-life and elevates Chk1 levels independently of ATM/ATR and p53; replication stress reduces Chk1 levels in a HUWE1-dependent manner.\",\n      \"method\": \"HUWE1 siRNA knockdown, in vitro ubiquitination assay with lysine mapping, Chk1 half-life measurement, replication stress (HU, camptothecin)\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ubiquitination with residue mapping plus cellular stability assay, single lab\",\n      \"pmids\": [\"31713291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HUWE1 interacts with AIM2, NLRP3, and NLRC4 through specific domain interactions (BH3 domain of HUWE1; HIN domain of AIM2; NACHT domains of NLRP3/NLRC4); HUWE1 mediates K27-linked polyubiquitination of these inflammasome sensors, promoting inflammasome assembly, ASC speck formation, and caspase-1 activation.\",\n      \"method\": \"Co-IP, domain-mapping experiments, ubiquitination assay (K27 linkage), HUWE1-KO macrophages, HUWE1 inhibitor (BI8622), in vivo bacterial infection models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-mapping co-IP, ubiquitin linkage determination, KO cells and in vivo models all converge on same mechanism\",\n      \"pmids\": [\"33104527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HUWE1-dependent neddylation of DNA-PKcs at its kinase domain, catalyzed by E2 UBE2M, is required for DNA-PKcs autophosphorylation at Ser2056 and efficient NHEJ repair.\",\n      \"method\": \"Co-IP, neddylation assay, HUWE1 siRNA, DNA-PKcs autophosphorylation assay, NHEJ efficiency measurement\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional autophosphorylation and NHEJ assays, single lab\",\n      \"pmids\": [\"32457294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HAPSTR1 (C16orf72) is required for HUWE1 nuclear localization; nuclear HUWE1 is needed for cell proliferation and modulation of p53 and NF-κB stress signaling pathways; unbiased quantitative proteomics shows HUWE1 substrate targeting is largely cell-type-specific.\",\n      \"method\": \"Quantitative proteomics, HAPSTR1 KO, subcellular fractionation, p53/NF-κB reporter assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased proteomics, genetic KO demonstrating localization-function link, multiple pathway readouts\",\n      \"pmids\": [\"37167062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structures of full-length HUWE1 reveal an alpha-solenoid assembly with a central pore decorated with protein interaction modules; the N-terminal ~3900 amino acids are indispensable for proper ligase function; neurodevelopmental disorder-associated variants and substrate binding were mapped onto the three-dimensional organization.\",\n      \"method\": \"Cryo-EM structure determination, functional analysis of deletion constructs, structure of disease-variant HUWE1 and substrate-bound complex\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — comprehensive cryo-EM with functional validation, disease variant structures, and substrate binding in single study\",\n      \"pmids\": [\"34314700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of Nematocida HUWE1 reveals a snake-like alpha-solenoid ring with the C-terminal HECT domain at the head; the ring structure is highly dynamic, allowing the flexible HECT domain to access substrate-presenting acceptor sites distributed around the ring; this explains how HUWE1 ubiquitinates diverse unrelated substrates.\",\n      \"method\": \"Crystal structure determination, integrative structural analysis, functional validation of ring architecture\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus integrative structural analysis, functional model validated\",\n      \"pmids\": [\"34294896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HUWE1 ubiquitinates ATG101 for proteasomal degradation; HUWE1-depleted cells have stable ATG101 and elevated autophagy; HUWE1 thus suppresses autophagy flux by targeting both WIPI2 and ATG101.\",\n      \"method\": \"CRISPR KO of ATG101, co-IP, siRNA knockdown, autophagy assays, ATG101 domain mapping of ubiquitination site\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, domain mapping, functional autophagy readout, single lab\",\n      \"pmids\": [\"34502089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HUWE1 directly ubiquitinates transferrin receptor 1 (TfR1) for proteasomal degradation, thereby regulating cellular iron metabolism; Huwe1 hepatocyte-specific knockout leads to TfR1 accumulation, excess iron uptake, lipid peroxidation, and ferroptosis in acute liver injury models.\",\n      \"method\": \"Hepatocyte-specific Huwe1 KO mice, ubiquitination assay, TfR1 protein level analysis, ferroptosis markers, chemical/genetic TfR1 inhibition rescue\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo hepatocyte KO, ubiquitination assay, epistasis rescue by TfR1 inhibition, multiple orthogonal methods\",\n      \"pmids\": [\"35260822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HUWE1 promotes ubiquitination and degradation of EGFR; HUWE1 physically interacts with EGFR; HUWE1 overexpression decreases EGFR levels and protects against TGF-β-induced renal tubular injury.\",\n      \"method\": \"Co-IP, ubiquitination assay, overexpression/knockdown in HK-2 cells, UUO mouse model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ubiquitination assay plus in vivo model, single lab\",\n      \"pmids\": [\"32017279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HUWE1 controls TTP (tristetraprolin) stability indirectly by regulating its phosphorylation; HUWE1 function requires phosphatase and E3 ligase binding sites in the TTP C-terminus; HUWE1-regulated phosphorylation is independent of MAPK-mediated S52/S178 phosphorylation and controls TTP stability at late stages of pro-inflammatory stimulation.\",\n      \"method\": \"Genetic screen, pharmacological kinase inhibition, TTP phosphorylation analysis, stability assays, domain-mapping\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen, pharmacological dissection, domain mapping, single lab\",\n      \"pmids\": [\"36961408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HUWE1 acts as a centrosomal E3 ligase targeting TTBK2 for degradation; TTBK2 stabilizes primary cilia and promotes GNP proliferation in response to SHH; HUWE1-mediated TTBK2 degradation triggers primary cilia disassembly, thereby driving GNP differentiation and migration in cerebellar development.\",\n      \"method\": \"Conditional knockout mouse (cerebellar GNPs), co-IP, ubiquitination assay, TTBK2 overexpression/depletion, primary cilia dynamics assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO plus ubiquitination assay plus primary cilia functional assays, mechanistic pathway ordered by genetic epistasis\",\n      \"pmids\": [\"38879724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CDK9 recruits HUWE1 to ubiquitinate and degrade RARα; pharmacological CDK9 inhibition stabilizes RARα and synergizes with ATRA in suppressing CTCL tumor growth.\",\n      \"method\": \"Co-IP (CDK9–HUWE1 interaction), RARα ubiquitination assay, CDK9 PROTAC, in vitro and xenograft combination treatment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating CDK9–HUWE1 interaction, ubiquitination assay, functional in vivo validation, single lab\",\n      \"pmids\": [\"39632829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HUWE1 possesses a Ubiquitin-Directed ubiquitin Ligase (UDL) activity: it recognizes substrates and aggregates/condensates bearing high-density pre-existing ubiquitin chains and amplifies ubiquitin modifications on them; this recruits p97/VCP for processing and downstream proteasomal or autophagic clearance.\",\n      \"method\": \"Biochemical reconstitution, aggregate clearance assays, p97 co-recruitment assay, cell-cycle transition analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — reconstitution with novel UDL activity, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"37398461\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HUWE1 interacts with and promotes proteasomal degradation of BRCA1 via the ubiquitin-proteasome pathway; HUWE1 binds BRCA1 through an N-terminal degron domain; HUWE1 depletion increases BRCA1 levels and resistance to ionizing radiation and mitomycin.\",\n      \"method\": \"Co-IP, siRNA knockdown, BRCA1 half-life assay, clonogenic survival after IR/mitomycin\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus functional radiation sensitivity assay, single lab, two publications reporting same finding\",\n      \"pmids\": [\"24472556\", \"24342616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRL4B E3 ligase with the DCAF adaptor RBBP7 polyubiquitinates HUWE1 and targets it for proteasomal degradation; RBBP7 depletion stabilizes HUWE1 and accelerates MCL-1 and BRCA1 degradation.\",\n      \"method\": \"Co-IP identifying RBBP7 as DCAF, in vitro ubiquitination assay, RBBP7 overexpression/depletion, MCL-1/BRCA1 levels\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vitro ubiquitination identifying specific adaptor, functional substrate effects measured, single lab\",\n      \"pmids\": [\"29738775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HUWE1 ubiquitinates the GlyR-α1 subunit of glycine receptors in the spinal cord dorsal horn, reducing surface expression of GlyRs via endocytosis; HUWE1 knockdown blunts GlyR-α1 ubiquitination, potentiates glycinergic transmission, and attenuates inflammatory pain in mice.\",\n      \"method\": \"Co-IP (HUWE1–GlyR-α1 interaction), ubiquitination assay, shRNA knockdown in dorsal horn, surface expression assay, electrophysiology, CFA pain model\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, ubiquitination assay, in vivo knockdown with defined electrophysiological and behavioral phenotypes\",\n      \"pmids\": [\"30721695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HUWE1 also functions as an E3 ligase for Ezh1α (PRC2 complex component): reduced HUWE1 association is required for Ezh1α stabilization under oxidative stress in skeletal muscle cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, kinase inhibition of Ser560 phosphorylation on Ezh1β\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP observation with limited mechanistic follow-up, single lab\",\n      \"pmids\": [\"31856907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HUWE1 ubiquitinates Ets-1 transcription factor for proteasomal degradation; this interaction is regulated by Ets-1 Thr38 phosphorylation status; HUWE1-mediated Ets-1 degradation reduces Treg cell differentiation and function, causing immune imbalance in ITP.\",\n      \"method\": \"Co-IP, cycloheximide-chase assay, ubiquitination assay, Thr38 phosphorylation analysis, HUWE1 overexpression/knockdown, in vivo ITP mouse model with HUWE1 inhibitor\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, phosphosite analysis, in vivo pharmacological rescue, single lab\",\n      \"pmids\": [\"34900980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Increased HUWE1 dosage in Drosophila (modeling human duplication) decreases Dishevelled (dsh) protein levels ~50% and causes axon branching defects in dorsal cluster neurons; fz2 and dsh mutants phenocopy this; constitutively active β-catenin (armadillo) partially rescues, placing HUWE1 upstream of Wnt/β-catenin in axon development.\",\n      \"method\": \"Drosophila overexpression model, protein level quantification, genetic epistasis with dsh/fz2/armadillo mutants\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo Drosophila genetic epistasis with substrate level measurement, consistent with Wnt pathway finding in vertebrates\",\n      \"pmids\": [\"24303071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HUWE1 ubiquitinates TOMM20 directly and also regulates TOMM20 degradation via a PARKIN-mediated pathway; HUWE1 overexpression impairs mitochondrial ATP production and membrane potential, increasing ROS and apoptosis, sensitizing CRC cells to oxaliplatin.\",\n      \"method\": \"Co-IP, ubiquitination assay, mitochondrial function assays (ATP, membrane potential), HUWE1 overexpression, oxaliplatin sensitivity assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ubiquitination assay plus functional mitochondrial readouts, single lab\",\n      \"pmids\": [\"38184713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HUWE1 ubiquitinates and degrades MUTYH (DNA repair glycosylase) in renal tubular epithelial cells; upregulation of HUWE1 during AKI causes MUTYH depletion, leading to 8-oxoG accumulation, mitochondrial dysfunction, and tubular injury; HUWE1 KO or BI8622 treatment protects against cisplatin-induced AKI.\",\n      \"method\": \"Co-IP, ubiquitination assay, HUWE1 KO mice, BI8622 pharmacological inhibition, cisplatin/FA AKI models, 8-oxoG quantification, mitochondrial function assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo KO and pharmacological rescue, multiple functional readouts, single lab\",\n      \"pmids\": [\"39921445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"UREB1 (HUWE1) inhibits p53 transcriptional activity when co-expressed; this inhibitory effect requires tyrosine phosphorylation of UREB1, as a tyrosine-to-alanine mutation in the phosphorylation site significantly attenuates suppression.\",\n      \"method\": \"Transient co-transfection in p53-null H1299 cells, p53 reporter assay, tyrosine phosphorylation site mutagenesis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single reporter assay with mutagenesis, no direct ubiquitination demonstrated, early study predating molecular characterization\",\n      \"pmids\": [\"7478539\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HUWE1 is a giant HECT-domain E3 ubiquitin ligase that adopts an alpha-solenoid ring structure presenting multiple substrate-binding modules to a flexible catalytic HECT domain; its activity is regulated by auto-inhibitory dimerization (relieved by ARF) and by phosphorylation/neddylation-controlled substrate recruitment. HUWE1 ubiquitinates a wide array of substrates—including N-Myc/c-Myc (K48-linked, for proteasomal degradation controlling neural stem cell differentiation and HSC quiescence), Mcl-1 (pro-apoptotic), p53 (degradation), Cdc6 (DNA-damage-responsive degradation), WIPI2 and ATG101 (mTORC1-directed autophagy suppression), Dishevelled (K63-linked, Wnt negative feedback), Atoh1/Shh-dependent phosphodegron (neural progenitor differentiation), TfR1 (iron metabolism/ferroptosis suppression), TTBK2 (primary cilia disassembly), GlyR-α1 (synaptic inhibition), H2AX (mono-ubiquitination at stalled forks), histone H1 (UV-damage DDR priming), DNA-PKcs (neddylation for NHEJ), and many unassembled soluble nuclear proteins (protein quality control); HUWE1 is itself regulated by CRL4B(RBBP7)-mediated ubiquitination after DNA damage, and its nuclear functions are gated by the adaptor HAPSTR1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HUWE1 is a giant HECT-domain E3 ubiquitin ligase that uses a dynamic alpha-solenoid ring architecture to present a flexible catalytic HECT domain to a structurally diverse repertoire of substrates, thereby acting as a master regulator of protein turnover across development, the DNA-damage response, cell death, and metabolism [#38, #39]. Cryo-EM and crystallographic studies show that the N-terminal ~3900 residues form a ring decorated with substrate-interaction modules whose mobility relative to the HECT head explains how a single ligase ubiquitinates unrelated targets [#38, #39], and its catalytic output is tuned by an N-terminal HECT helix that differentially controls self- versus substrate-ubiquitination [#3] and by an auto-inhibited asymmetric dimer that is relieved upon dimer disruption and modulated by p14ARF binding [#27]. A dominant theme is control of MYC-family oncoproteins: HUWE1 catalyzes K48-linked polyubiquitination and proteasomal degradation of N-Myc to drive cell-cycle exit and neuronal differentiation, acting upstream of the N-Myc–DLL3–Notch axis in neural stem cells and of N-myc-dependent HSC quiescence [#0, #4, #24], while in intestinal and skin epithelia it suppresses c-Myc/Miz1 transcriptional output and functions as a bona fide tumor suppressor whose cancer-associated mutations inactivate its ligase activity [#11, #15, #25]. HUWE1 broadly governs apoptosis and the DNA-damage response by degrading Mcl-1 [#10], p53 [#1], and Cdc6 [#2], by mono-ubiquitinating H2AX at stalled forks via PCNA [#19, #23], and by priming histone H1 for RNF8-dependent chain elongation to recruit RNF168/53BP1 [#26]. It additionally suppresses autophagy by degrading WIPI2 and ATG101 downstream of mTORC1 [#30, #40], drives AMBRA1/MCL1-dependent mitophagy [#29, #33], imposes negative feedback on Wnt signaling through K63-linked ubiquitination of Dishevelled [#12, #52], controls iron homeostasis and ferroptosis via TfR1 degradation [#41], promotes inflammasome assembly through K27-linked ubiquitination of AIM2/NLRP3/NLRC4 [#35], and performs cytoplasmic protein quality control of unassembled hydrophobic-segment-bearing proteins in a p97/NPL4-dependent manner [#22]. HUWE1 itself is a regulated node: it is targeted for degradation by CRL4B/CUL4B(RBBP7) in a NEDD8-dependent, DNA-damage-responsive manner [#20, #48], and its nuclear localization and stress-pathway functions are gated by the adaptor HAPSTR1 [#37].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established the earliest functional link between HUWE1 (UREB1) and p53, indicating it negatively regulates p53 transcriptional activity before any ligase mechanism was known.\",\n      \"evidence\": \"Co-transfection p53 reporter assay with tyrosine phosphosite mutagenesis in p53-null cells\",\n      \"pmids\": [\"7478539\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No ubiquitination demonstrated\", \"Mechanism of phosphorylation-dependent suppression not defined\", \"Single reporter readout, not independently confirmed at the time\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved that HUWE1 acts as an E3 ligase for p53, showing the HECT catalytic domain drives p53 proteasomal degradation.\",\n      \"evidence\": \"Overexpression, p53 reporter, MG132 rescue, and HECT catalytic point mutants\",\n      \"pmids\": [\"15567145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage type on p53 not defined\", \"Degron on p53 unmapped\", \"Overexpression-based, physiological context limited\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated a DNA-damage-responsive substrate role by showing HUWE1 directly polyubiquitinates Cdc6 for degradation, with evolutionary conservation in the yeast ortholog Tom1.\",\n      \"evidence\": \"In vitro polyubiquitination, co-IP, siRNA, and yeast genetics\",\n      \"pmids\": [\"17567951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage on Cdc6 not specified\", \"Signal coupling damage to Cdc6 recognition unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the canonical developmental role of HUWE1: K48-linked ubiquitination and degradation of N-Myc enforces cell-cycle exit and neuronal differentiation, validated by genetic rescue.\",\n      \"evidence\": \"In vitro ubiquitination with K48 linkage, knockout/RNAi, and N-myc-silencing rescue in ES cells and mouse cortex\",\n      \"pmids\": [\"18488021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron/phosphorylation requirements for N-Myc recognition not defined here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placed HUWE1 in a defined developmental signaling circuit (N-Myc–DLL3–Notch) and provided structural insight into how the HECT domain self-regulates activity.\",\n      \"evidence\": \"Conditional knockout mouse with pathway epistasis; crystal structure plus deletion mutagenesis and in vitro activity assays\",\n      \"pmids\": [\"19686682\", \"20007713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length regulatory architecture not yet resolved\", \"How HECT helix discriminates substrates mechanistically unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established HUWE1 as a context-dependent tumor suppressor that restrains c-Myc/Miz1 oncogenic transcription, and revealed reciprocal regulation by MDM2.\",\n      \"evidence\": \"Conditional knockout chemical carcinogenesis with c-Myc/p53/p19Arf co-knockout epistasis; co-IP, ubiquitination, and HER2+ xenograft analyses\",\n      \"pmids\": [\"23699408\", \"23652204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of tumor-suppressor versus oncogenic outcome across tissues unresolved\", \"MDM2-HUWE1 hierarchy in normal physiology unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Broadly expanded the substrate landscape into signaling and adhesion control, including K63-linked Dishevelled ubiquitination as Wnt negative feedback, TIAM1 degradation at junctions, MIZ1 turnover required for MYC transactivation, and metabolic/transcriptional targets.\",\n      \"evidence\": \"Cross-species RNAi, mass-spectrometry linkage determination, co-IP, ubiquitination assays, HUWE1 small-molecule inhibitors, and diGly proteomics\",\n      \"pmids\": [\"24643799\", \"25543140\", \"25253726\", \"25147182\", \"25022756\", \"22277673\", \"24472556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How distinct chain linkages are selected per substrate unresolved\", \"Cell-type specificity of substrate choice not explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined HUWE1's protein quality-control function for unassembled cytoplasmic hydrophobic-segment proteins and its role in replication stress via PCNA-coupled H2AX mono-ubiquitination.\",\n      \"evidence\": \"SILAC proteomics with p97/nuclear-import inhibition; knockout cells, DNA fiber analysis, and PCNA co-IP\",\n      \"pmids\": [\"27867533\", \"27146073\", \"26711340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recognition code for orphan hydrophobic segments not defined\", \"Coordination of fork-localized activity with bulk QC unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the autoregulatory mechanism: HUWE1 forms an auto-inhibited asymmetric dimer relieved by an intramolecular segment and modulated by p14ARF, and extended H1-based DDR priming and tissue-specific Atoh1 control.\",\n      \"evidence\": \"Crystal structure with dimerization and activation assays; diGly proteomics with ordered RNF168/53BP1 recruitment; in vivo cochlear knockout\",\n      \"pmids\": [\"28193319\", \"29127375\", \"27542412\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell triggers of dimer disruption not identified\", \"Quantitative contribution of ARF in vivo unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected HUWE1 to autophagy/mitophagy and metabolic regulation, showing mTORC1-phosphorylated WIPI2 degradation suppresses autophagy and IKKα/AMBRA1 phosphorylation drives PINK1/PARKIN-independent mitophagy.\",\n      \"evidence\": \"In vitro kinase and ubiquitination assays, co-IP, phospho-mutant analysis, and in vivo liver silencing\",\n      \"pmids\": [\"30340022\", \"30217973\", \"29331071\", \"30026863\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Switch between autophagy suppression and mitophagy promotion not reconciled\", \"Substrate selection under nutrient stress incompletely mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified non-degradative and atypical-linkage activities (K27 ubiquitination of inflammasome sensors, neddylation of DNA-PKcs) and the adaptor HAPSTR1 gating nuclear HUWE1 function.\",\n      \"evidence\": \"Domain-mapping co-IP, K27 linkage determination, KO macrophages and infection models; neddylation assays; quantitative proteomics with HAPSTR1 KO and fractionation\",\n      \"pmids\": [\"33104527\", \"32457294\", \"37167062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for switching between ubiquitin and NEDD8 transfer unknown\", \"How HAPSTR1 directs nuclear substrate selection unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Delivered the full-length architectural model: alpha-solenoid ring with a mobile HECT head presenting substrates around the ring, explaining promiscuous substrate engagement and mapping neurodevelopmental disorder variants.\",\n      \"evidence\": \"Cryo-EM and crystal structures with deletion-construct functional validation and substrate-bound complexes\",\n      \"pmids\": [\"34314700\", \"34294896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How specific substrate modules are licensed in cells unresolved\", \"Conformational dynamics during catalysis not directly visualized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended HUWE1's physiological reach to iron homeostasis and ferroptosis through TfR1 degradation, with hepatocyte-specific genetic and epistatic validation.\",\n      \"evidence\": \"Hepatocyte-specific knockout mice, ubiquitination assays, and TfR1 inhibition rescue\",\n      \"pmids\": [\"35260822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal coupling HUWE1 activity to iron status not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a centrosomal role controlling ciliary dynamics and SHH-driven proliferation by degrading TTBK2 to trigger primary cilia disassembly during cerebellar development.\",\n      \"evidence\": \"Conditional knockout in cerebellar granule precursors, co-IP, ubiquitination, and cilia dynamics assays\",\n      \"pmids\": [\"38879724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How HUWE1 is recruited to the centrosome unresolved\", \"Spatial control of TTBK2 access not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HUWE1 achieves substrate, linkage, and tissue specificity from a single promiscuous ring-shaped ligase—and how its proposed ubiquitin-directed ligase activity integrates with adaptor- and phosphorylation-gated recruitment—remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking conformational state to substrate choice\", \"UDL activity reported only in preprint, awaiting peer review\", \"Adaptor inventory beyond HAPSTR1/PAQR3 incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 35, 36]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 19, 23, 35]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 12, 35]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 2, 30]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [27, 37]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [22, 23, 26, 37]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [29, 33, 53]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [44]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [13, 49]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 22]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 19, 23, 26, 36]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 20, 33]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [29, 30, 40]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 4, 5, 17, 44]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 14, 52]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [35, 51]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 15, 25]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MYCN\", \"MYC\", \"MIZ1\", \"MCL1\", \"TP53\", \"DVL\", \"PCNA\", \"HAPSTR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}