{"gene":"MIB2","run_date":"2026-04-28T18:30:28","timeline":{"discoveries":[{"year":2003,"finding":"MIB2 (skeletrophin) binds to actin monomer, as demonstrated by yeast two-hybrid screening and co-immunoprecipitation experiments; it contains a cysteine-rich zinc-finger motif and five ankyrin repeats.","method":"Yeast two-hybrid screen + co-immunoprecipitation","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal interaction confirmed by two methods (Y2H + Co-IP), single lab","pmids":["14507647"],"is_preprint":false},{"year":2005,"finding":"MIB2 (skeletrophin) is a RING finger-dependent E3 ubiquitin ligase that binds the intracellular region of Notch ligand Jagged-2 (but not Delta-1, -3, -4, or Jagged-1) and catalyzes its ubiquitination; RING-mutated MIB2 loses this activity. Exogenous MIB2, but not its RING mutant, induced Hes-1 expression in stromal cells through Notch signaling.","method":"In vitro autoubiquitination assay with recombinant proteins; RING mutant analysis; cell-based Hes-1 reporter","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with RING mutant controls, replicated in cell-based assay","pmids":["15920166"],"is_preprint":false},{"year":2006,"finding":"MIB2 (skeletrophin) reduces melanoma cell invasion in vitro and in vivo and suppresses colony formation in soft agar in a RING motif-dependent manner; it also downregulates transcription of the Met oncogene and increases Hes1 transcription.","method":"Loss-of-function/gain-of-function with RING mutant; invasion assay; soft agar colony formation; gene expression analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — RING mutant controls used, multiple readouts, single lab","pmids":["16715130"],"is_preprint":false},{"year":2006,"finding":"Zebrafish Mib2, like Mib, has C-terminal-most RING finger-dependent E3 ubiquitin ligase activity; Mib and Mib2 are reciprocal E3 ubiquitin ligases and substrates of each other. They share DeltaC as a common substrate for ubiquitylation and endocytosis, but differ in DeltaD internalization. Mib and Mib2 bind differently to extracellular and intracellular parts of DeltaA and DeltaC.","method":"In vitro ubiquitination assay; RING mutant analysis; Delta internalization assay; binding assays in transfected cells","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro ubiquitination with RING mutants, multiple substrates tested, ortholog consistent with mammalian MIB2","pmids":["17196985"],"is_preprint":false},{"year":2007,"finding":"Zebrafish Mib2 is colocalized with Mib in transfected cells and functions redundantly with Mib in regulating Notch signaling in embryos. Dominant-negative Mib alleles suppress Mib2 function in a dosage-dependent manner, indicating competitive interaction. Notch signaling negatively regulates mib expression in a Su(H)-dependent negative feedback loop.","method":"Colocalization in transfected cells; genetic epistasis in zebrafish embryos; dominant-negative analysis; Su(H) reporter assay","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple alleles and colocalization, replicated in vivo, consistent with mammalian function","pmids":["17331493"],"is_preprint":false},{"year":2007,"finding":"Targeted disruption of Mib2 in mice causes exencephaly (failure to close cranial neural tube) with variable penetrance dependent on genetic background, establishing a role for Mib2 in neural tube closure in vivo.","method":"Targeted gene knockout in mice; morphological phenotypic analysis","journal":"Genesis (New York, N.Y. : 2000)","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined phenotype, but molecular mechanism not detailed beyond Notch pathway membership","pmids":["17987667"],"is_preprint":false},{"year":2011,"finding":"MIB2 is a component of the activated BCL10 signaling complex; it directly interacts with BCL10 (shown by in vitro translation/pulldown), promotes autoubiquitination and ubiquitination of IKKγ/NEMO, recruits and activates TAK1, and thereby controls BCL10-dependent NF-κB activation. MIB2 knockdown inhibits BCL10-dependent NF-κB activation.","method":"Proteomic identification; in vitro pulldown; overexpression; siRNA knockdown; NF-κB reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct in vitro binding, ubiquitination assays, KD phenotype, multiple orthogonal methods","pmids":["21896478"],"is_preprint":false},{"year":2014,"finding":"CYLD (deubiquitinating enzyme) interacts with MIB2 (E3 ubiquitin ligase); coexpression of CYLD and MIB2 results in stabilization of MIB2 protein levels and reduced levels of JAG2, linking MIB2 to regulation of Notch signaling through CYLD.","method":"Proteomics-based interaction screen; co-expression experiments; siRNA knockdown of CYLD; Notch target gene expression analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 3 — interaction identified by proteomics and validated in cells, functional consequence shown, single lab","pmids":["25565632"],"is_preprint":false},{"year":2017,"finding":"In Drosophila, the ankyrin repeats (in their entirety) and the MIB-specific domains of Mib2 are essential for its function in maintaining integrity of larval skeletal and visceral muscles. The RING finger domain is required for adult flight muscle development. Missense mutations in the MIB domain and RING finger cause flightless hypomorphic phenotypes, establishing domain-specific functional requirements.","method":"Domain deletion/mutagenesis in vivo; forward genetic screen; in vivo imaging of flight muscles","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — systematic domain dissection with multiple alleles in vivo, consistent with mammalian MIB2 domain architecture","pmids":["28282454"],"is_preprint":false},{"year":2017,"finding":"MIB2 missense variant p.V742G shows reduced ubiquitination activity in vitro and is associated with reduced NOTCH signaling (decreased HES1 and NOTCH3 expression) in white blood cells; MIB2 variants affect NOTCH signaling, proliferation, and apoptosis in primary rat cardiomyocytes, establishing MIB2 as a regulator of NOTCH signaling relevant to cardiac trabeculation.","method":"In vitro ubiquitination assay with purified variant protein; whole exome sequencing; gene expression analysis; primary cardiomyocyte functional assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1 — in vitro activity assay with variant protein, corroborated by cell-based and patient data","pmids":["28013292"],"is_preprint":false},{"year":2018,"finding":"MIB2 mediates Lys-63-linked ubiquitination of GABAB1 subunit of GABAB receptors, sorting them to lysosomes for degradation. CaMKIIβ (but not CaMKIIα) promotes MIB2-mediated K63-linked ubiquitination of GABAB1 at multiple sites by phosphorylating GABAB1 at Ser-867; phosphomimetic S867D mutation increases K63-ubiquitination while S867A mutation reduces it and increases surface expression.","method":"Overexpression of CaMKII isoforms; phosphomimetic/phospho-null mutagenesis; K63-ubiquitination assay; surface GABAB receptor quantification in cortical neurons","journal":"Molecular neurobiology","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of phosphorylation site, K63-ubiquitination assay, multiple orthogonal methods, defined PTM writer identified","pmids":["29881949"],"is_preprint":false},{"year":2019,"finding":"MIB2 promotes proteasomal degradation of the deubiquitinating enzyme CYLD by catalyzing Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. The ankyrin repeat of MIB2 interacts with the third CAP domain of CYLD. MIB2-dependent CYLD degradation activates NF-κB signaling via TNFα and LUBAC. Mib2-knockout mice showed suppressed arthritic inflammation and reduced serum IL-6.","method":"Cell-free AlphaScreen and pulldown assays; immunofluorescence; Mib2 KO cells and mice; site-specific ubiquitination mapping; arthritis model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro binding, site-specific ubiquitination with K→R mutants, KO mice with defined inflammatory phenotype, multiple methods","pmids":["31366726"],"is_preprint":false},{"year":2021,"finding":"Gm364 (a multi-pass transmembrane protein) directly binds and anchors MIB2 on the membrane; membrane-localized MIB2 ubiquitinates and activates DLL3, which activates Notch2, leading to production of NICD2 that activates AKT to regulate oocyte meiosis and quality.","method":"Knockout mouse model; co-IP; oocyte phenotypic analysis (ROS, mitochondrial membrane potential, aneuploidy); epistasis experiments","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — KO phenotype with pathway epistasis and Co-IP, single lab","pmids":["34635817"],"is_preprint":false},{"year":2023,"finding":"MIB2 is required for translocation of PD-L1 from the trans-Golgi network (TGN) to the plasma membrane. Mechanistically, MIB2 catalyzes nonproteolytic K63-linked ubiquitination of PD-L1, facilitating its trafficking via RAB8-mediated exocytosis from TGN to plasma membrane. MIB2 deficiency reduces PD-L1 surface expression and promotes antitumor T-cell immunity in mice.","method":"MIB2 knockdown/knockout; K63-ubiquitination assay; RAB8 epistasis; surface PD-L1 flow cytometry; in vivo mouse tumor models","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 — K63-ubiquitination assay, RAB8 epistasis, KO mouse model, multiple orthogonal methods","pmids":["36719382"],"is_preprint":false},{"year":2023,"finding":"FAT1 acts as an upstream regulator of MIB2 in endothelial cells: FAT1 interacts with MIB2 (identified by interactome analysis), and together they promote ubiquitination and proteasomal degradation of YAP/TAZ. Loss of MIB2 in endothelial cells recapitulates FAT1 depletion, causing decreased YAP/TAZ degradation, increased YAP/TAZ signaling, and increased endothelial cell proliferation and angiogenesis.","method":"Co-IP/interactome analysis; MIB2 KD in vitro and in vivo; YAP/TAZ ubiquitination assay; angiogenesis models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — interactome identification, ubiquitination assay, KD with defined cellular and in vivo phenotypes, multiple orthogonal methods","pmids":["37031213"],"is_preprint":false},{"year":2023,"finding":"MIB2 interacts with CARD6 and promotes K48-linked CARD6 polyubiquitination and proteasomal degradation in hepatocytes under high fructose conditions; MIB2 knockdown reverses CARD6 downregulation and lipid accumulation, establishing MIB2 as an upstream regulator of CARD6 in hepatic lipid metabolism.","method":"Immunoprecipitation; immunofluorescence; siRNA knockdown; immunoblotting","journal":"Food & function","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP interaction and KD phenotype, single lab, no in vitro ubiquitination reconstitution","pmids":["37186242"],"is_preprint":false},{"year":2024,"finding":"MIB2 expression is increased in SN (surrounded nucleolus)-stage oocytes; depletion of MIB2 in SN oocytes disrupts meiotic apparatus and increases aneuploidy, while overexpression of MIB2 in NSN oocytes facilitates chromatin configuration transition from NSN to SN and mitigates spindle/chromosome disorganization.","method":"Quantitative proteomics; MIB2 depletion and overexpression in oocytes; meiotic phenotype analysis (spindle assembly, aneuploidy)","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 2 — loss- and gain-of-function with defined cellular phenotypes, single lab","pmids":["39019259"],"is_preprint":false},{"year":2025,"finding":"MIB2 directly interacts with and ubiquitinates SUZ12 (a PRC2 complex component), controlling SUZ12 stability and H3K27me3 levels; the MIB/HERC and ZZ-type domains of MIB2 mediate interaction with SUZ12. MIB2 knockdown reduces SUZ12 protein and H3K27me3, upregulates PRC2 target genes, and decreases cell proliferation.","method":"Immunoprecipitation + mass spectrometry; siRNA knockdown; ubiquitination assay; RNA-seq; flow cytometry; colony formation assay","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP/MS interaction, ubiquitination assay, KD phenotype, single lab","pmids":["40478202"],"is_preprint":false},{"year":2025,"finding":"MIB2 directly interacts with Runx2 and ubiquitinates it for degradation, thereby inhibiting Hmgcs2 transcription and impairing fatty acid metabolic processes in cardiomyocytes. Cardiac-specific overexpression of Mib2 in ob/ob mice worsens cardiac dysfunction and lipid accumulation.","method":"Immunoprecipitation; dual luciferase reporter assay; proteomic analysis; AAV9-mediated cardiac overexpression in mice","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, luciferase reporter, in vivo overexpression, single lab","pmids":["40159625"],"is_preprint":false},{"year":2025,"finding":"FAT1 loss in tumor cells (including HNSCC) decreases YAP/TAZ ubiquitination and degradation mediated by MIB2; suppression of MIB2 alone phenocopies FAT1 loss, reducing YAP/TAZ ubiquitination and increasing tumor cell proliferation in vitro and tumor growth in vivo.","method":"FAT1/MIB2 KD in tumor cells; YAP/TAZ ubiquitination assay; tumor xenograft in vivo; interactome analysis of FAT1 cytoplasmic domain","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 — ubiquitination assay, KD with in vivo xenograft validation, interactome identification, multiple cell lines","pmids":["40478800"],"is_preprint":false},{"year":2025,"finding":"Ebola virus VP35 contains an NNLNS motif (residues 201–205) that serves as a direct binding site for MIB2; VP35 binding to MIB2 via this motif inhibits MIB2-mediated interferon induction and also suppresses EBOV minigenome RNA synthesis activity.","method":"Mutagenesis of NNLNS motif; minigenome assay; interferon induction assay; interaction mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis of binding motif with functional readouts (IFN induction, minigenome), single lab","pmids":["40982696"],"is_preprint":false},{"year":2025,"finding":"MIB2 promotes ubiquitin-mediated degradation of GPX4 by interacting with GPX4, an interaction regulated by Nrf2; taraxerol treatment reduces MIB2-mediated GPX4 ubiquitination by targeting Nrf2/MIB2 interaction, triggering ferroptosis in breast cancer cells.","method":"Co-IP; dual-luciferase reporter assay; ubiquitination assay; xenograft in vivo model","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, ubiquitination assay, in vivo xenograft, single lab","pmids":["40592077"],"is_preprint":false}],"current_model":"MIB2 is a RING finger-dependent E3 ubiquitin ligase whose catalytic activity requires its C-terminal RING domain and whose substrate recruitment involves ankyrin repeats and MIB-specific domains; it catalyzes K63-linked ubiquitination of Notch ligands (Jagged-2, Delta) to promote their endocytosis and Notch pathway activation, K63-linked ubiquitination of PD-L1 to drive RAB8-mediated exocytosis to the plasma membrane, K63-linked ubiquitination of GABAB1 (primed by CaMKIIβ phosphorylation at Ser-867) for lysosomal sorting, and K48-linked ubiquitination of CYLD, CARD6, SUZ12, Runx2, and YAP/TAZ for proteasomal degradation; MIB2 is recruited to substrates by interacting partners including FAT1 (for YAP/TAZ) and Gm364 (for DLL3/Notch2), and its activity is counteracted by CYLD, placing MIB2 at the intersection of Notch, NF-κB, YAP/TAZ, and immune checkpoint signaling pathways."},"narrative":{"teleology":[{"year":2003,"claim":"Identification of MIB2 (skeletrophin) as a zinc-finger and ankyrin-repeat protein that binds actin monomer established its initial molecular identity and suggested cytoskeletal association.","evidence":"Yeast two-hybrid screen and co-immunoprecipitation in mammalian cells","pmids":["14507647"],"confidence":"Medium","gaps":["Actin binding not confirmed by reciprocal pull-down with purified components","Functional significance of actin interaction never followed up","No catalytic activity demonstrated at this stage"]},{"year":2005,"claim":"Demonstrating that MIB2 is a RING-dependent E3 ubiquitin ligase that ubiquitinates Jagged-2 and activates Notch signaling resolved the catalytic function and first substrate specificity of MIB2.","evidence":"In vitro autoubiquitination assay with recombinant MIB2 and RING mutant; cell-based Hes-1 reporter","pmids":["15920166"],"confidence":"High","gaps":["Whether MIB2 ubiquitinates other Notch ligands in mammals remained open","Ubiquitin chain linkage type not determined","In vivo relevance not yet tested"]},{"year":2006,"claim":"Zebrafish studies established that MIB2 and MIB are reciprocal E3 ligases sharing Delta substrates but differing in specificity, revealing functional redundancy and divergence within the MIB family for Notch ligand regulation.","evidence":"In vitro ubiquitination and Delta internalization assays with RING mutants in zebrafish; genetic epistasis in embryos","pmids":["17196985","17331493"],"confidence":"High","gaps":["Relative contributions of MIB vs MIB2 in mammalian tissues unresolved","Structural basis for substrate selectivity differences unknown"]},{"year":2007,"claim":"Mouse Mib2 knockout causing exencephaly established an in vivo requirement for MIB2 in neural tube closure, extending its role beyond cell-autonomous Notch activation.","evidence":"Targeted gene knockout in mice with morphological phenotyping","pmids":["17987667"],"confidence":"Medium","gaps":["Molecular substrates responsible for neural tube phenotype not identified","Variable penetrance suggests modifier effects not characterized","Notch-dependence of the phenotype not formally tested"]},{"year":2011,"claim":"Identification of MIB2 in the BCL10 signaling complex, where it ubiquitinates NEMO and recruits TAK1, revealed a Notch-independent role for MIB2 in NF-κB pathway activation.","evidence":"Proteomic identification of MIB2 in BCL10 complex; in vitro pulldown; siRNA knockdown with NF-κB reporter","pmids":["21896478"],"confidence":"High","gaps":["Ubiquitin chain type on NEMO not specified","Physiological contexts requiring MIB2-dependent NF-κB activation not defined","Relationship between NF-κB and Notch functions of MIB2 unclear"]},{"year":2017,"claim":"Domain dissection in Drosophila showed ankyrin repeats and MIB-specific domains are essential for muscle integrity while the RING finger is specifically required for flight muscle development, establishing domain-specific functional requirements.","evidence":"Systematic domain deletion and missense mutagenesis in Drosophila with in vivo muscle phenotyping","pmids":["28282454"],"confidence":"Medium","gaps":["Substrates mediating muscle phenotypes not identified","Whether domain requirements are conserved in mammalian muscle unknown"]},{"year":2018,"claim":"Showing that CaMKIIβ phosphorylation of GABAB1 at Ser-867 primes MIB2-mediated K63-linked ubiquitination for lysosomal sorting established a phosphorylation-dependent substrate recognition mechanism and a neuronal function for MIB2.","evidence":"Phosphomimetic/phospho-null mutagenesis; K63-ubiquitination assay; surface receptor quantification in cortical neurons","pmids":["29881949"],"confidence":"High","gaps":["Whether MIB2 directly recognizes the phospho-degron or requires an adaptor unclear","In vivo neuronal phenotype of MIB2 loss not examined"]},{"year":2019,"claim":"Demonstrating that MIB2 K48-ubiquitinates CYLD at defined lysines for proteasomal degradation, and that Mib2 KO mice are protected from arthritis, established a direct MIB2–CYLD antagonism controlling NF-κB-driven inflammation in vivo.","evidence":"In vitro binding (AlphaScreen); site-specific K→R mutagenesis; Mib2 KO mice in arthritis model","pmids":["31366726"],"confidence":"High","gaps":["How MIB2 itself is regulated in inflammatory contexts not defined","Relative contribution of CYLD degradation vs. direct NEMO ubiquitination to NF-κB activation unclear"]},{"year":2023,"claim":"Discovery that MIB2 K63-ubiquitinates PD-L1 for RAB8-mediated TGN-to-plasma membrane trafficking, and that MIB2 deficiency enhances antitumor immunity, established MIB2 as a druggable node in immune checkpoint regulation.","evidence":"MIB2 KO; K63-ubiquitination assay; RAB8 epistasis; surface PD-L1 flow cytometry; mouse tumor models","pmids":["36719382"],"confidence":"High","gaps":["Specific PD-L1 lysine residues ubiquitinated not mapped","Whether MIB2 affects other immune checkpoint molecules unknown"]},{"year":2023,"claim":"Showing that FAT1 recruits MIB2 to ubiquitinate YAP/TAZ for proteasomal degradation in endothelial and tumor cells linked MIB2 to Hippo pathway regulation and angiogenesis control.","evidence":"Co-IP/interactome analysis; MIB2 KD in vitro and in vivo; YAP/TAZ ubiquitination assay; angiogenesis and xenograft models","pmids":["37031213","40478800"],"confidence":"High","gaps":["Ubiquitin chain type on YAP/TAZ not determined","Whether FAT1-MIB2 interaction is direct or scaffold-mediated not resolved"]},{"year":2025,"claim":"Recent work expanded MIB2 substrates to include SUZ12 (controlling H3K27me3 and PRC2 target gene silencing), Runx2 (controlling fatty acid metabolism in cardiomyocytes), and GPX4 (regulating ferroptosis), broadening MIB2's role into chromatin regulation, cardiac metabolism, and cell death.","evidence":"Co-IP/mass spectrometry; ubiquitination assays; siRNA knockdown with RNA-seq; AAV9-mediated cardiac overexpression in mice; xenograft models","pmids":["40478202","40159625","40592077"],"confidence":"Medium","gaps":["SUZ12 and Runx2 ubiquitination sites not mapped","Chain linkage type for GPX4 ubiquitination not determined","Each substrate identified by single lab without independent replication"]},{"year":2025,"claim":"Ebola virus VP35 binds MIB2 via an NNLNS motif to suppress MIB2-mediated interferon induction, revealing viral exploitation of MIB2 in innate immune evasion.","evidence":"Mutagenesis of VP35 NNLNS motif; minigenome and interferon induction assays","pmids":["40982696"],"confidence":"Medium","gaps":["MIB2 substrates mediating interferon induction not identified","Whether other viral proteins similarly target MIB2 unknown","Single lab finding"]},{"year":null,"claim":"How MIB2 achieves substrate selectivity across its numerous targets (K48 vs K63 linkage, degradative vs trafficking outcomes), and what upstream signals regulate MIB2 activity and expression in different tissues, remain major unresolved questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of MIB2 with any substrate","Post-translational regulation of MIB2 catalytic activity largely unexplored","Tissue-specific substrate hierarchies not systematically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,3,6,10,11,13,14,15,17,18,19,21]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,3,11]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[13]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[12,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,4,9,14,19]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,11,13,20]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,10,11,13,15,17,18,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[21]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,9]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[17]}],"complexes":[],"partners":["JAG2","CYLD","BCL10","FAT1","SUZ12","RUNX2","YAP1","GPX4"],"other_free_text":[]},"mechanistic_narrative":"MIB2 is a RING finger-dependent E3 ubiquitin ligase that regulates diverse cellular processes—including Notch signaling, NF-κB activation, YAP/TAZ turnover, immune checkpoint trafficking, and receptor sorting—by catalyzing both K63- and K48-linked ubiquitination of distinct substrate classes. MIB2 ubiquitinates Notch ligands (Jagged-2, Delta family members) to promote their endocytosis and activate Notch signaling, a function conserved from zebrafish to mammals and required for neural tube closure and cardiac trabeculation [PMID:15920166, PMID:17196985, PMID:17987667, PMID:28013292]. Through K48-linked ubiquitination, MIB2 targets CYLD, YAP/TAZ, SUZ12, Runx2, and CARD6 for proteasomal degradation, thereby modulating NF-κB-driven inflammation, Hippo pathway output, PRC2-dependent chromatin silencing, and metabolic gene expression [PMID:31366726, PMID:37031213, PMID:40478202, PMID:40159625]. MIB2 also catalyzes nonproteolytic K63-linked ubiquitination to direct PD-L1 from the trans-Golgi network to the plasma membrane via RAB8-mediated exocytosis and to sort GABAB receptors to lysosomes following CaMKIIβ-dependent phosphorylation [PMID:36719382, PMID:29881949]."},"prefetch_data":{"uniprot":{"accession":"Q96AX9","full_name":"E3 ubiquitin-protein ligase MIB2","aliases":["Mind bomb homolog 2","Novel zinc finger protein","Novelzin","Putative NF-kappa-B-activating protein 002N","RING-type E3 ubiquitin transferase MIB2","Skeletrophin","Zinc finger ZZ type with ankyrin repeat domain protein 1"],"length_aa":955,"mass_kda":103.7,"function":"E3 ubiquitin-protein ligase that mediates ubiquitination of Delta receptors, which act as ligands of Notch proteins. Positively regulates the Delta-mediated Notch signaling by ubiquitinating the intracellular domain of Delta, leading to endocytosis of Delta receptors","subcellular_location":"Cytoplasm; Endosome","url":"https://www.uniprot.org/uniprotkb/Q96AX9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MIB2","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MIB2","total_profiled":1310},"omim":[{"mim_id":"611141","title":"MIB E3 UBIQUITIN PROTEIN LIGASE 2; MIB2","url":"https://www.omim.org/entry/611141"},{"mim_id":"608677","title":"MIB E3 UBIQUITIN PROTEIN LIGASE 1; MIB1","url":"https://www.omim.org/entry/608677"},{"mim_id":"603453","title":"RECEPTOR-INTERACTING SERINE/THREONINE KINASE 1; RIPK1","url":"https://www.omim.org/entry/603453"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":46.9}],"url":"https://www.proteinatlas.org/search/MIB2"},"hgnc":{"alias_symbol":["skeletrophin","ZZZ5","FLJ39787"],"prev_symbol":["ZZANK1"]},"alphafold":{"accession":"Q96AX9","domains":[{"cath_id":"-","chopping":"67-307","consensus_level":"medium","plddt":89.4585,"start":67,"end":307},{"cath_id":"2.30.30,2.30.30","chopping":"391-454","consensus_level":"medium","plddt":85.86,"start":391,"end":454},{"cath_id":"1.25.40.20","chopping":"461-613","consensus_level":"high","plddt":88.2117,"start":461,"end":613},{"cath_id":"1.25.40","chopping":"745-855","consensus_level":"high","plddt":84.0335,"start":745,"end":855},{"cath_id":"3.30.40.10","chopping":"888-933","consensus_level":"high","plddt":89.4211,"start":888,"end":933},{"cath_id":"3.30.40.10","chopping":"967-1011","consensus_level":"high","plddt":89.5316,"start":967,"end":1011},{"cath_id":"2.30.30","chopping":"320-383","consensus_level":"medium","plddt":85.1064,"start":320,"end":383}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96AX9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96AX9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96AX9-F1-predicted_aligned_error_v6.png","plddt_mean":85.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MIB2","jax_strain_url":"https://www.jax.org/strain/search?query=MIB2"},"sequence":{"accession":"Q96AX9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96AX9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96AX9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96AX9"}},"corpus_meta":[{"pmid":"35904542","id":"PMC_35904542","title":"MIB2: metal ion-binding site prediction and modeling server.","date":"2022","source":"Bioinformatics (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/35904542","citation_count":88,"is_preprint":false},{"pmid":"36719382","id":"PMC_36719382","title":"PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.","date":"2023","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/36719382","citation_count":50,"is_preprint":false},{"pmid":"15920166","id":"PMC_15920166","title":"Skeletrophin, a novel ubiquitin ligase to the intracellular region of Jagged-2, is aberrantly expressed in multiple myeloma.","date":"2005","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/15920166","citation_count":49,"is_preprint":false},{"pmid":"17331493","id":"PMC_17331493","title":"The characterization of zebrafish antimorphic mib alleles reveals that Mib and Mind bomb-2 (Mib2) function redundantly.","date":"2007","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17331493","citation_count":45,"is_preprint":false},{"pmid":"21896478","id":"PMC_21896478","title":"The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21896478","citation_count":41,"is_preprint":false},{"pmid":"17196985","id":"PMC_17196985","title":"Zebrafish Mib and Mib2 are mutual E3 ubiquitin ligases with common and specific delta substrates.","date":"2006","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17196985","citation_count":34,"is_preprint":false},{"pmid":"16715130","id":"PMC_16715130","title":"A ubiquitin ligase, skeletrophin, is a negative regulator of melanoma 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signalling.","date":"2014","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/25565632","citation_count":26,"is_preprint":false},{"pmid":"14507647","id":"PMC_14507647","title":"Down-regulation of a novel actin-binding molecule, skeletrophin, in malignant melanoma.","date":"2003","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/14507647","citation_count":24,"is_preprint":false},{"pmid":"29881949","id":"PMC_29881949","title":"Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) β-Dependent Phosphorylation of GABAB1 Triggers Lysosomal Degradation of GABAB Receptors via Mind Bomb-2 (MIB2)-Mediated Lys-63-Linked Ubiquitination.","date":"2018","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/29881949","citation_count":22,"is_preprint":false},{"pmid":"34635817","id":"PMC_34635817","title":"Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation.","date":"2021","source":"Cell death and 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phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40592077","citation_count":5,"is_preprint":false},{"pmid":"40159625","id":"PMC_40159625","title":"Mib2 Regulates Lipid Metabolism in Heart Failure With Preserved Ejection Fraction via the Runx2-Hmgcs2 Axis.","date":"2025","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40159625","citation_count":3,"is_preprint":false},{"pmid":"40478800","id":"PMC_40478800","title":"The tumor suppressor FAT1 controls YAP/TAZ protein degradation and tumor cell proliferation through E3 ligase MIB2.","date":"2025","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/40478800","citation_count":2,"is_preprint":false},{"pmid":"40982696","id":"PMC_40982696","title":"Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40982696","citation_count":1,"is_preprint":false},{"pmid":"37436668","id":"PMC_37436668","title":"MIB2 promotes the progression of non-small cell lung cancer by regulating cell cycle control pathways.","date":"2023","source":"Genes & genomics","url":"https://pubmed.ncbi.nlm.nih.gov/37436668","citation_count":1,"is_preprint":false},{"pmid":"40777377","id":"PMC_40777377","title":"Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40777377","citation_count":1,"is_preprint":false},{"pmid":"39019259","id":"PMC_39019259","title":"MIB2 Functions in Oocyte Meiosis by Modulating Chromatin Configuration.","date":"2024","source":"Molecular & cellular proteomics : 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\"confidence_rationale\": \"Tier 2 — reciprocal interaction confirmed by two methods (Y2H + Co-IP), single lab\",\n      \"pmids\": [\"14507647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MIB2 (skeletrophin) is a RING finger-dependent E3 ubiquitin ligase that binds the intracellular region of Notch ligand Jagged-2 (but not Delta-1, -3, -4, or Jagged-1) and catalyzes its ubiquitination; RING-mutated MIB2 loses this activity. Exogenous MIB2, but not its RING mutant, induced Hes-1 expression in stromal cells through Notch signaling.\",\n      \"method\": \"In vitro autoubiquitination assay with recombinant proteins; RING mutant analysis; cell-based Hes-1 reporter\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with RING mutant controls, replicated in cell-based assay\",\n      \"pmids\": [\"15920166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MIB2 (skeletrophin) reduces melanoma cell invasion in vitro and in vivo and suppresses colony formation in soft agar in a RING motif-dependent manner; it also downregulates transcription of the Met oncogene and increases Hes1 transcription.\",\n      \"method\": \"Loss-of-function/gain-of-function with RING mutant; invasion assay; soft agar colony formation; gene expression analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RING mutant controls used, multiple readouts, single lab\",\n      \"pmids\": [\"16715130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Zebrafish Mib2, like Mib, has C-terminal-most RING finger-dependent E3 ubiquitin ligase activity; Mib and Mib2 are reciprocal E3 ubiquitin ligases and substrates of each other. They share DeltaC as a common substrate for ubiquitylation and endocytosis, but differ in DeltaD internalization. Mib and Mib2 bind differently to extracellular and intracellular parts of DeltaA and DeltaC.\",\n      \"method\": \"In vitro ubiquitination assay; RING mutant analysis; Delta internalization assay; binding assays in transfected cells\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination with RING mutants, multiple substrates tested, ortholog consistent with mammalian MIB2\",\n      \"pmids\": [\"17196985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Zebrafish Mib2 is colocalized with Mib in transfected cells and functions redundantly with Mib in regulating Notch signaling in embryos. Dominant-negative Mib alleles suppress Mib2 function in a dosage-dependent manner, indicating competitive interaction. Notch signaling negatively regulates mib expression in a Su(H)-dependent negative feedback loop.\",\n      \"method\": \"Colocalization in transfected cells; genetic epistasis in zebrafish embryos; dominant-negative analysis; Su(H) reporter assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple alleles and colocalization, replicated in vivo, consistent with mammalian function\",\n      \"pmids\": [\"17331493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Targeted disruption of Mib2 in mice causes exencephaly (failure to close cranial neural tube) with variable penetrance dependent on genetic background, establishing a role for Mib2 in neural tube closure in vivo.\",\n      \"method\": \"Targeted gene knockout in mice; morphological phenotypic analysis\",\n      \"journal\": \"Genesis (New York, N.Y. : 2000)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined phenotype, but molecular mechanism not detailed beyond Notch pathway membership\",\n      \"pmids\": [\"17987667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MIB2 is a component of the activated BCL10 signaling complex; it directly interacts with BCL10 (shown by in vitro translation/pulldown), promotes autoubiquitination and ubiquitination of IKKγ/NEMO, recruits and activates TAK1, and thereby controls BCL10-dependent NF-κB activation. MIB2 knockdown inhibits BCL10-dependent NF-κB activation.\",\n      \"method\": \"Proteomic identification; in vitro pulldown; overexpression; siRNA knockdown; NF-κB reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct in vitro binding, ubiquitination assays, KD phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"21896478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CYLD (deubiquitinating enzyme) interacts with MIB2 (E3 ubiquitin ligase); coexpression of CYLD and MIB2 results in stabilization of MIB2 protein levels and reduced levels of JAG2, linking MIB2 to regulation of Notch signaling through CYLD.\",\n      \"method\": \"Proteomics-based interaction screen; co-expression experiments; siRNA knockdown of CYLD; Notch target gene expression analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — interaction identified by proteomics and validated in cells, functional consequence shown, single lab\",\n      \"pmids\": [\"25565632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Drosophila, the ankyrin repeats (in their entirety) and the MIB-specific domains of Mib2 are essential for its function in maintaining integrity of larval skeletal and visceral muscles. The RING finger domain is required for adult flight muscle development. Missense mutations in the MIB domain and RING finger cause flightless hypomorphic phenotypes, establishing domain-specific functional requirements.\",\n      \"method\": \"Domain deletion/mutagenesis in vivo; forward genetic screen; in vivo imaging of flight muscles\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic domain dissection with multiple alleles in vivo, consistent with mammalian MIB2 domain architecture\",\n      \"pmids\": [\"28282454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MIB2 missense variant p.V742G shows reduced ubiquitination activity in vitro and is associated with reduced NOTCH signaling (decreased HES1 and NOTCH3 expression) in white blood cells; MIB2 variants affect NOTCH signaling, proliferation, and apoptosis in primary rat cardiomyocytes, establishing MIB2 as a regulator of NOTCH signaling relevant to cardiac trabeculation.\",\n      \"method\": \"In vitro ubiquitination assay with purified variant protein; whole exome sequencing; gene expression analysis; primary cardiomyocyte functional assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro activity assay with variant protein, corroborated by cell-based and patient data\",\n      \"pmids\": [\"28013292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MIB2 mediates Lys-63-linked ubiquitination of GABAB1 subunit of GABAB receptors, sorting them to lysosomes for degradation. CaMKIIβ (but not CaMKIIα) promotes MIB2-mediated K63-linked ubiquitination of GABAB1 at multiple sites by phosphorylating GABAB1 at Ser-867; phosphomimetic S867D mutation increases K63-ubiquitination while S867A mutation reduces it and increases surface expression.\",\n      \"method\": \"Overexpression of CaMKII isoforms; phosphomimetic/phospho-null mutagenesis; K63-ubiquitination assay; surface GABAB receptor quantification in cortical neurons\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of phosphorylation site, K63-ubiquitination assay, multiple orthogonal methods, defined PTM writer identified\",\n      \"pmids\": [\"29881949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MIB2 promotes proteasomal degradation of the deubiquitinating enzyme CYLD by catalyzing Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. The ankyrin repeat of MIB2 interacts with the third CAP domain of CYLD. MIB2-dependent CYLD degradation activates NF-κB signaling via TNFα and LUBAC. Mib2-knockout mice showed suppressed arthritic inflammation and reduced serum IL-6.\",\n      \"method\": \"Cell-free AlphaScreen and pulldown assays; immunofluorescence; Mib2 KO cells and mice; site-specific ubiquitination mapping; arthritis model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding, site-specific ubiquitination with K→R mutants, KO mice with defined inflammatory phenotype, multiple methods\",\n      \"pmids\": [\"31366726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Gm364 (a multi-pass transmembrane protein) directly binds and anchors MIB2 on the membrane; membrane-localized MIB2 ubiquitinates and activates DLL3, which activates Notch2, leading to production of NICD2 that activates AKT to regulate oocyte meiosis and quality.\",\n      \"method\": \"Knockout mouse model; co-IP; oocyte phenotypic analysis (ROS, mitochondrial membrane potential, aneuploidy); epistasis experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO phenotype with pathway epistasis and Co-IP, single lab\",\n      \"pmids\": [\"34635817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIB2 is required for translocation of PD-L1 from the trans-Golgi network (TGN) to the plasma membrane. Mechanistically, MIB2 catalyzes nonproteolytic K63-linked ubiquitination of PD-L1, facilitating its trafficking via RAB8-mediated exocytosis from TGN to plasma membrane. MIB2 deficiency reduces PD-L1 surface expression and promotes antitumor T-cell immunity in mice.\",\n      \"method\": \"MIB2 knockdown/knockout; K63-ubiquitination assay; RAB8 epistasis; surface PD-L1 flow cytometry; in vivo mouse tumor models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — K63-ubiquitination assay, RAB8 epistasis, KO mouse model, multiple orthogonal methods\",\n      \"pmids\": [\"36719382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FAT1 acts as an upstream regulator of MIB2 in endothelial cells: FAT1 interacts with MIB2 (identified by interactome analysis), and together they promote ubiquitination and proteasomal degradation of YAP/TAZ. Loss of MIB2 in endothelial cells recapitulates FAT1 depletion, causing decreased YAP/TAZ degradation, increased YAP/TAZ signaling, and increased endothelial cell proliferation and angiogenesis.\",\n      \"method\": \"Co-IP/interactome analysis; MIB2 KD in vitro and in vivo; YAP/TAZ ubiquitination assay; angiogenesis models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — interactome identification, ubiquitination assay, KD with defined cellular and in vivo phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"37031213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIB2 interacts with CARD6 and promotes K48-linked CARD6 polyubiquitination and proteasomal degradation in hepatocytes under high fructose conditions; MIB2 knockdown reverses CARD6 downregulation and lipid accumulation, establishing MIB2 as an upstream regulator of CARD6 in hepatic lipid metabolism.\",\n      \"method\": \"Immunoprecipitation; immunofluorescence; siRNA knockdown; immunoblotting\",\n      \"journal\": \"Food & function\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP interaction and KD phenotype, single lab, no in vitro ubiquitination reconstitution\",\n      \"pmids\": [\"37186242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MIB2 expression is increased in SN (surrounded nucleolus)-stage oocytes; depletion of MIB2 in SN oocytes disrupts meiotic apparatus and increases aneuploidy, while overexpression of MIB2 in NSN oocytes facilitates chromatin configuration transition from NSN to SN and mitigates spindle/chromosome disorganization.\",\n      \"method\": \"Quantitative proteomics; MIB2 depletion and overexpression in oocytes; meiotic phenotype analysis (spindle assembly, aneuploidy)\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss- and gain-of-function with defined cellular phenotypes, single lab\",\n      \"pmids\": [\"39019259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MIB2 directly interacts with and ubiquitinates SUZ12 (a PRC2 complex component), controlling SUZ12 stability and H3K27me3 levels; the MIB/HERC and ZZ-type domains of MIB2 mediate interaction with SUZ12. MIB2 knockdown reduces SUZ12 protein and H3K27me3, upregulates PRC2 target genes, and decreases cell proliferation.\",\n      \"method\": \"Immunoprecipitation + mass spectrometry; siRNA knockdown; ubiquitination assay; RNA-seq; flow cytometry; colony formation assay\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP/MS interaction, ubiquitination assay, KD phenotype, single lab\",\n      \"pmids\": [\"40478202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MIB2 directly interacts with Runx2 and ubiquitinates it for degradation, thereby inhibiting Hmgcs2 transcription and impairing fatty acid metabolic processes in cardiomyocytes. Cardiac-specific overexpression of Mib2 in ob/ob mice worsens cardiac dysfunction and lipid accumulation.\",\n      \"method\": \"Immunoprecipitation; dual luciferase reporter assay; proteomic analysis; AAV9-mediated cardiac overexpression in mice\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, luciferase reporter, in vivo overexpression, single lab\",\n      \"pmids\": [\"40159625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FAT1 loss in tumor cells (including HNSCC) decreases YAP/TAZ ubiquitination and degradation mediated by MIB2; suppression of MIB2 alone phenocopies FAT1 loss, reducing YAP/TAZ ubiquitination and increasing tumor cell proliferation in vitro and tumor growth in vivo.\",\n      \"method\": \"FAT1/MIB2 KD in tumor cells; YAP/TAZ ubiquitination assay; tumor xenograft in vivo; interactome analysis of FAT1 cytoplasmic domain\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ubiquitination assay, KD with in vivo xenograft validation, interactome identification, multiple cell lines\",\n      \"pmids\": [\"40478800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ebola virus VP35 contains an NNLNS motif (residues 201–205) that serves as a direct binding site for MIB2; VP35 binding to MIB2 via this motif inhibits MIB2-mediated interferon induction and also suppresses EBOV minigenome RNA synthesis activity.\",\n      \"method\": \"Mutagenesis of NNLNS motif; minigenome assay; interferon induction assay; interaction mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of binding motif with functional readouts (IFN induction, minigenome), single lab\",\n      \"pmids\": [\"40982696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MIB2 promotes ubiquitin-mediated degradation of GPX4 by interacting with GPX4, an interaction regulated by Nrf2; taraxerol treatment reduces MIB2-mediated GPX4 ubiquitination by targeting Nrf2/MIB2 interaction, triggering ferroptosis in breast cancer cells.\",\n      \"method\": \"Co-IP; dual-luciferase reporter assay; ubiquitination assay; xenograft in vivo model\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, ubiquitination assay, in vivo xenograft, single lab\",\n      \"pmids\": [\"40592077\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MIB2 is a RING finger-dependent E3 ubiquitin ligase whose catalytic activity requires its C-terminal RING domain and whose substrate recruitment involves ankyrin repeats and MIB-specific domains; it catalyzes K63-linked ubiquitination of Notch ligands (Jagged-2, Delta) to promote their endocytosis and Notch pathway activation, K63-linked ubiquitination of PD-L1 to drive RAB8-mediated exocytosis to the plasma membrane, K63-linked ubiquitination of GABAB1 (primed by CaMKIIβ phosphorylation at Ser-867) for lysosomal sorting, and K48-linked ubiquitination of CYLD, CARD6, SUZ12, Runx2, and YAP/TAZ for proteasomal degradation; MIB2 is recruited to substrates by interacting partners including FAT1 (for YAP/TAZ) and Gm364 (for DLL3/Notch2), and its activity is counteracted by CYLD, placing MIB2 at the intersection of Notch, NF-κB, YAP/TAZ, and immune checkpoint signaling pathways.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MIB2 is a RING finger-dependent E3 ubiquitin ligase that regulates diverse cellular processes—including Notch signaling, NF-κB activation, YAP/TAZ turnover, immune checkpoint trafficking, and receptor sorting—by catalyzing both K63- and K48-linked ubiquitination of distinct substrate classes. MIB2 ubiquitinates Notch ligands (Jagged-2, Delta family members) to promote their endocytosis and activate Notch signaling, a function conserved from zebrafish to mammals and required for neural tube closure and cardiac trabeculation [PMID:15920166, PMID:17196985, PMID:17987667, PMID:28013292]. Through K48-linked ubiquitination, MIB2 targets CYLD, YAP/TAZ, SUZ12, Runx2, and CARD6 for proteasomal degradation, thereby modulating NF-κB-driven inflammation, Hippo pathway output, PRC2-dependent chromatin silencing, and metabolic gene expression [PMID:31366726, PMID:37031213, PMID:40478202, PMID:40159625]. MIB2 also catalyzes nonproteolytic K63-linked ubiquitination to direct PD-L1 from the trans-Golgi network to the plasma membrane via RAB8-mediated exocytosis and to sort GABAB receptors to lysosomes following CaMKIIβ-dependent phosphorylation [PMID:36719382, PMID:29881949].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of MIB2 (skeletrophin) as a zinc-finger and ankyrin-repeat protein that binds actin monomer established its initial molecular identity and suggested cytoskeletal association.\",\n      \"evidence\": \"Yeast two-hybrid screen and co-immunoprecipitation in mammalian cells\",\n      \"pmids\": [\"14507647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Actin binding not confirmed by reciprocal pull-down with purified components\", \"Functional significance of actin interaction never followed up\", \"No catalytic activity demonstrated at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrating that MIB2 is a RING-dependent E3 ubiquitin ligase that ubiquitinates Jagged-2 and activates Notch signaling resolved the catalytic function and first substrate specificity of MIB2.\",\n      \"evidence\": \"In vitro autoubiquitination assay with recombinant MIB2 and RING mutant; cell-based Hes-1 reporter\",\n      \"pmids\": [\"15920166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MIB2 ubiquitinates other Notch ligands in mammals remained open\", \"Ubiquitin chain linkage type not determined\", \"In vivo relevance not yet tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Zebrafish studies established that MIB2 and MIB are reciprocal E3 ligases sharing Delta substrates but differing in specificity, revealing functional redundancy and divergence within the MIB family for Notch ligand regulation.\",\n      \"evidence\": \"In vitro ubiquitination and Delta internalization assays with RING mutants in zebrafish; genetic epistasis in embryos\",\n      \"pmids\": [\"17196985\", \"17331493\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of MIB vs MIB2 in mammalian tissues unresolved\", \"Structural basis for substrate selectivity differences unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mouse Mib2 knockout causing exencephaly established an in vivo requirement for MIB2 in neural tube closure, extending its role beyond cell-autonomous Notch activation.\",\n      \"evidence\": \"Targeted gene knockout in mice with morphological phenotyping\",\n      \"pmids\": [\"17987667\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrates responsible for neural tube phenotype not identified\", \"Variable penetrance suggests modifier effects not characterized\", \"Notch-dependence of the phenotype not formally tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of MIB2 in the BCL10 signaling complex, where it ubiquitinates NEMO and recruits TAK1, revealed a Notch-independent role for MIB2 in NF-κB pathway activation.\",\n      \"evidence\": \"Proteomic identification of MIB2 in BCL10 complex; in vitro pulldown; siRNA knockdown with NF-κB reporter\",\n      \"pmids\": [\"21896478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on NEMO not specified\", \"Physiological contexts requiring MIB2-dependent NF-κB activation not defined\", \"Relationship between NF-κB and Notch functions of MIB2 unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Domain dissection in Drosophila showed ankyrin repeats and MIB-specific domains are essential for muscle integrity while the RING finger is specifically required for flight muscle development, establishing domain-specific functional requirements.\",\n      \"evidence\": \"Systematic domain deletion and missense mutagenesis in Drosophila with in vivo muscle phenotyping\",\n      \"pmids\": [\"28282454\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrates mediating muscle phenotypes not identified\", \"Whether domain requirements are conserved in mammalian muscle unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing that CaMKIIβ phosphorylation of GABAB1 at Ser-867 primes MIB2-mediated K63-linked ubiquitination for lysosomal sorting established a phosphorylation-dependent substrate recognition mechanism and a neuronal function for MIB2.\",\n      \"evidence\": \"Phosphomimetic/phospho-null mutagenesis; K63-ubiquitination assay; surface receptor quantification in cortical neurons\",\n      \"pmids\": [\"29881949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MIB2 directly recognizes the phospho-degron or requires an adaptor unclear\", \"In vivo neuronal phenotype of MIB2 loss not examined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that MIB2 K48-ubiquitinates CYLD at defined lysines for proteasomal degradation, and that Mib2 KO mice are protected from arthritis, established a direct MIB2–CYLD antagonism controlling NF-κB-driven inflammation in vivo.\",\n      \"evidence\": \"In vitro binding (AlphaScreen); site-specific K→R mutagenesis; Mib2 KO mice in arthritis model\",\n      \"pmids\": [\"31366726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MIB2 itself is regulated in inflammatory contexts not defined\", \"Relative contribution of CYLD degradation vs. direct NEMO ubiquitination to NF-κB activation unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that MIB2 K63-ubiquitinates PD-L1 for RAB8-mediated TGN-to-plasma membrane trafficking, and that MIB2 deficiency enhances antitumor immunity, established MIB2 as a druggable node in immune checkpoint regulation.\",\n      \"evidence\": \"MIB2 KO; K63-ubiquitination assay; RAB8 epistasis; surface PD-L1 flow cytometry; mouse tumor models\",\n      \"pmids\": [\"36719382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific PD-L1 lysine residues ubiquitinated not mapped\", \"Whether MIB2 affects other immune checkpoint molecules unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing that FAT1 recruits MIB2 to ubiquitinate YAP/TAZ for proteasomal degradation in endothelial and tumor cells linked MIB2 to Hippo pathway regulation and angiogenesis control.\",\n      \"evidence\": \"Co-IP/interactome analysis; MIB2 KD in vitro and in vivo; YAP/TAZ ubiquitination assay; angiogenesis and xenograft models\",\n      \"pmids\": [\"37031213\", \"40478800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on YAP/TAZ not determined\", \"Whether FAT1-MIB2 interaction is direct or scaffold-mediated not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Recent work expanded MIB2 substrates to include SUZ12 (controlling H3K27me3 and PRC2 target gene silencing), Runx2 (controlling fatty acid metabolism in cardiomyocytes), and GPX4 (regulating ferroptosis), broadening MIB2's role into chromatin regulation, cardiac metabolism, and cell death.\",\n      \"evidence\": \"Co-IP/mass spectrometry; ubiquitination assays; siRNA knockdown with RNA-seq; AAV9-mediated cardiac overexpression in mice; xenograft models\",\n      \"pmids\": [\"40478202\", \"40159625\", \"40592077\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUZ12 and Runx2 ubiquitination sites not mapped\", \"Chain linkage type for GPX4 ubiquitination not determined\", \"Each substrate identified by single lab without independent replication\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Ebola virus VP35 binds MIB2 via an NNLNS motif to suppress MIB2-mediated interferon induction, revealing viral exploitation of MIB2 in innate immune evasion.\",\n      \"evidence\": \"Mutagenesis of VP35 NNLNS motif; minigenome and interferon induction assays\",\n      \"pmids\": [\"40982696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MIB2 substrates mediating interferon induction not identified\", \"Whether other viral proteins similarly target MIB2 unknown\", \"Single lab finding\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MIB2 achieves substrate selectivity across its numerous targets (K48 vs K63 linkage, degradative vs trafficking outcomes), and what upstream signals regulate MIB2 activity and expression in different tissues, remain major unresolved questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of MIB2 with any substrate\", \"Post-translational regulation of MIB2 catalytic activity largely unexplored\", \"Tissue-specific substrate hierarchies not systematically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 3, 6, 10, 11, 13, 14, 15, 17, 18, 19, 21]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 3, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [12, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 4, 9, 14, 19]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 11, 13, 20]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 10, 11, 13, 15, 17, 18, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [21]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"JAG2\",\n      \"CYLD\",\n      \"BCL10\",\n      \"FAT1\",\n      \"SUZ12\",\n      \"RUNX2\",\n      \"YAP1\",\n      \"GPX4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}