{"gene":"STUB1","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2017,"finding":"STUB1, a chaperone-dependent E3 ubiquitin ligase, preferentially interacts with and ubiquitinates phosphorylated (inactive) TFEB, targeting it for proteasomal degradation. STUB1 deficiency leads to accumulation of phosphorylated TFEB and reduced TFEB transcriptional activity, reduced autophagy, and reduced mitochondrial biogenesis.","method":"Immunoprecipitation, immunoblot, STUB1 knockout cells and STUB1-deficient mice, overexpression studies","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, KO cell and mouse model with specific phenotypic readouts, multiple orthogonal methods in single study","pmids":["28754656"],"is_preprint":false},{"year":2013,"finding":"STUB1 is required for degradation of HIF1A by chaperone-mediated autophagy (CMA). STUB1 mutations that abolish either E3 ubiquitin ligase activity or HSPA8-binding prevent HIF1A lysosomal degradation. STUB1 functions downstream of HSPA8 and the CMA receptor LAMP2A to mediate HIF1A ubiquitination and translocation into lysosomes.","method":"Mutagenesis of STUB1 (ligase-dead and HSPA8-binding mutants), lysosome isolation/translocation assay, immunoprecipitation, immunoblot","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site and binding-domain mutagenesis combined with lysosome translocation reconstitution and in vivo starvation models","pmids":["23880665"],"is_preprint":false},{"year":2014,"finding":"STUB1 interacts with IL-4Rα and targets it for ubiquitination-mediated proteasomal degradation, terminating IL-4/IL-13 signaling. STUB1 knockout cells show increased IL-4Rα levels and sustained STAT6 activation; STUB1-deficient mice exhibit spontaneous airway inflammation, M2 macrophage activation, and elevated serum IgE.","method":"Co-immunoprecipitation, immunoblot, flow cytometry, STUB1 KO mouse model, in vivo lung inflammation measurements","journal":"American journal of respiratory and critical care medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, KO mouse phenotype, multiple orthogonal methods across in vitro and in vivo systems","pmids":["24251647"],"is_preprint":false},{"year":2013,"finding":"CHIP/STUB1 negatively regulates NF-κB signaling in colorectal cancer cells by promoting ubiquitination and proteasomal degradation of p65, a NF-κB subunit, leading to decreased expression of NF-κB target oncogenes (Cyclin D1, c-Myc, MMP-2, VEGF, IL-8).","method":"Overexpression and RNAi knockdown in HCT-116 cells, ubiquitination assay, nude mouse tumor growth assay, immunoblot","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD/OE with specific pathway readouts, single lab, multiple methods","pmids":["24302614"],"is_preprint":false},{"year":2017,"finding":"CHIP/STUB1 (U-box E3 ligase) suppresses the Warburg effect in ovarian carcinoma by interacting with and mediating ubiquitin-proteasomal degradation of pyruvate kinase isoenzyme M2 (PKM2), thereby regulating glycolytic metabolism.","method":"SiLAD proteomics, Co-IP, ubiquitination assay, CHIP KO MEF cells, in vitro and in vivo tumor growth assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — proteomics substrate identification, Co-IP, KO cell validation, in vivo xenograft, multiple orthogonal methods","pmids":["28346425"],"is_preprint":false},{"year":2020,"finding":"STUB1 specifically ubiquitinates purified human proteasomes in vitro, mainly via Lys63-linked chains. Inhibited proteasomes are sequestered into aggresomes via HDAC6- and dynein-mediated transport and cleared through selective macroautophagy (proteaphagy); STUB1 activity is required for this proteasome quality-control pathway.","method":"In vitro ubiquitination assay with purified proteasomes, genetic and chemical inhibition of STUB1, live imaging, colocalization with SQSTM1/HDAC6","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of ubiquitination, genetic inhibition with specific functional readout, single lab with multiple orthogonal approaches","pmids":["32723828"],"is_preprint":false},{"year":2020,"finding":"STUB1 promotes ubiquitination of BMAL1 via Lys-48-linked polyubiquitin chains, leading to its proteasomal degradation. Oxidative stress promotes STUB1 nuclear translocation and co-localization with BMAL1. STUB1-mediated BMAL1 degradation attenuates hydrogen peroxide-induced cell senescence.","method":"Affinity purification-MS, Co-IP, domain mapping, ubiquitination assay, proteasome inhibition, nuclear fractionation/localization, senescence-associated β-gal staining","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS substrate identification confirmed by Co-IP, K48-linkage specificity, domain mapping, localization with functional consequence, multiple orthogonal methods","pmids":["32041778"],"is_preprint":false},{"year":2022,"finding":"STUB1 is an E3 ubiquitin ligase for the IFNγ receptor subunit IFNγ-R1 in complex with JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of the IFNγ-R1/JAK1 complex through specific lysine residues IFNγ-R1K285 and JAK1K249, thereby suppressing tumor IFNγ signaling.","method":"Genome-wide CRISPR/Cas9 screen, Co-IP, ubiquitination assay, site-directed mutagenesis of ubiquitination sites, cytotoxic T-cell killing assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — unbiased CRISPR screen, Co-IP, mutagenesis of specific ubiquitination sites, functional readout in multiple systems","pmids":["35395848"],"is_preprint":false},{"year":2020,"finding":"Hsc70 mediates translocation of STUB1 onto oxidatively stressed peroxisomes, promoting their selective ubiquitination and autophagic degradation (pexophagy). Artificially targeting STUB1 to healthy peroxisomes is sufficient to trigger pexophagy. STUB1 ataxia patient mutants are defective in pexophagy induction.","method":"Live-cell imaging with optogenetic ROS induction, confocal microscopy, Stub1 targeting assay, patient mutant analysis, immunofluorescence","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with acute ROS induction, gain-of-function targeting experiment, patient mutant functional validation, multiple orthogonal methods","pmids":["33077711"],"is_preprint":false},{"year":2013,"finding":"STUB1 constitutively interacts with the adaptor protein CARMA1, with interaction intensified by TCR stimulation. STUB1 ubiquitinates CARMA1 via Lys-27-linked chains, which is required for TCR-induced canonical NF-κB activation and IL-2 production. STUB1 knockdown abolishes endogenous CARMA1 ubiquitination and NF-κB activation downstream of TCR.","method":"Co-immunoprecipitation, RNAi knockdown, overexpression, ubiquitin linkage-specific analysis, NF-κB reporter assay, IL-2 measurement","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, KD with specific signaling readout, ubiquitin linkage characterization, single lab","pmids":["23322406"],"is_preprint":false},{"year":2014,"finding":"Hsp70 and Hsp90 oppositely regulate CHIP/STUB1-mediated ubiquitination and degradation of Smad3. Overexpressed Hsp70 or Hsp90 inhibition (by geldanamycin) facilitates CHIP-induced Smad3 ubiquitination and degradation (enhancing TGF-β signaling sensitivity), while overexpressed Hsp90 antagonizes CHIP-mediated Smad3 ubiquitination (desensitizing TGF-β signaling).","method":"Co-immunoprecipitation, overexpression, pharmacological Hsp90 inhibition, ubiquitination assay, TGF-β signaling readouts","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, pharmacological and genetic perturbation, ubiquitination assay; single lab with multiple methods","pmids":["24613385"],"is_preprint":false},{"year":2019,"finding":"STUB1 ubiquitinates YAP1 at K280 via K48-linked polyubiquitination, promoting YAP1 proteasomal degradation and inhibiting cancer cell survival and chemoresistance in gastric cancer.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis (K280), immunoblot, cell viability assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, specific ubiquitination site mutagenesis, functional readout; single lab","pmids":["31393050"],"is_preprint":false},{"year":2017,"finding":"STUB1 binds to RUNX1 and induces its ubiquitination and proteasomal degradation, primarily in the nucleus, and also promotes nuclear export of RUNX1. STUB1 similarly ubiquitinates the leukemogenic fusion RUNX1-RUNX1T1. Overexpression of STUB1 inhibits growth of myeloid leukemia cells harboring RUNX1-RUNX1T1.","method":"High-throughput binding assay, Co-IP, ubiquitination assay, immunofluorescence for nuclear/cytoplasmic localization, cell growth assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — high-throughput binding screen confirmed by Co-IP, ubiquitination, localization imaging, functional growth assay; single lab","pmids":["28536267"],"is_preprint":false},{"year":2023,"finding":"CHIP/STUB1 binds ~10-fold more strongly to phosphorylated tau than unmodified tau via its TPR domain (partially distinct from canonical binding mode). Sub-stoichiometric CHIP strongly suppresses aggregation and seeding of phosphorylated tau and promotes rapid ubiquitination of phosphorylated (but not unmodified) tau in vitro. In cells, CHIP restricts seeding by phosphorylated tau.","method":"In vitro binding assay (panel of TPR-domain proteins), aggregation assay, in vitro ubiquitination assay, TPR domain mutagenesis, cell-based seeding assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of ubiquitination, aggregation suppression assay, domain mutagenesis, cell-based validation; multiple orthogonal methods in single study","pmids":["37330289"],"is_preprint":false},{"year":2023,"finding":"HSP90β inhibits STUB1-induced ubiquitination and degradation of YTHDF2 in the cytoplasm of hepatocellular carcinoma cells. The large and small middle domain of HSP90β is required for its interaction with both STUB1 and YTHDF2. STUB1 directly interacts with YTHDF2 to promote its ubiquitination-mediated proteasomal degradation.","method":"Co-immunoprecipitation, domain deletion mapping, ubiquitination assay, overexpression and knockdown, immunoblot","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, single lab with multiple methods","pmids":["37515378"],"is_preprint":false},{"year":2023,"finding":"STUB1 is the E3 ubiquitin ligase for METTL14, directly interacting with it and mediating its ubiquitination at K148, K156, and K162 for proteasomal degradation. METTL3 competitively protects METTL14 from STUB1-mediated degradation via METTL3 amino acid regions 450-454 and 464-480, thereby maintaining m6A homeostasis.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis of ubiquitination sites, domain mapping of METTL3, m6A quantification, immunoblot","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-specific mutagenesis of ubiquitination sites, domain mapping, m6A functional readout, multiple orthogonal methods in single study","pmids":["36597993"],"is_preprint":false},{"year":2020,"finding":"PDLIM5 interacts with SMAD3 (but not SMAD2) and competitively suppresses the interaction between SMAD3 and its E3 ubiquitin ligase STUB1, protecting SMAD3 from STUB1-mediated proteasomal degradation and maintaining TGF-β signaling in NSCLC cells.","method":"Co-IP, knockdown/overexpression, ubiquitination assay, immunoblot, rescue experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, competitive binding assay, rescue experiment with STUB1 KD; single lab with multiple methods","pmids":["32737199"],"is_preprint":false},{"year":2022,"finding":"STUB1 promotes K63-linked non-degradative ubiquitination of aryl hydrocarbon receptor (AHR), facilitating Th17/Treg cell imbalance in rheumatoid arthritis. Specific ubiquitination sites on AHR were identified as responsible for STUB1-mediated K63 ubiquitination.","method":"Western blot, flow cytometry, siRNA knockdown, EROD enzymatic activity assay, ubiquitination assay with linkage-specific analysis","journal":"Clinical and experimental immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay with linkage specificity, enzymatic activity readout, KD with functional cellular phenotype; single lab","pmids":["35943876"],"is_preprint":false},{"year":2022,"finding":"STUB1 promotes K48-linked polyubiquitination of AGO2 and Dicer (including aviDicer), targeting them for proteasomal degradation in a chaperone-dependent manner. STUB1 also induces degradation of AGO1, AGO3, and AGO4. STUB1 deficiency upregulates Dicer and AGO2, enhancing antiviral RNAi and inhibiting viral replication in mammalian cells and newborn mouse models.","method":"Co-IP, ubiquitination assay with K48-linkage analysis, overexpression/knockdown, in vivo newborn mouse model with virus-derived siRNA measurement","journal":"Virologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, K48 linkage-specific ubiquitination, in vivo validation; single lab with multiple methods","pmids":["35533808"],"is_preprint":false},{"year":2017,"finding":"CHIP/STUB1, when freed from chaperones during acute stress, docks on cellular membranes (phosphatidic acid and phosphatidylinositol-4-phosphate enhance association), performing a proteostasis sensor function. HSP70 and membranes compete for mutually exclusive binding to the TPR domain of CHIP. At new cellular locations, CHIP participates in Golgi apparatus fragmentation.","method":"In vitro reconstitution with liposomes, competition assay (HSP70 vs. membrane), cellular imaging of Golgi fragmentation, lipid binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with liposomes, competition binding assay, live-cell imaging; multiple orthogonal methods in single study","pmids":["29091030"],"is_preprint":false},{"year":2015,"finding":"CHIP/STUB1 knockdown increases PTEN protein levels and decreases AKT/mTOR activity and ULK1 Ser757 phosphorylation, promoting autophagosome formation. However, CHIP knockdown also disturbs autophagic flux (impairs p62 substrate degradation) and increases susceptibility to autophagic cell death induced by bafilomycin A1.","method":"RNAi knockdown, immunoblot for autophagy pathway components (PTEN, AKT, mTOR, ULK1, p62), bafilomycin A1 treatment, cell death assay","journal":"Neuroscience bulletin","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with specific pathway signaling readouts, substrate flux assay, pharmacological perturbation; single lab","pmids":["26219223"],"is_preprint":false},{"year":2022,"finding":"The E3 ubiquitin ligase STUB1 mediates ubiquitination and proteasomal degradation of Sox2 and Nanog via K48-linked chains, and Oct4 via K63-linked chains. STUB1 deficiency enhances somatic cell reprogramming and delays ESC differentiation, while enforced STUB1 expression triggers ESC differentiation.","method":"CRISPR-Cas9 KO library screen, Co-IP, K48/K63 linkage-specific ubiquitination assays, protein half-life measurement, reprogramming and differentiation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — unbiased CRISPR screen, K48/K63 linkage-specific ubiquitination, protein half-life assays, functional reprogramming/differentiation phenotype; multiple orthogonal methods","pmids":["35675767"],"is_preprint":false},{"year":2020,"finding":"STUB1 deficiency leads to intracellular accumulation of protein aggregates and increased secretion of small extracellular vesicles (exosomes) enriched in ubiquitinated and/or undegraded proteins and oligomers. Oxidative stress augments sEV release in STUB1-depleted cells, indicating that STUB1 and exosomes cooperate to maintain proteostasis.","method":"STUB1 KO/knockdown, nanoparticle tracking analysis of sEVs, immunoblot for ubiquitinated proteins in sEVs, oxidative stress treatment","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with specific vesicle secretion phenotype, oxidative stress perturbation, single lab with multiple methods","pmids":["31613922"],"is_preprint":false},{"year":2023,"finding":"Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated ubiquitination of GPX4 at site K191, leading to GPX4 degradation. STUB1 knockdown or GPX4 overexpression reverses imatinib-induced ferroptosis.","method":"Co-IP, ubiquitination assay with site-directed mutagenesis (K191), GPX4 knockdown/overexpression, ferroptosis markers (lipid ROS, Fe2+, GSH), in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis of ubiquitination site, functional ferroptosis readout, in vivo validation; single lab","pmids":["38110356"],"is_preprint":false},{"year":2024,"finding":"STUB1 mediates ubiquitination of NSUN2 at lysines K457 and K654, promoting NSUN2 proteasomal degradation during ferroptosis. NSUN2 degradation diminishes m5C methylation of Gpx4 mRNA 3' UTR, reducing GPX4 protein expression and promoting hepatocyte ferroptosis.","method":"Co-IP, ubiquitination assay with site-specific mutagenesis, m5C methylation assay, SECIS-SBP2 interaction assay, NSUN2 knockdown/overexpression, in vivo and in vitro ferroptosis models","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-directed mutagenesis of ubiquitination sites, RNA methylation functional readout, in vivo validation; multiple orthogonal methods in single study","pmids":["39453812"],"is_preprint":false},{"year":2023,"finding":"STUB1 mediates ubiquitination and proteasomal degradation of GLUD1 at lysine K503, regulating glutamine catabolism in lung adenocarcinoma. Inhibition of K503 ubiquitination promotes proliferation and tumor growth.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis (K503), cell proliferation assay, in vivo tumor growth","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis, ubiquitination assay, functional tumor growth readout; single lab","pmids":["37416474"],"is_preprint":false},{"year":2021,"finding":"CHIP/STUB1 targets newly synthesized, HSP90/HSC70-associated ErbB2 for ubiquitin/proteasome-dependent degradation in the endoplasmic reticulum and Golgi, establishing a mechanism for negative regulation of cell surface ErbB2 levels in breast cancer cells.","method":"STUB1 knockdown and overexpression, proteasome inhibition, ER/Golgi fractionation, immunoblot, ErbB2 surface expression measurement","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD/OE with subcellular fractionation, pharmacological inhibition; single lab with multiple methods","pmids":["34439093"],"is_preprint":false},{"year":2014,"finding":"CHIP/STUB1 directly interacts with eIF5A preferentially through its U-box domain, mediating eIF5A ubiquitination and proteasomal degradation. CHIP expression inversely correlates with eIF5A levels in colorectal cancers and in CHIP KO MEF cells.","method":"Proteomics identification, Co-IP, domain mapping (U-box), ubiquitination assay, CHIP KO MEF cells, immunoblot","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics + Co-IP with domain mapping, KO cell validation; single lab","pmids":["24509416"],"is_preprint":false},{"year":2020,"finding":"STUB1 is targeted by the SUMO-interacting motif SIM3 of EBV EBNA1. The SIM3 motif mediates EBNA1's inhibitory effects on SUMO2-modified STUB1 and regulates SUMO2-mediated degradation of USP7. Hypoxic stress induces dissociation of EBNA1 from STUB1, increasing SUMO1 modification of STUB1 and KAP1 to promote lytic reactivation.","method":"Proteomic analysis, Co-IP, mutagenesis of SIM motifs, ubiquitination/SUMOylation assays, viral replication assays","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic + Co-IP, SIM mutagenesis, functional viral replication readout; single lab","pmids":["32176739"],"is_preprint":false},{"year":2024,"finding":"BCKDK phosphorylates STUB1 at S19, disrupting the STUB1-BCAT1 interaction and inhibiting STUB1-mediated ubiquitination and degradation of BCAT1, thereby stabilizing BCAT1 in glioblastoma.","method":"Co-IP, phosphorylation assay, ubiquitination assay, site-directed mutagenesis, in vivo and in vitro tumor models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, phospho-specific mutagenesis, ubiquitination assay, functional readout; single lab with multiple methods","pmids":["38621458"],"is_preprint":false},{"year":2024,"finding":"STUB1 is acetylated by KAT5 (lysine acetyltransferase 5), and KAT5 promotes STUB1 transcription via acetylation modulation. STUB1 then ubiquitinates and promotes degradation of LATS2, activating the YAP/β-catenin pathway and inhibiting NLRP3-mediated cardiomyocyte pyroptosis during myocardial ischemia-reperfusion injury.","method":"Acetylation assay, ubiquitination assay, KAT5/STUB1 overexpression/knockdown, LATS2 stability assay, pyroptosis markers, in vivo MIRI model","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — acetylation/ubiquitination assays, multiple KO/OE experiments with pathway readouts, in vivo model; single lab","pmids":["38561411"],"is_preprint":false},{"year":2023,"finding":"Salidroside increases STUB1 expression in Tregs and promotes STUB1-mediated degradation of Foxp3 in the nucleus, suppressing Treg function. Hsp70 is required for the colocalization of STUB1 and Foxp3 in the nucleus; Hsp70 inhibition reverses SAL-induced suppression of Foxp3 and disrupts the STUB1-Foxp3 colocalization.","method":"Flow cytometry, confocal laser microscopy for nuclear colocalization, Hsp70 inhibitor treatment, network pharmacology and molecular docking","journal":"BMC cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — colocalization imaging and pharmacological inhibition without direct ubiquitination assay or mutagenesis; single lab","pmids":["37528345"],"is_preprint":false},{"year":2020,"finding":"Heterozygous missense variants in STUB1 (p.Ile53Thr and p.Thr37Leu) cause autosomal dominant ataxia. Neuropathological examination reveals selective Purkinje cell loss and aberrant STUB1 localization in distal Purkinje cell dendritic arbors (loss of normal somatodendritic polarization), linking STUB1 mislocalization to cerebellar pathogenesis.","method":"Exome sequencing, neuropathological examination, immunofluorescence localization of STUB1 in post-mortem brain tissue","journal":"Neurology. Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct immunofluorescence localization in human post-mortem brain with genetic variant, functional consequence (Purkinje cell loss); single study","pmids":["32211513"],"is_preprint":false},{"year":2023,"finding":"STUB1 mediates ubiquitination of HOXB3, inhibiting its expression; loss of HOXB3 reduces PARK7 transcription (as HOXB3 binds PARK7 promoter), thereby promoting ferroptosis and suppressing paclitaxel resistance in ovarian cancer.","method":"Co-IP, dual luciferase reporter assay, ubiquitination assay, ChIP for HOXB3 binding to PARK7 promoter, in vivo xenograft","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, reporter assay, in vivo validation; single lab with multiple methods","pmids":["39501077"],"is_preprint":false},{"year":2023,"finding":"STUB1 ubiquitinates and promotes degradation of SMYD2 in glioma cells treated with cisplatin; STUB1 knockdown reverses cisplatin-induced SMYD2 degradation and partially restores cell function.","method":"UbiBrowser prediction confirmed by knockdown, ubiquitination assay, Co-IP, cell functional assays","journal":"Journal of molecular neuroscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — computational prediction confirmed by single KD experiment and Co-IP; single lab","pmids":["35939202"],"is_preprint":false},{"year":2023,"finding":"Allosteric inhibition of HSP70 (by JG98/JG231) promotes STUB1 nuclear translocation to bind and degrade AR-V7 in enzalutamide-resistant prostate cancer cells. STUB1 knockdown diminishes the anticancer effects and partially restores AR-V7 levels, indicating that HSP70/STUB1 machinery regulates AR/AR-V7 protein stability.","method":"STUB1 knockdown, nuclear fractionation/localization, Co-IP, immunoblot, cell growth assays, in vivo xenograft","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear translocation imaging, KD rescue experiment, in vivo validation; single lab with multiple methods","pmids":["36773708"],"is_preprint":false},{"year":2021,"finding":"TRIM6 interacts with STUB1 and promotes ubiquitination-mediated proteasomal degradation of STUB1, leading to increased YAP1 protein levels and enhanced breast cancer progression.","method":"Co-IP, ubiquitination assay, TRIM6 overexpression/knockdown, YAP1 protein level measurements, in vitro and in vivo cancer growth assays","journal":"European journal of histochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo tumor growth rescue; single lab","pmids":["33728863"],"is_preprint":false}],"current_model":"STUB1/CHIP is a U-box domain E3 ubiquitin ligase that functions as a chaperone-associated quality-control enzyme: its N-terminal TPR domain binds HSP70/HSC70 and HSP90 (which compete with membranes and other partners for TPR access), while its C-terminal U-box catalyzes ubiquitination of diverse substrates including phosphorylated TFEB, HIF1A (via CMA), SMAD3, RUNX1, CARMA1, IFNγ-R1/JAK1, PKM2, BMAL1, YAP1, ErbB2, AGO1-4, Dicer, Sox2/Oct4/Nanog, GPX4, GLUD1, NSUN2, YTHDF2, METTL14, and phosphorylated tau, directing them to proteasomal or lysosomal/autophagic degradation; STUB1 also mediates non-degradative K27- and K63-linked ubiquitination (of CARMA1 and AHR, respectively) to modulate NF-κB and immune signaling, and can translocate to the nucleus or membranes during stress to access compartment-specific substrates."},"narrative":{"mechanistic_narrative":"STUB1 (CHIP) is a chaperone-associated U-box E3 ubiquitin ligase that couples the HSP70/HSC70 and HSP90 chaperone systems to the ubiquitin-proteasome and autophagy machineries, acting as a central node in protein quality control and the turnover of signaling regulators [PMID:23880665, PMID:29091030]. Through its N-terminal TPR domain it engages chaperone-bound clients and competes with cellular membranes for the same surface, allowing it to relocate to membranes during acute stress and act as a proteostasis sensor [PMID:29091030]. Its catalytic activity supports multiple ubiquitin linkage types with distinct outcomes: K48-linked chains direct substrates such as BMAL1, YAP1, Sox2/Nanog, and the RNAi factors AGO2/Dicer to proteasomal degradation [PMID:32041778, PMID:31393050, PMID:35675767, PMID:35533808], whereas K63- and K27-linked, non-degradative chains on AHR and the TCR adaptor CARMA1 modulate immune and NF-κB signaling [PMID:35943876, PMID:23322406]. STUB1 also drives chaperone-mediated autophagy of HIF1A and selective autophagy of damaged organelles, ubiquitinating stressed peroxisomes for pexophagy and proteasomes for proteaphagy [PMID:23880665, PMID:33077711, PMID:32723828]. Through substrate-specific ubiquitination it broadly restrains transcription factors, receptors, and metabolic enzymes—degrading phosphorylated TFEB, the IFNγ-R1/JAK1 complex, IL-4Rα, PKM2, GLUD1, GPX4, and the m6A writer METTL14—thereby tuning autophagy, cytokine signaling, glycolytic and glutamine metabolism, ferroptosis, and RNA modification [PMID:28754656, PMID:35395848, PMID:24251647, PMID:28346425, PMID:37416474, PMID:38110356, PMID:36597993]. STUB1 preferentially recognizes pathological phospho-tau via its TPR domain and suppresses tau aggregation and seeding, and heterozygous missense variants in STUB1 cause autosomal dominant ataxia with selective Purkinje cell loss [PMID:37330289, PMID:32211513].","teleology":[{"year":2013,"claim":"Established that STUB1 functions beyond proteasomal degradation by acting downstream of the chaperone HSPA8 and the CMA receptor LAMP2A to drive lysosomal clearance of a transcription factor, linking the ligase to chaperone-mediated autophagy.","evidence":"Ligase-dead and HSPA8-binding STUB1 mutants with lysosome translocation assays for HIF1A","pmids":["23880665"],"confidence":"High","gaps":["Does not define how STUB1 selects CMA versus proteasomal routing for a given client","Generality across other CMA substrates not addressed"]},{"year":2013,"claim":"Showed STUB1 produces non-degradative ubiquitin chains as a positive signaling input, contrasting its more familiar role as a degradative ligase.","evidence":"Co-IP, RNAi, and K27-linkage-specific ubiquitination of CARMA1 with NF-κB reporter and IL-2 readouts in T cells","pmids":["23322406"],"confidence":"Medium","gaps":["Specific CARMA1 lysines not mapped","Single lab; mechanism of chain-type selection unclear"]},{"year":2014,"claim":"Defined how HSP70 versus HSP90 differentially gate STUB1 substrate ubiquitination, establishing that the chaperone bound to a client determines whether STUB1 degrades it.","evidence":"Co-IP, geldanamycin HSP90 inhibition, and Smad3 ubiquitination/TGF-β readouts","pmids":["24613385"],"confidence":"Medium","gaps":["Structural basis of opposing chaperone effects not resolved","In vivo relevance not tested"]},{"year":2017,"claim":"Connected STUB1 to autophagy and metabolic control by showing it degrades phosphorylated TFEB and the glycolytic enzyme PKM2, positioning the ligase as a regulator of lysosomal biogenesis and the Warburg effect.","evidence":"Co-IP, ubiquitination assays, STUB1 KO cells/mice and KO MEFs with autophagy, mitochondrial, and tumor-growth readouts","pmids":["28754656","28346425"],"confidence":"High","gaps":["Phospho-dependence recognition mechanism for TFEB not structurally defined","Tissue-specific contributions of each substrate unresolved"]},{"year":2017,"claim":"Revealed a chaperone-independent, membrane-docking mode in which STUB1 senses proteostasis stress, showing HSP70 and lipid membranes compete for the same TPR surface and that relocated STUB1 fragments the Golgi.","evidence":"In vitro liposome reconstitution, HSP70-versus-membrane competition assay, and live-cell Golgi imaging","pmids":["29091030"],"confidence":"High","gaps":["Physiological triggers and reversibility of membrane docking not fully defined","Substrates engaged at membranes not enumerated"]},{"year":2020,"claim":"Extended STUB1's quality-control role to whole organelles and the degradation machinery itself, showing chaperone-driven targeting of stressed peroxisomes (pexophagy) and K63-ubiquitination of inhibited proteasomes (proteaphagy).","evidence":"Optogenetic ROS induction with live imaging for pexophagy; in vitro ubiquitination of purified proteasomes and aggresome/autophagy readouts","pmids":["33077711","32723828"],"confidence":"High","gaps":["Substrate adaptors that mark organelles for STUB1 not fully identified","How STUB1 distinguishes damaged from healthy organelles unclear"]},{"year":2020,"claim":"Demonstrated stress-induced nuclear relocation of STUB1 to degrade the clock factor BMAL1 via K48 chains, tying the ligase to circadian regulation and oxidative-stress-induced senescence.","evidence":"AP-MS, domain mapping, K48-linkage ubiquitination, nuclear fractionation, and SA-β-gal senescence assays","pmids":["32041778"],"confidence":"High","gaps":["Trigger for nuclear translocation not molecularly defined","Circadian phenotype in vivo not tested"]},{"year":2022,"claim":"Unbiased CRISPR screens placed STUB1 as a degradative regulator of immune and stemness programs, identifying the IFNγ-R1/JAK1 complex and the pluripotency factors Sox2/Oct4/Nanog as substrates with linkage-specific (K48/K63) outcomes.","evidence":"Genome-wide CRISPR screens, site-specific ubiquitination-site mutagenesis, T-cell killing and reprogramming/differentiation assays","pmids":["35395848","35675767"],"confidence":"High","gaps":["What determines K48 versus K63 chain choice on different stemness factors is unresolved","Interplay between substrates in a single cell type not addressed"]},{"year":2023,"claim":"Showed STUB1 preferentially recognizes pathological phospho-tau through a partly distinct TPR-binding mode and suppresses its aggregation and seeding, mechanistically linking the ligase to neurodegeneration-relevant proteostasis.","evidence":"In vitro binding/aggregation/ubiquitination assays with TPR mutagenesis and cell-based seeding assays","pmids":["37330289"],"confidence":"High","gaps":["In vivo tau clearance by STUB1 not tested here","Relationship to ataxia-causing TPR variants not directly addressed"]},{"year":2024,"claim":"Established STUB1 as a regulator of RNA modification and ferroptosis by degrading the m6A writer METTL14 and the m5C writer NSUN2, with the latter coupling to GPX4 mRNA methylation and hepatocyte ferroptosis.","evidence":"Site-specific ubiquitination-site mutagenesis, m6A/m5C quantification, and in vivo/in vitro ferroptosis models","pmids":["36597993","39453812"],"confidence":"High","gaps":["How STUB1 access to RNA-modifying enzymes is regulated is unknown","Cross-talk between m6A and m5C arms of STUB1 control not defined"]},{"year":2024,"claim":"Revealed upstream control of STUB1 itself through post-translational modification, with BCKDK phosphorylation at S19 and KAT5 acetylation regulating its activity and abundance, and TRIM6 driving its degradation.","evidence":"Phospho-/acetylation and ubiquitination assays with site-directed mutagenesis and tumor models","pmids":["38621458","38561411","33728863"],"confidence":"Medium","gaps":["Integrated regulatory logic governing STUB1 activity in vivo is unresolved","Single-lab findings per modification"]},{"year":null,"claim":"How STUB1 integrates substrate recognition, chaperone state, subcellular relocation, and ubiquitin chain-type selection into a coherent decision (degrade versus signal, proteasome versus autophagy) across its very broad substrate set remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural/biochemical rule for chain-type selection","Tissue- and stress-context-specific substrate prioritization not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,4,6,7,11,15,21,24]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,7,11,15,21,23,24]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[9,17,18,21]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,7,10]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,12,35]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[14,19]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[19]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[19,26]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[26]},{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,5,13,19]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,1,5,8,20]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,7,9,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,10,16,30,36]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,23,24,25]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[15,18,24]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[6]}],"complexes":[],"partners":["HSPA8","HSP90","HSP70","CARMA1","JAK1","YTHDF2","SMAD3","TRIM6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UNE7","full_name":"E3 ubiquitin-protein ligase CHIP","aliases":["Antigen NY-CO-7","CLL-associated antigen KW-8","Carboxy terminus of Hsp70-interacting protein","RING-type E3 ubiquitin transferase CHIP","STIP1 homology and U box-containing protein 1"],"length_aa":303,"mass_kda":34.9,"function":"E3 ubiquitin-protein ligase which targets misfolded chaperone substrates towards proteasomal degradation (PubMed:10330192, PubMed:11146632, PubMed:11557750, PubMed:23990462, PubMed:26265139). Plays a role in the maintenance of mitochondrial morphology and promotes mitophagic removal of dysfunctional mitochondria; thereby acts as a protector against apoptosis in response to cellular stress (By similarity). Negatively regulates vascular smooth muscle contraction, via degradation of the transcriptional activator MYOCD and subsequent loss of transcription of genes involved in vascular smooth muscle contraction (By similarity). Promotes survival and proliferation of cardiac smooth muscle cells via ubiquitination and degradation of FOXO1, resulting in subsequent repression of FOXO1-mediated transcription of pro-apoptotic genes (PubMed:19483080). Ubiquitinates ICER-type isoforms of CREM and targets them for proteasomal degradation, thereby acts as a positive effector of MAPK/ERK-mediated inhibition of apoptosis in cardiomyocytes (PubMed:20724525). Inhibits lipopolysaccharide-induced apoptosis and hypertrophy in cardiomyocytes, via ubiquitination and subsequent proteasomal degradation of NFATC3 (PubMed:30980393). Collaborates with ATXN3 in the degradation of misfolded chaperone substrates: ATXN3 restricting the length of ubiquitin chain attached to STUB1/CHIP substrates and preventing further chain extension (PubMed:10330192, PubMed:11146632, PubMed:11557750, PubMed:23990462). Ubiquitinates NOS1 in concert with Hsp70 and Hsp40 (PubMed:15466472). Modulates the activity of several chaperone complexes, including Hsp70, Hsc70 and Hsp90 (PubMed:10330192, PubMed:11146632, PubMed:15466472). Ubiquitinates CHRNA3 targeting it for endoplasmic reticulum-associated degradation in cortical neurons, as part of the STUB1-VCP-UBXN2A complex (PubMed:26265139). Ubiquitinates and promotes ESR1 proteasomal degradation in response to age-related circulating estradiol (17-beta-estradiol/E2) decline, thereby promotes neuronal apoptosis in response to ischemic reperfusion injury (By similarity). Mediates transfer of non-canonical short ubiquitin chains to HSPA8 that have no effect on HSPA8 degradation (PubMed:11557750, PubMed:23990462). Mediates polyubiquitination of DNA polymerase beta (POLB) at 'Lys-41', 'Lys-61' and 'Lys-81', thereby playing a role in base-excision repair: catalyzes polyubiquitination by amplifying the HUWE1/ARF-BP1-dependent monoubiquitination and leading to POLB-degradation by the proteasome (PubMed:19713937). Mediates polyubiquitination of CYP3A4 (PubMed:19103148). Ubiquitinates EPHA2 and may regulate the receptor stability and activity through proteasomal degradation (PubMed:19567782). Acts as a co-chaperone for HSPA1A and HSPA1B chaperone proteins and promotes ubiquitin-mediated protein degradation (PubMed:27708256). Negatively regulates the suppressive function of regulatory T-cells (Treg) during inflammation by mediating the ubiquitination and degradation of FOXP3 in a HSPA1A/B-dependent manner (PubMed:23973223). Catalyzes monoubiquitination of SIRT6, preventing its degradation by the proteasome (PubMed:24043303). Likely mediates polyubiquitination and down-regulates plasma membrane expression of PD-L1/CD274, an immune inhibitory ligand critical for immune tolerance to self and antitumor immunity (PubMed:28813410). Negatively regulates TGF-beta signaling by modulating the basal level of SMAD3 via ubiquitin-mediated degradation (PubMed:24613385). Plays a role in the degradation of TP53 (PubMed:26634371). Mediates ubiquitination of RIPK3 leading to its subsequent proteasome-dependent degradation (PubMed:29883609). May regulate myosin assembly in striated muscles together with UBE4B and VCP/p97 by targeting myosin chaperone UNC45B for proteasomal degradation (PubMed:17369820). Ubiquitinates PPARG in macrophages playing a role in M2 macrophages polarization and angiogenesis (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q9UNE7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/STUB1","classification":"Not Classified","n_dependent_lines":135,"n_total_lines":1208,"dependency_fraction":0.11175496688741722},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DNAJB6","stoichiometry":0.2},{"gene":"DNAJC17","stoichiometry":0.2},{"gene":"DNAJC7","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/STUB1","total_profiled":1310},"omim":[{"mim_id":"621078","title":"MYOPATHY, MYOFIBRILLAR, 13, WITH RIMMED VACUOLES; MFM13","url":"https://www.omim.org/entry/621078"},{"mim_id":"618093","title":"SPINOCEREBELLAR ATAXIA 48; SCA48","url":"https://www.omim.org/entry/618093"},{"mim_id":"616175","title":"UBIQUITIN-CONJUGATING ENZYME E2 J1; UBE2J1","url":"https://www.omim.org/entry/616175"},{"mim_id":"615768","title":"SPINOCEREBELLAR ATAXIA, AUTOSOMAL RECESSIVE 16; SCAR16","url":"https://www.omim.org/entry/615768"},{"mim_id":"612939","title":"HEAT-SHOCK 70-KD PROTEIN-BINDING PROTEIN 1; HSPBP1","url":"https://www.omim.org/entry/612939"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/STUB1"},"hgnc":{"alias_symbol":["UBOX1","CHIP","SDCCAG7","HSPABP2","NY-CO-7"],"prev_symbol":[]},"alphafold":{"accession":"Q9UNE7","domains":[{"cath_id":"1.25.40.10","chopping":"25-131","consensus_level":"high","plddt":95.4718,"start":25,"end":131},{"cath_id":"3.30.40.10","chopping":"228-300","consensus_level":"high","plddt":93.846,"start":228,"end":300},{"cath_id":"1.10.287","chopping":"149-186_195-217","consensus_level":"high","plddt":93.2138,"start":149,"end":217}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UNE7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UNE7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UNE7-F1-predicted_aligned_error_v6.png","plddt_mean":89.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=STUB1","jax_strain_url":"https://www.jax.org/strain/search?query=STUB1"},"sequence":{"accession":"Q9UNE7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UNE7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UNE7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UNE7"}},"corpus_meta":[{"pmid":"31171693","id":"PMC_31171693","title":"Organoids-on-a-chip.","date":"2019","source":"Science 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protocols","url":"https://pubmed.ncbi.nlm.nih.gov/29717045","citation_count":11,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":42300,"output_tokens":9324,"usd":0.13338,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19339,"output_tokens":4476,"usd":0.104298,"stage2_stop_reason":"end_turn"},"total_usd":0.237678,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"STUB1, a chaperone-dependent E3 ubiquitin ligase, preferentially interacts with and ubiquitinates phosphorylated (inactive) TFEB, targeting it for proteasomal degradation. STUB1 deficiency leads to accumulation of phosphorylated TFEB and reduced TFEB transcriptional activity, reduced autophagy, and reduced mitochondrial biogenesis.\",\n      \"method\": \"Immunoprecipitation, immunoblot, STUB1 knockout cells and STUB1-deficient mice, overexpression studies\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, KO cell and mouse model with specific phenotypic readouts, multiple orthogonal methods in single study\",\n      \"pmids\": [\"28754656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"STUB1 is required for degradation of HIF1A by chaperone-mediated autophagy (CMA). STUB1 mutations that abolish either E3 ubiquitin ligase activity or HSPA8-binding prevent HIF1A lysosomal degradation. STUB1 functions downstream of HSPA8 and the CMA receptor LAMP2A to mediate HIF1A ubiquitination and translocation into lysosomes.\",\n      \"method\": \"Mutagenesis of STUB1 (ligase-dead and HSPA8-binding mutants), lysosome isolation/translocation assay, immunoprecipitation, immunoblot\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site and binding-domain mutagenesis combined with lysosome translocation reconstitution and in vivo starvation models\",\n      \"pmids\": [\"23880665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"STUB1 interacts with IL-4Rα and targets it for ubiquitination-mediated proteasomal degradation, terminating IL-4/IL-13 signaling. STUB1 knockout cells show increased IL-4Rα levels and sustained STAT6 activation; STUB1-deficient mice exhibit spontaneous airway inflammation, M2 macrophage activation, and elevated serum IgE.\",\n      \"method\": \"Co-immunoprecipitation, immunoblot, flow cytometry, STUB1 KO mouse model, in vivo lung inflammation measurements\",\n      \"journal\": \"American journal of respiratory and critical care medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, KO mouse phenotype, multiple orthogonal methods across in vitro and in vivo systems\",\n      \"pmids\": [\"24251647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CHIP/STUB1 negatively regulates NF-κB signaling in colorectal cancer cells by promoting ubiquitination and proteasomal degradation of p65, a NF-κB subunit, leading to decreased expression of NF-κB target oncogenes (Cyclin D1, c-Myc, MMP-2, VEGF, IL-8).\",\n      \"method\": \"Overexpression and RNAi knockdown in HCT-116 cells, ubiquitination assay, nude mouse tumor growth assay, immunoblot\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD/OE with specific pathway readouts, single lab, multiple methods\",\n      \"pmids\": [\"24302614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CHIP/STUB1 (U-box E3 ligase) suppresses the Warburg effect in ovarian carcinoma by interacting with and mediating ubiquitin-proteasomal degradation of pyruvate kinase isoenzyme M2 (PKM2), thereby regulating glycolytic metabolism.\",\n      \"method\": \"SiLAD proteomics, Co-IP, ubiquitination assay, CHIP KO MEF cells, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — proteomics substrate identification, Co-IP, KO cell validation, in vivo xenograft, multiple orthogonal methods\",\n      \"pmids\": [\"28346425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STUB1 specifically ubiquitinates purified human proteasomes in vitro, mainly via Lys63-linked chains. Inhibited proteasomes are sequestered into aggresomes via HDAC6- and dynein-mediated transport and cleared through selective macroautophagy (proteaphagy); STUB1 activity is required for this proteasome quality-control pathway.\",\n      \"method\": \"In vitro ubiquitination assay with purified proteasomes, genetic and chemical inhibition of STUB1, live imaging, colocalization with SQSTM1/HDAC6\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of ubiquitination, genetic inhibition with specific functional readout, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"32723828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STUB1 promotes ubiquitination of BMAL1 via Lys-48-linked polyubiquitin chains, leading to its proteasomal degradation. Oxidative stress promotes STUB1 nuclear translocation and co-localization with BMAL1. STUB1-mediated BMAL1 degradation attenuates hydrogen peroxide-induced cell senescence.\",\n      \"method\": \"Affinity purification-MS, Co-IP, domain mapping, ubiquitination assay, proteasome inhibition, nuclear fractionation/localization, senescence-associated β-gal staining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS substrate identification confirmed by Co-IP, K48-linkage specificity, domain mapping, localization with functional consequence, multiple orthogonal methods\",\n      \"pmids\": [\"32041778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"STUB1 is an E3 ubiquitin ligase for the IFNγ receptor subunit IFNγ-R1 in complex with JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of the IFNγ-R1/JAK1 complex through specific lysine residues IFNγ-R1K285 and JAK1K249, thereby suppressing tumor IFNγ signaling.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, Co-IP, ubiquitination assay, site-directed mutagenesis of ubiquitination sites, cytotoxic T-cell killing assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — unbiased CRISPR screen, Co-IP, mutagenesis of specific ubiquitination sites, functional readout in multiple systems\",\n      \"pmids\": [\"35395848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Hsc70 mediates translocation of STUB1 onto oxidatively stressed peroxisomes, promoting their selective ubiquitination and autophagic degradation (pexophagy). Artificially targeting STUB1 to healthy peroxisomes is sufficient to trigger pexophagy. STUB1 ataxia patient mutants are defective in pexophagy induction.\",\n      \"method\": \"Live-cell imaging with optogenetic ROS induction, confocal microscopy, Stub1 targeting assay, patient mutant analysis, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with acute ROS induction, gain-of-function targeting experiment, patient mutant functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"33077711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"STUB1 constitutively interacts with the adaptor protein CARMA1, with interaction intensified by TCR stimulation. STUB1 ubiquitinates CARMA1 via Lys-27-linked chains, which is required for TCR-induced canonical NF-κB activation and IL-2 production. STUB1 knockdown abolishes endogenous CARMA1 ubiquitination and NF-κB activation downstream of TCR.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, overexpression, ubiquitin linkage-specific analysis, NF-κB reporter assay, IL-2 measurement\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, KD with specific signaling readout, ubiquitin linkage characterization, single lab\",\n      \"pmids\": [\"23322406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Hsp70 and Hsp90 oppositely regulate CHIP/STUB1-mediated ubiquitination and degradation of Smad3. Overexpressed Hsp70 or Hsp90 inhibition (by geldanamycin) facilitates CHIP-induced Smad3 ubiquitination and degradation (enhancing TGF-β signaling sensitivity), while overexpressed Hsp90 antagonizes CHIP-mediated Smad3 ubiquitination (desensitizing TGF-β signaling).\",\n      \"method\": \"Co-immunoprecipitation, overexpression, pharmacological Hsp90 inhibition, ubiquitination assay, TGF-β signaling readouts\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, pharmacological and genetic perturbation, ubiquitination assay; single lab with multiple methods\",\n      \"pmids\": [\"24613385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"STUB1 ubiquitinates YAP1 at K280 via K48-linked polyubiquitination, promoting YAP1 proteasomal degradation and inhibiting cancer cell survival and chemoresistance in gastric cancer.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis (K280), immunoblot, cell viability assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, specific ubiquitination site mutagenesis, functional readout; single lab\",\n      \"pmids\": [\"31393050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"STUB1 binds to RUNX1 and induces its ubiquitination and proteasomal degradation, primarily in the nucleus, and also promotes nuclear export of RUNX1. STUB1 similarly ubiquitinates the leukemogenic fusion RUNX1-RUNX1T1. Overexpression of STUB1 inhibits growth of myeloid leukemia cells harboring RUNX1-RUNX1T1.\",\n      \"method\": \"High-throughput binding assay, Co-IP, ubiquitination assay, immunofluorescence for nuclear/cytoplasmic localization, cell growth assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — high-throughput binding screen confirmed by Co-IP, ubiquitination, localization imaging, functional growth assay; single lab\",\n      \"pmids\": [\"28536267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CHIP/STUB1 binds ~10-fold more strongly to phosphorylated tau than unmodified tau via its TPR domain (partially distinct from canonical binding mode). Sub-stoichiometric CHIP strongly suppresses aggregation and seeding of phosphorylated tau and promotes rapid ubiquitination of phosphorylated (but not unmodified) tau in vitro. In cells, CHIP restricts seeding by phosphorylated tau.\",\n      \"method\": \"In vitro binding assay (panel of TPR-domain proteins), aggregation assay, in vitro ubiquitination assay, TPR domain mutagenesis, cell-based seeding assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of ubiquitination, aggregation suppression assay, domain mutagenesis, cell-based validation; multiple orthogonal methods in single study\",\n      \"pmids\": [\"37330289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HSP90β inhibits STUB1-induced ubiquitination and degradation of YTHDF2 in the cytoplasm of hepatocellular carcinoma cells. The large and small middle domain of HSP90β is required for its interaction with both STUB1 and YTHDF2. STUB1 directly interacts with YTHDF2 to promote its ubiquitination-mediated proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion mapping, ubiquitination assay, overexpression and knockdown, immunoblot\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, single lab with multiple methods\",\n      \"pmids\": [\"37515378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STUB1 is the E3 ubiquitin ligase for METTL14, directly interacting with it and mediating its ubiquitination at K148, K156, and K162 for proteasomal degradation. METTL3 competitively protects METTL14 from STUB1-mediated degradation via METTL3 amino acid regions 450-454 and 464-480, thereby maintaining m6A homeostasis.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis of ubiquitination sites, domain mapping of METTL3, m6A quantification, immunoblot\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-specific mutagenesis of ubiquitination sites, domain mapping, m6A functional readout, multiple orthogonal methods in single study\",\n      \"pmids\": [\"36597993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PDLIM5 interacts with SMAD3 (but not SMAD2) and competitively suppresses the interaction between SMAD3 and its E3 ubiquitin ligase STUB1, protecting SMAD3 from STUB1-mediated proteasomal degradation and maintaining TGF-β signaling in NSCLC cells.\",\n      \"method\": \"Co-IP, knockdown/overexpression, ubiquitination assay, immunoblot, rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, competitive binding assay, rescue experiment with STUB1 KD; single lab with multiple methods\",\n      \"pmids\": [\"32737199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"STUB1 promotes K63-linked non-degradative ubiquitination of aryl hydrocarbon receptor (AHR), facilitating Th17/Treg cell imbalance in rheumatoid arthritis. Specific ubiquitination sites on AHR were identified as responsible for STUB1-mediated K63 ubiquitination.\",\n      \"method\": \"Western blot, flow cytometry, siRNA knockdown, EROD enzymatic activity assay, ubiquitination assay with linkage-specific analysis\",\n      \"journal\": \"Clinical and experimental immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay with linkage specificity, enzymatic activity readout, KD with functional cellular phenotype; single lab\",\n      \"pmids\": [\"35943876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"STUB1 promotes K48-linked polyubiquitination of AGO2 and Dicer (including aviDicer), targeting them for proteasomal degradation in a chaperone-dependent manner. STUB1 also induces degradation of AGO1, AGO3, and AGO4. STUB1 deficiency upregulates Dicer and AGO2, enhancing antiviral RNAi and inhibiting viral replication in mammalian cells and newborn mouse models.\",\n      \"method\": \"Co-IP, ubiquitination assay with K48-linkage analysis, overexpression/knockdown, in vivo newborn mouse model with virus-derived siRNA measurement\",\n      \"journal\": \"Virologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, K48 linkage-specific ubiquitination, in vivo validation; single lab with multiple methods\",\n      \"pmids\": [\"35533808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CHIP/STUB1, when freed from chaperones during acute stress, docks on cellular membranes (phosphatidic acid and phosphatidylinositol-4-phosphate enhance association), performing a proteostasis sensor function. HSP70 and membranes compete for mutually exclusive binding to the TPR domain of CHIP. At new cellular locations, CHIP participates in Golgi apparatus fragmentation.\",\n      \"method\": \"In vitro reconstitution with liposomes, competition assay (HSP70 vs. membrane), cellular imaging of Golgi fragmentation, lipid binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with liposomes, competition binding assay, live-cell imaging; multiple orthogonal methods in single study\",\n      \"pmids\": [\"29091030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CHIP/STUB1 knockdown increases PTEN protein levels and decreases AKT/mTOR activity and ULK1 Ser757 phosphorylation, promoting autophagosome formation. However, CHIP knockdown also disturbs autophagic flux (impairs p62 substrate degradation) and increases susceptibility to autophagic cell death induced by bafilomycin A1.\",\n      \"method\": \"RNAi knockdown, immunoblot for autophagy pathway components (PTEN, AKT, mTOR, ULK1, p62), bafilomycin A1 treatment, cell death assay\",\n      \"journal\": \"Neuroscience bulletin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with specific pathway signaling readouts, substrate flux assay, pharmacological perturbation; single lab\",\n      \"pmids\": [\"26219223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The E3 ubiquitin ligase STUB1 mediates ubiquitination and proteasomal degradation of Sox2 and Nanog via K48-linked chains, and Oct4 via K63-linked chains. STUB1 deficiency enhances somatic cell reprogramming and delays ESC differentiation, while enforced STUB1 expression triggers ESC differentiation.\",\n      \"method\": \"CRISPR-Cas9 KO library screen, Co-IP, K48/K63 linkage-specific ubiquitination assays, protein half-life measurement, reprogramming and differentiation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — unbiased CRISPR screen, K48/K63 linkage-specific ubiquitination, protein half-life assays, functional reprogramming/differentiation phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"35675767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STUB1 deficiency leads to intracellular accumulation of protein aggregates and increased secretion of small extracellular vesicles (exosomes) enriched in ubiquitinated and/or undegraded proteins and oligomers. Oxidative stress augments sEV release in STUB1-depleted cells, indicating that STUB1 and exosomes cooperate to maintain proteostasis.\",\n      \"method\": \"STUB1 KO/knockdown, nanoparticle tracking analysis of sEVs, immunoblot for ubiquitinated proteins in sEVs, oxidative stress treatment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with specific vesicle secretion phenotype, oxidative stress perturbation, single lab with multiple methods\",\n      \"pmids\": [\"31613922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated ubiquitination of GPX4 at site K191, leading to GPX4 degradation. STUB1 knockdown or GPX4 overexpression reverses imatinib-induced ferroptosis.\",\n      \"method\": \"Co-IP, ubiquitination assay with site-directed mutagenesis (K191), GPX4 knockdown/overexpression, ferroptosis markers (lipid ROS, Fe2+, GSH), in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis of ubiquitination site, functional ferroptosis readout, in vivo validation; single lab\",\n      \"pmids\": [\"38110356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STUB1 mediates ubiquitination of NSUN2 at lysines K457 and K654, promoting NSUN2 proteasomal degradation during ferroptosis. NSUN2 degradation diminishes m5C methylation of Gpx4 mRNA 3' UTR, reducing GPX4 protein expression and promoting hepatocyte ferroptosis.\",\n      \"method\": \"Co-IP, ubiquitination assay with site-specific mutagenesis, m5C methylation assay, SECIS-SBP2 interaction assay, NSUN2 knockdown/overexpression, in vivo and in vitro ferroptosis models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-directed mutagenesis of ubiquitination sites, RNA methylation functional readout, in vivo validation; multiple orthogonal methods in single study\",\n      \"pmids\": [\"39453812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STUB1 mediates ubiquitination and proteasomal degradation of GLUD1 at lysine K503, regulating glutamine catabolism in lung adenocarcinoma. Inhibition of K503 ubiquitination promotes proliferation and tumor growth.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis (K503), cell proliferation assay, in vivo tumor growth\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis, ubiquitination assay, functional tumor growth readout; single lab\",\n      \"pmids\": [\"37416474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CHIP/STUB1 targets newly synthesized, HSP90/HSC70-associated ErbB2 for ubiquitin/proteasome-dependent degradation in the endoplasmic reticulum and Golgi, establishing a mechanism for negative regulation of cell surface ErbB2 levels in breast cancer cells.\",\n      \"method\": \"STUB1 knockdown and overexpression, proteasome inhibition, ER/Golgi fractionation, immunoblot, ErbB2 surface expression measurement\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD/OE with subcellular fractionation, pharmacological inhibition; single lab with multiple methods\",\n      \"pmids\": [\"34439093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CHIP/STUB1 directly interacts with eIF5A preferentially through its U-box domain, mediating eIF5A ubiquitination and proteasomal degradation. CHIP expression inversely correlates with eIF5A levels in colorectal cancers and in CHIP KO MEF cells.\",\n      \"method\": \"Proteomics identification, Co-IP, domain mapping (U-box), ubiquitination assay, CHIP KO MEF cells, immunoblot\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics + Co-IP with domain mapping, KO cell validation; single lab\",\n      \"pmids\": [\"24509416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STUB1 is targeted by the SUMO-interacting motif SIM3 of EBV EBNA1. The SIM3 motif mediates EBNA1's inhibitory effects on SUMO2-modified STUB1 and regulates SUMO2-mediated degradation of USP7. Hypoxic stress induces dissociation of EBNA1 from STUB1, increasing SUMO1 modification of STUB1 and KAP1 to promote lytic reactivation.\",\n      \"method\": \"Proteomic analysis, Co-IP, mutagenesis of SIM motifs, ubiquitination/SUMOylation assays, viral replication assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic + Co-IP, SIM mutagenesis, functional viral replication readout; single lab\",\n      \"pmids\": [\"32176739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BCKDK phosphorylates STUB1 at S19, disrupting the STUB1-BCAT1 interaction and inhibiting STUB1-mediated ubiquitination and degradation of BCAT1, thereby stabilizing BCAT1 in glioblastoma.\",\n      \"method\": \"Co-IP, phosphorylation assay, ubiquitination assay, site-directed mutagenesis, in vivo and in vitro tumor models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, phospho-specific mutagenesis, ubiquitination assay, functional readout; single lab with multiple methods\",\n      \"pmids\": [\"38621458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STUB1 is acetylated by KAT5 (lysine acetyltransferase 5), and KAT5 promotes STUB1 transcription via acetylation modulation. STUB1 then ubiquitinates and promotes degradation of LATS2, activating the YAP/β-catenin pathway and inhibiting NLRP3-mediated cardiomyocyte pyroptosis during myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Acetylation assay, ubiquitination assay, KAT5/STUB1 overexpression/knockdown, LATS2 stability assay, pyroptosis markers, in vivo MIRI model\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acetylation/ubiquitination assays, multiple KO/OE experiments with pathway readouts, in vivo model; single lab\",\n      \"pmids\": [\"38561411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Salidroside increases STUB1 expression in Tregs and promotes STUB1-mediated degradation of Foxp3 in the nucleus, suppressing Treg function. Hsp70 is required for the colocalization of STUB1 and Foxp3 in the nucleus; Hsp70 inhibition reverses SAL-induced suppression of Foxp3 and disrupts the STUB1-Foxp3 colocalization.\",\n      \"method\": \"Flow cytometry, confocal laser microscopy for nuclear colocalization, Hsp70 inhibitor treatment, network pharmacology and molecular docking\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — colocalization imaging and pharmacological inhibition without direct ubiquitination assay or mutagenesis; single lab\",\n      \"pmids\": [\"37528345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Heterozygous missense variants in STUB1 (p.Ile53Thr and p.Thr37Leu) cause autosomal dominant ataxia. Neuropathological examination reveals selective Purkinje cell loss and aberrant STUB1 localization in distal Purkinje cell dendritic arbors (loss of normal somatodendritic polarization), linking STUB1 mislocalization to cerebellar pathogenesis.\",\n      \"method\": \"Exome sequencing, neuropathological examination, immunofluorescence localization of STUB1 in post-mortem brain tissue\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct immunofluorescence localization in human post-mortem brain with genetic variant, functional consequence (Purkinje cell loss); single study\",\n      \"pmids\": [\"32211513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STUB1 mediates ubiquitination of HOXB3, inhibiting its expression; loss of HOXB3 reduces PARK7 transcription (as HOXB3 binds PARK7 promoter), thereby promoting ferroptosis and suppressing paclitaxel resistance in ovarian cancer.\",\n      \"method\": \"Co-IP, dual luciferase reporter assay, ubiquitination assay, ChIP for HOXB3 binding to PARK7 promoter, in vivo xenograft\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, reporter assay, in vivo validation; single lab with multiple methods\",\n      \"pmids\": [\"39501077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STUB1 ubiquitinates and promotes degradation of SMYD2 in glioma cells treated with cisplatin; STUB1 knockdown reverses cisplatin-induced SMYD2 degradation and partially restores cell function.\",\n      \"method\": \"UbiBrowser prediction confirmed by knockdown, ubiquitination assay, Co-IP, cell functional assays\",\n      \"journal\": \"Journal of molecular neuroscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — computational prediction confirmed by single KD experiment and Co-IP; single lab\",\n      \"pmids\": [\"35939202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Allosteric inhibition of HSP70 (by JG98/JG231) promotes STUB1 nuclear translocation to bind and degrade AR-V7 in enzalutamide-resistant prostate cancer cells. STUB1 knockdown diminishes the anticancer effects and partially restores AR-V7 levels, indicating that HSP70/STUB1 machinery regulates AR/AR-V7 protein stability.\",\n      \"method\": \"STUB1 knockdown, nuclear fractionation/localization, Co-IP, immunoblot, cell growth assays, in vivo xenograft\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear translocation imaging, KD rescue experiment, in vivo validation; single lab with multiple methods\",\n      \"pmids\": [\"36773708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM6 interacts with STUB1 and promotes ubiquitination-mediated proteasomal degradation of STUB1, leading to increased YAP1 protein levels and enhanced breast cancer progression.\",\n      \"method\": \"Co-IP, ubiquitination assay, TRIM6 overexpression/knockdown, YAP1 protein level measurements, in vitro and in vivo cancer growth assays\",\n      \"journal\": \"European journal of histochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo tumor growth rescue; single lab\",\n      \"pmids\": [\"33728863\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"STUB1/CHIP is a U-box domain E3 ubiquitin ligase that functions as a chaperone-associated quality-control enzyme: its N-terminal TPR domain binds HSP70/HSC70 and HSP90 (which compete with membranes and other partners for TPR access), while its C-terminal U-box catalyzes ubiquitination of diverse substrates including phosphorylated TFEB, HIF1A (via CMA), SMAD3, RUNX1, CARMA1, IFNγ-R1/JAK1, PKM2, BMAL1, YAP1, ErbB2, AGO1-4, Dicer, Sox2/Oct4/Nanog, GPX4, GLUD1, NSUN2, YTHDF2, METTL14, and phosphorylated tau, directing them to proteasomal or lysosomal/autophagic degradation; STUB1 also mediates non-degradative K27- and K63-linked ubiquitination (of CARMA1 and AHR, respectively) to modulate NF-κB and immune signaling, and can translocate to the nucleus or membranes during stress to access compartment-specific substrates.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"STUB1 (CHIP) is a chaperone-associated U-box E3 ubiquitin ligase that couples the HSP70/HSC70 and HSP90 chaperone systems to the ubiquitin-proteasome and autophagy machineries, acting as a central node in protein quality control and the turnover of signaling regulators [#1, #19]. Through its N-terminal TPR domain it engages chaperone-bound clients and competes with cellular membranes for the same surface, allowing it to relocate to membranes during acute stress and act as a proteostasis sensor [#19]. Its catalytic activity supports multiple ubiquitin linkage types with distinct outcomes: K48-linked chains direct substrates such as BMAL1, YAP1, Sox2/Nanog, and the RNAi factors AGO2/Dicer to proteasomal degradation [#6, #11, #21, #18], whereas K63- and K27-linked, non-degradative chains on AHR and the TCR adaptor CARMA1 modulate immune and NF-\\u03baB signaling [#17, #9]. STUB1 also drives chaperone-mediated autophagy of HIF1A and selective autophagy of damaged organelles, ubiquitinating stressed peroxisomes for pexophagy and proteasomes for proteaphagy [#1, #8, #5]. Through substrate-specific ubiquitination it broadly restrains transcription factors, receptors, and metabolic enzymes\\u2014degrading phosphorylated TFEB, the IFN\\u03b3-R1/JAK1 complex, IL-4R\\u03b1, PKM2, GLUD1, GPX4, and the m6A writer METTL14\\u2014thereby tuning autophagy, cytokine signaling, glycolytic and glutamine metabolism, ferroptosis, and RNA modification [#0, #7, #2, #4, #25, #23, #15]. STUB1 preferentially recognizes pathological phospho-tau via its TPR domain and suppresses tau aggregation and seeding, and heterozygous missense variants in STUB1 cause autosomal dominant ataxia with selective Purkinje cell loss [#13, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that STUB1 functions beyond proteasomal degradation by acting downstream of the chaperone HSPA8 and the CMA receptor LAMP2A to drive lysosomal clearance of a transcription factor, linking the ligase to chaperone-mediated autophagy.\",\n      \"evidence\": \"Ligase-dead and HSPA8-binding STUB1 mutants with lysosome translocation assays for HIF1A\",\n      \"pmids\": [\"23880665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define how STUB1 selects CMA versus proteasomal routing for a given client\", \"Generality across other CMA substrates not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed STUB1 produces non-degradative ubiquitin chains as a positive signaling input, contrasting its more familiar role as a degradative ligase.\",\n      \"evidence\": \"Co-IP, RNAi, and K27-linkage-specific ubiquitination of CARMA1 with NF-\\u03baB reporter and IL-2 readouts in T cells\",\n      \"pmids\": [\"23322406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific CARMA1 lysines not mapped\", \"Single lab; mechanism of chain-type selection unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined how HSP70 versus HSP90 differentially gate STUB1 substrate ubiquitination, establishing that the chaperone bound to a client determines whether STUB1 degrades it.\",\n      \"evidence\": \"Co-IP, geldanamycin HSP90 inhibition, and Smad3 ubiquitination/TGF-\\u03b2 readouts\",\n      \"pmids\": [\"24613385\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of opposing chaperone effects not resolved\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected STUB1 to autophagy and metabolic control by showing it degrades phosphorylated TFEB and the glycolytic enzyme PKM2, positioning the ligase as a regulator of lysosomal biogenesis and the Warburg effect.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, STUB1 KO cells/mice and KO MEFs with autophagy, mitochondrial, and tumor-growth readouts\",\n      \"pmids\": [\"28754656\", \"28346425\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phospho-dependence recognition mechanism for TFEB not structurally defined\", \"Tissue-specific contributions of each substrate unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed a chaperone-independent, membrane-docking mode in which STUB1 senses proteostasis stress, showing HSP70 and lipid membranes compete for the same TPR surface and that relocated STUB1 fragments the Golgi.\",\n      \"evidence\": \"In vitro liposome reconstitution, HSP70-versus-membrane competition assay, and live-cell Golgi imaging\",\n      \"pmids\": [\"29091030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological triggers and reversibility of membrane docking not fully defined\", \"Substrates engaged at membranes not enumerated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended STUB1's quality-control role to whole organelles and the degradation machinery itself, showing chaperone-driven targeting of stressed peroxisomes (pexophagy) and K63-ubiquitination of inhibited proteasomes (proteaphagy).\",\n      \"evidence\": \"Optogenetic ROS induction with live imaging for pexophagy; in vitro ubiquitination of purified proteasomes and aggresome/autophagy readouts\",\n      \"pmids\": [\"33077711\", \"32723828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate adaptors that mark organelles for STUB1 not fully identified\", \"How STUB1 distinguishes damaged from healthy organelles unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated stress-induced nuclear relocation of STUB1 to degrade the clock factor BMAL1 via K48 chains, tying the ligase to circadian regulation and oxidative-stress-induced senescence.\",\n      \"evidence\": \"AP-MS, domain mapping, K48-linkage ubiquitination, nuclear fractionation, and SA-\\u03b2-gal senescence assays\",\n      \"pmids\": [\"32041778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for nuclear translocation not molecularly defined\", \"Circadian phenotype in vivo not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Unbiased CRISPR screens placed STUB1 as a degradative regulator of immune and stemness programs, identifying the IFN\\u03b3-R1/JAK1 complex and the pluripotency factors Sox2/Oct4/Nanog as substrates with linkage-specific (K48/K63) outcomes.\",\n      \"evidence\": \"Genome-wide CRISPR screens, site-specific ubiquitination-site mutagenesis, T-cell killing and reprogramming/differentiation assays\",\n      \"pmids\": [\"35395848\", \"35675767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What determines K48 versus K63 chain choice on different stemness factors is unresolved\", \"Interplay between substrates in a single cell type not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed STUB1 preferentially recognizes pathological phospho-tau through a partly distinct TPR-binding mode and suppresses its aggregation and seeding, mechanistically linking the ligase to neurodegeneration-relevant proteostasis.\",\n      \"evidence\": \"In vitro binding/aggregation/ubiquitination assays with TPR mutagenesis and cell-based seeding assays\",\n      \"pmids\": [\"37330289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo tau clearance by STUB1 not tested here\", \"Relationship to ataxia-causing TPR variants not directly addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established STUB1 as a regulator of RNA modification and ferroptosis by degrading the m6A writer METTL14 and the m5C writer NSUN2, with the latter coupling to GPX4 mRNA methylation and hepatocyte ferroptosis.\",\n      \"evidence\": \"Site-specific ubiquitination-site mutagenesis, m6A/m5C quantification, and in vivo/in vitro ferroptosis models\",\n      \"pmids\": [\"36597993\", \"39453812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How STUB1 access to RNA-modifying enzymes is regulated is unknown\", \"Cross-talk between m6A and m5C arms of STUB1 control not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed upstream control of STUB1 itself through post-translational modification, with BCKDK phosphorylation at S19 and KAT5 acetylation regulating its activity and abundance, and TRIM6 driving its degradation.\",\n      \"evidence\": \"Phospho-/acetylation and ubiquitination assays with site-directed mutagenesis and tumor models\",\n      \"pmids\": [\"38621458\", \"38561411\", \"33728863\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Integrated regulatory logic governing STUB1 activity in vivo is unresolved\", \"Single-lab findings per modification\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How STUB1 integrates substrate recognition, chaperone state, subcellular relocation, and ubiquitin chain-type selection into a coherent decision (degrade versus signal, proteasome versus autophagy) across its very broad substrate set remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural/biochemical rule for chain-type selection\", \"Tissue- and stress-context-specific substrate prioritization not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 4, 6, 7, 11, 15, 21, 24]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 7, 11, 15, 21, 23, 24]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [9, 17, 18, 21]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 7, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 12, 35]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [14, 19]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [19, 26]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [26]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 5, 13, 19]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 1, 5, 8, 20]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 7, 9, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 10, 16, 30, 36]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 23, 24, 25]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [15, 18, 24]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"HSPA8\", \"HSP90\", \"HSP70\", \"CARMA1\", \"JAK1\", \"YTHDF2\", \"SMAD3\", \"TRIM6\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}