{"gene":"OTUB1","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2010,"finding":"OTUB1 suppresses RNF168-dependent K63-linked poly-ubiquitination at DNA double-strand breaks independently of its catalytic deubiquitinase activity by binding to and inhibiting UBC13 (UBE2N), the cognate E2 enzyme for RNF168. OTUB1 also binds E2s of the UBE2D and UBE2E subfamilies through the same non-canonical mechanism.","method":"Co-immunoprecipitation, in vitro ubiquitination assays, siRNA knockdown with DSB repair readouts, catalytic mutant analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, in vitro assays with catalytic-dead mutant, replicated across multiple E2 families, published in Nature","pmids":["20725033"],"is_preprint":false},{"year":2012,"finding":"Crystal structures of OTUB1–UBC13 and OTUB1 bound to ubiquitin aldehyde plus a chemical UBC13~Ub conjugate reveal that free ubiquitin allosterically binds a distal site on OTUB1, triggering conformational changes that form an N-terminal ubiquitin-binding helix, increase affinity for donor ubiquitin in the E2~Ub thiolester, occlude the RING E3 binding site on UBC13, and block acceptor-ubiquitin attack on the thiolester—thereby inhibiting K63-linked ubiquitin chain synthesis.","method":"X-ray crystallography, biochemical ubiquitination assays, structure-guided mutagenesis, surface plasmon resonance","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus reconstituted biochemistry and mutagenesis in a single rigorous study","pmids":["22367539"],"is_preprint":false},{"year":2012,"finding":"OTUB1 co-opts its canonical Lys48-linked distal ubiquitin-binding site to recognize charged E2~Ub thioester intermediates; free ubiquitin binding at this site promotes formation of the inhibited E2 complex, with the donor-ubiquitin Lys48 positioned near the OTUB1 catalytic site in a configuration mimicking K48-linked chain cleavage products.","method":"X-ray crystallography, biochemical E2 inhibition assays, mutagenesis, structural analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation, independent confirmation of the same mechanism as PMID:22367539","pmids":["22325355"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of human OTUB1 in complex with UBC13 and MMS2 at 3.15 Å resolution reveals atomic-level contacts required for inhibition of K63-linked ubiquitin chain formation; structure-based OTUB1 mutants fail to inhibit K63 chain formation in vitro and histone ubiquitination and 53BP1 foci in vivo.","method":"X-ray crystallography, surface plasmon resonance, structure-guided mutagenesis, in vitro ubiquitination assays, cell-based 53BP1 foci assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical and cellular validation, multiple orthogonal methods","pmids":["22679021"],"is_preprint":false},{"year":2013,"finding":"E2 ubiquitin-conjugating enzymes (UBC13, UBCH5) stimulate OTUB1 catalytic cleavage of K48-linked polyubiquitin by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix; this stimulation is regulated by the ratio of charged to uncharged E2 and by concentrations of K48 polyubiquitin and free ubiquitin, establishing bidirectional cross-regulation between OTUB1 and its E2 partners.","method":"Biochemical DUB activity assays, X-ray crystallography of human and worm OTUB1/UBCH5B, mutagenesis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus reconstituted biochemistry across two species with mutagenesis","pmids":["23955022"],"is_preprint":false},{"year":2009,"finding":"OTUB1 interacts with TRAF3 and TRAF6 by co-immunoprecipitation and mediates their deubiquitination, thereby negatively regulating virus-triggered type I interferon induction and IRF3/NF-κB activation.","method":"Co-immunoprecipitation, overexpression/knockdown with reporter assays for IFN-β transcription, antiviral response assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with functional knockdown/overexpression data, single lab, two orthogonal readouts","pmids":["19996094"],"is_preprint":false},{"year":2009,"finding":"OTUB1 directly deubiquitinates estrogen receptor alpha (ERα) in vitro and in cells; it interacts with ERα (identified by mass spectrometry of an ERα complex) and negatively regulates ERα-mediated transcription while stabilizing ERα protein on chromatin.","method":"Mass spectrometry of ERα complex, in vitro deubiquitination assay, Co-IP, transcriptional reporter assays, ChIP","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro DUB assay plus co-IP and functional reporter assays, single lab","pmids":["19383985"],"is_preprint":false},{"year":2013,"finding":"OTUB1 is recruited to the active phospho-SMAD2/3 complex upon TGFβ stimulation; it inhibits ubiquitylation and proteasomal degradation of phospho-SMAD2/3 via non-catalytic E2 inhibition (independent of its isopeptidase activity), thereby sustaining TGFβ-mediated gene transcription and cell migration. Depletion of OTUB1 causes rapid loss of phospho-SMAD2/3 rescued by the proteasomal inhibitor bortezomib.","method":"Co-immunoprecipitation (signal-dependent), siRNA knockdown, catalytic mutant analysis, proteasome inhibitor rescue, TGFβ reporter assays, migration assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, catalytic-dead mutant, proteasome inhibitor rescue, functional migration assay; multiple orthogonal methods in one study","pmids":["24071738"],"is_preprint":false},{"year":2013,"finding":"OTUB1 associates with c-IAP1 within the TWEAK receptor signalling complex, disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo, stabilises c-IAP1, and thereby sustains TWEAK-induced canonical NF-κB and MAPK signalling while preventing caspase activation and cell death.","method":"Co-immunoprecipitation, in vitro and in vivo deubiquitination assays, siRNA knockdown with caspase/NF-κB readouts, zebrafish c-IAP knockdown model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro DUB assay, reciprocal Co-IP, in vivo zebrafish validation, multiple functional readouts","pmids":["23524849"],"is_preprint":false},{"year":2014,"finding":"Otub1 suppresses MDM2-mediated p53 ubiquitination and stabilises p53 independently of its deubiquitinase activity by non-canonically inhibiting the E2 enzyme UbcH5; monoubiquitination of Otub1 (primarily at Lys59 and Lys109 by UbcH5) is required for Otub1 to suppress UbcH5 and stabilise p53, as a lysine-free Otub1 mutant (K0) fails to achieve these functions.","method":"In vitro ubiquitination/monoubiquitination assays, Co-IP, cycloheximide chase, apoptosis assays, cell proliferation assays, UbcH5 binding studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutants, multiple functional cellular readouts, mechanistic dissection of auto-regulation","pmids":["24403071"],"is_preprint":false},{"year":2015,"finding":"Casein kinase 2 (CK2) phosphorylates OTUB1 at Ser16, promoting its nuclear accumulation; pharmacological CK2 inhibition or genetic ablation blocks Ser16 phosphorylation and causes nuclear exclusion of OTUB1. Ser16-phosphorylated OTUB1 is detected exclusively in the nucleus and promotes 53BP1 DNA repair foci formation after ionising radiation.","method":"In vitro CK2 phosphorylation assay, phospho-specific antibody, CK2 inhibitor/KO, subcellular fractionation/immunofluorescence, 53BP1 foci assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay, genetic and pharmacological CK2 ablation, direct localization linked to functional DNA repair outcome","pmids":["25872870"],"is_preprint":false},{"year":2015,"finding":"OTUB1 inhibits ubiquitination and proteasomal degradation of FOXM1 through its catalytic (C91-dependent) deubiquitinase activity; the C91S catalytic mutant fails to stabilise FOXM1, increase its half-life, or enhance proliferative and epirubicin-resistance phenotypes.","method":"Co-immunoprecipitation, siRNA knockdown and overexpression, cycloheximide chase, catalytic mutant (C91S), K48-ubiquitin linkage analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, catalytic mutant, cycloheximide chase, multiple functional assays in one study","pmids":["26148240"],"is_preprint":false},{"year":2016,"finding":"OTUB1 inhibits RAS mono- and di-ubiquitination independently of its catalytic activity, resulting in sequestration of RAS on the plasma membrane and enhanced RAS activation and ERK1/2 signalling, thereby promoting tumorigenesis in wild-type RAS cells.","method":"In vitro ubiquitination assays, catalytic mutant analysis, confocal microscopy for RAS membrane localisation, RAS-GTP pull-down, tumour xenograft assays","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro assays with catalytic mutant, RAS membrane localisation by imaging, functional in vivo xenograft data, single lab","pmids":["26881969"],"is_preprint":false},{"year":2016,"finding":"FIH (factor inhibiting HIF) hydroxylates OTUB1 at asparagine 22; mutation of N22 profoundly alters OTUB1 protein interaction network and impairs cellular metabolic processes, identifying OTUB1 as a non-HIF substrate whose activity is regulated by oxygen-dependent hydroxylation.","method":"In vitro hydroxylation assays, N22A mutant co-immunoprecipitation proteomics, cellular metabolic assays (oxygen consumption, glycolysis)","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro hydroxylation assay plus interactome and metabolic readouts with N22A mutant, single lab","pmids":["26752685"],"is_preprint":false},{"year":2018,"finding":"OTUB1 directly interacts with DEPTOR via its N-terminal domain, deubiquitinates DEPTOR in a Cys91-independent but Asp88-dependent (non-canonical) manner, stabilises DEPTOR, and thereby suppresses amino-acid-induced mTORC1 activation, cellular autophagy, and cell proliferation/size.","method":"In vitro deubiquitination assay, co-immunoprecipitation, catalytic-dead and D88A mutant analysis, mTORC1 activity assays (S6K phosphorylation), autophagy assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro DUB assay, D88A and C91S mutants dissect mechanism, multiple mTOR pathway readouts, single lab","pmids":["29382726"],"is_preprint":false},{"year":2018,"finding":"OTUB1 stabilises Snail by preventing its ubiquitination and proteasomal degradation, facilitating epithelial-mesenchymal transition and metastasis of esophageal squamous cell carcinoma cells.","method":"Co-immunoprecipitation, siRNA/overexpression, ubiquitination assays, invasion/migration assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional invasion readout, single lab","pmids":["29559747"],"is_preprint":false},{"year":2018,"finding":"OTUB1 non-canonically stabilises the E2 enzyme UBE2E1 by suppressing its autoubiquitination in vitro and in cells, preventing proteasomal degradation of UBE2E1; this function is independent of OTUB1 catalytic activity but requires its ability to bind UBE2E1. Otub1 knockout mice exhibit late-stage embryonic lethality.","method":"In vitro autoubiquitination assay, OTUB1 KO cell lines (mouse and human), cycloheximide chase, catalytic mutant and binding-defective mutant analysis, mouse knockout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution, genetic KO in two species, catalytic vs. binding mutant dissection, replicated in mouse and human cells","pmids":["30282802"],"is_preprint":false},{"year":2019,"finding":"OTUB1 directly interacts with SLC7A11 (xCT), a key component of the cystine-glutamate antiporter, and stabilises SLC7A11 protein. CD44 overexpression enhances the OTUB1–SLC7A11 interaction, while CD44 depletion partially abrogates it. OTUB1 knockdown destabilises SLC7A11 and promotes ferroptosis in cancer xenografts.","method":"Biochemical purification/mass spectrometry to identify OTUB1 as SLC7A11-binding partner, Co-IP, siRNA knockdown, xenograft tumour growth assays, ferroptosis markers","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — mass-spec identification, reciprocal Co-IP, in vivo xenograft validation, CD44 epistasis, multiple orthogonal methods","pmids":["30709928"],"is_preprint":false},{"year":2019,"finding":"FIH forms an oxygen-sensitive, likely covalent (co-translational amide) bond with OTUB1 within cells, generating a heterotrimeric complex of two FIH and one OTUB1; this complexation regulates OTUB1 deubiquitinase activity in an oxygen-dependent manner, providing a mechanism for hypoxia adaptation.","method":"Biochemical co-precipitation, mass spectrometry analysis of bond, co-expression in mammalian and bacterial cells, DUB activity assays, oxygen-dependency experiments","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods, novel covalent bond proposed with supporting MS data, single lab","pmids":["31299612"],"is_preprint":false},{"year":2019,"finding":"IL-15 induces membrane recruitment of Otub1, which then non-canonically inhibits ubiquitin-dependent AKT activation, setting a threshold for CD8+ T cell and NK cell activation; Otub1 deficiency causes AKT hyperactivation, enhanced T cell metabolic reprogramming, and augmented anti-cancer immunity.","method":"Conditional Otub1 KO mice, IL-15 stimulation assays, membrane fractionation, ubiquitination assays of AKT, T cell and NK cell functional assays, tumour challenge models","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo, membrane recruitment localization, ubiquitination mechanistic readout, multiple immune cell functional assays","pmids":["31182807"],"is_preprint":false},{"year":2019,"finding":"Otub1 stabilises p100 (NF-κB precursor) by preventing its ubiquitination and degradation; Otub1 deficiency causes both increased signal-induced p100 processing (non-canonical NF-κB) and steady-state p100 degradation (leading to aberrant canonical NF-κB activation), resulting in B-cell hyperplasia and lupus-like autoimmunity.","method":"B cell-conditional Otub1 KO mice, ubiquitination assays, NF-κB pathway activation readouts, immunophenotyping, autoimmunity assessment","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo, ubiquitination mechanistic assays, multiple NF-κB pathway readouts and autoimmune phenotype","pmids":["31086255"],"is_preprint":false},{"year":2019,"finding":"The ubiquitin-like modifier FAT10 non-covalently interacts with OTUB1 and stimulates its K48-linked DUB activity; FAT10 also strengthens OTUB1's non-catalytic suppression of K63 polyubiquitylation on TRAF3 and increases OTUB1 interaction with UBCH5B, while covalent FAT10ylation leads to OTUB1 proteasomal degradation.","method":"In vitro DUB activity assays, Co-IP, ubiquitination assays of TRAF3, FAT10 covalent vs. non-covalent interaction analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro DUB assays, Co-IP, functional ubiquitination assays for TRAF3, single lab","pmids":["30718280"],"is_preprint":false},{"year":2020,"finding":"OTUB1 removes K48-linked ubiquitin chains from PD-L1 via its catalytic deubiquitinase activity, blocking ERAD-dependent PD-L1 degradation, stabilising PD-L1 at the tumour cell surface, and suppressing CD8+ T cell-mediated cytotoxicity.","method":"Co-immunoprecipitation, K48-ubiquitin chain-specific deubiquitination assays, OTUB1 KO/knockdown, PD-1 binding assay, PBMC cytotoxicity assay, in vivo tumour model with CD8+ T cell infiltration","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — DUB assay, K48-specific ubiquitin analysis, functional immune assays in vitro and in vivo, multiple orthogonal methods","pmids":["33328570"],"is_preprint":false},{"year":2020,"finding":"OTUB1 activates RIG-I antiviral signalling via a dual mechanism: hydrolysis of K48-linked polyubiquitin and formation of an E2-repressive complex with UBCH5c. Upon IAV infection, OTUB1 relocalises from the nucleus to mitochondrial membranes together with RIG-I. Influenza A NS1 protein triggers proteasomal degradation of OTUB1 to antagonise this signalling.","method":"Cell-free reconstitution system with [35S]IRF3, purified RIG-I, mitochondrial membranes, and OTUB1 variants; immunofluorescence for relocalization; OTUB1 CRISPR/knockdown with IFN readouts; NS1 degradation assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cell-free reconstitution system, localization experiment, functional antiviral assays, NS1 mechanistic study; single lab","pmids":["32023470"],"is_preprint":false},{"year":2020,"finding":"OTUB1 promotes NF-κB activity in dendritic cells through K48-linked deubiquitination and stabilisation of the E2 enzyme UBC13, which in turn increases K63-linked ubiquitination of IRAK1 and TRAF6, augmenting cytokine (IL-6, IL-12, TNF) production upon TLR stimulation.","method":"DC-specific Otub1 KO mice, ubiquitination assays for UBC13/IRAK1/TRAF6, cytokine measurement, T. gondii infection model","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo, mechanistic ubiquitination assays for multiple pathway components, in vivo infection model with functional outcome","pmids":["32024978"],"is_preprint":false},{"year":2020,"finding":"Otub1 directly interacts with c-Maf and abrogates K48-linked polyubiquitination of c-Maf, preventing its degradation and enhancing its transcriptional activity; this deubiquitination depends on Otub1's Lys71 and N terminus but is independent of UBE2O.","method":"Mass spectrometry identification of Otub1-c-Maf interaction, Co-IP, K48-ubiquitination assays, deletion/point mutant analysis, myeloma cell survival assays","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction, Co-IP, ubiquitination assays with structure-function mutants, single lab","pmids":["32842143"],"is_preprint":false},{"year":2021,"finding":"OTUB1 inhibits MSH2 ubiquitination by blocking E2 ubiquitin transfer activity (non-canonical mechanism), maintaining MSH2 protein stability; OTUB1 depletion promotes MSH2 ubiquitination, increases mutation frequency, and causes resistance to genotoxic agents.","method":"Functional ubiquitination and deubiquitination assays, OTUB1 knockdown, mutation frequency assays, drug sensitivity assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and cellular ubiquitination assays, functional mutation/drug resistance readouts, single lab","pmids":["33640455"],"is_preprint":false},{"year":2021,"finding":"OTUB1 stabilises SOCS1 via K48-linked deubiquitination, thereby inhibiting IFN-γ-induced JAK2/STAT1 signalling hyperactivation in astrocytes. Astrocyte-specific Otub1 KO mice develop more severe EAE due to astrocyte hyperactivation.","method":"Astrocyte-specific conditional Otub1 KO mice, K48-ubiquitination assays for SOCS1, JAK/STAT signalling readouts, EAE model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo, mechanistic ubiquitination assays, JAK/STAT pathway readouts, EAE functional phenotype","pmids":["30944096"],"is_preprint":false},{"year":2021,"finding":"OTUB1 prevents hepatocyte necroptosis by stabilising c-IAP1 through K48-deubiquitination, thereby maintaining K63-linked ubiquitination of RIPK1 and preventing RIPK1/RIPK3 necrosome formation and MLKL phosphorylation. OTUB1 deletion in liver parenchymal cells causes lethal necroptosis upon bacterial infection or TNF challenge.","method":"Hepatocyte-specific Otub1 KO mice (OTUB1LPC-KO), human HepG2 OTUB1-KO cells, K48-ubiquitination assay for c-IAP1, RIPK1 K63-ubiquitination, RIPK3/MLKL phosphorylation, necrostatin-1s rescue, MLKL KO rescue","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — hepatocyte-specific KO in vivo, human cell KO, mechanistic ubiquitination cascade dissected, genetic rescue with MLKL KO and necrostatin-1s","pmids":["33712742"],"is_preprint":false},{"year":2021,"finding":"OTUB1 directly deubiquitinates YAP protein via its OTU domain at multiple lysine sites (K90, K280, K343, K494, K497), stabilising YAP and promoting Hippo pathway suppression in gastric cancer.","method":"DUB siRNA library screen, Co-IP, in vitro and in vivo deubiquitination assays, site-directed mutagenesis of YAP lysines","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DUB screen followed by Co-IP and ubiquitination assays with defined lysine sites, single lab","pmids":["36271031"],"is_preprint":false},{"year":2021,"finding":"OTUB1 deubiquitinates and stabilises MYC at the K323 site, preventing its proteasomal degradation; stabilised MYC promotes HK2 expression and aerobic glycolysis in breast cancer. OTUB1-mediated MYC stability is confirmed in OTUB1-knockout mice.","method":"Co-IP, in vitro deubiquitination assay, site-directed mutagenesis (K323), OTUB1-KO mice, glycolysis assays, in vivo tumorigenesis","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro DUB assay, site-specific mutagenesis, OTUB1-KO mouse validation, functional metabolic readout","pmids":["35296795"],"is_preprint":false},{"year":2022,"finding":"CST1 recruits OTUB1 to GPX4, relieving GPX4 ubiquitination and improving its protein stability; this reduces intracellular ROS and inhibits ferroptosis in gastric cancer cells. The CST1–OTUB1–GPX4 interaction was confirmed by Co-IP combined with mass spectrometry.","method":"Co-immunoprecipitation combined with mass spectrometry, ubiquitination assay, siRNA knockdown, ROS assays, in vivo metastasis models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-confirmed Co-IP, ubiquitination assay, functional ferroptosis and metastasis readouts, single lab","pmids":["36369321"],"is_preprint":false},{"year":2022,"finding":"OTUB1 stabilises HIF-1α under hypoxia via non-canonical inhibition of K48-linked polyubiquitination (independent of PHDs/VHL/FIH); OTUB1 depletion reduces endogenous HIF-1α protein and suppresses hypoxia-induced glycolytic reprogramming.","method":"Co-IP, K48-ubiquitination assay, OTUB1 knockdown under hypoxia, HIF-1α protein stability assays, glycolysis metabolic assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional hypoxia/metabolic readouts, single lab","pmids":["35732631"],"is_preprint":false},{"year":2022,"finding":"OTUB1 non-canonically stabilises Raptor (mTORC1 component) by preventing its ubiquitination; phosphorylation of OTUB1 at Tyr26 by Src and SRMS kinases is required for the OTUB1–Raptor interaction, and dephosphorylation of OTUB1 at Y26 destabilises Raptor and sensitises cancer cells to anti-cancer drugs via mitochondrial ROS-mediated dysfunction.","method":"Biochemical interaction assays, Co-IP with Y26 mutant, in vitro kinase assays (Src/SRMS), ubiquitination assays of Raptor, drug sensitivity assays, mitochondrial ROS measurement","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro kinase assay, Y26 mutant dissects function, functional drug-sensitisation assay, single lab","pmids":["35927303"],"is_preprint":false},{"year":2022,"finding":"Endogenous H2S (produced by CTH) persulfidates OTUB1 at cysteine 91, promoting OTUB1 interaction with the E2 UBCH5A and thereby decreasing ubiquitination and proteasomal degradation of SLC7A11/xCT, increasing cellular glutathione; GLRX reverses this by deglutathionylation (at C23 or C204) of OTUB1.","method":"S-glutathionylation/persulfidation site-mapping, Co-IP of OTUB1-UBCH5A, ubiquitination and stability assays of SLC7A11, GLRX knockout/overexpression, GSH measurement","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific PTM mapping, Co-IP, ubiquitination assay, GLRX KO functional validation, single lab","pmids":["37703360"],"is_preprint":false},{"year":2023,"finding":"SET7 lysine monomethylase directly interacts with OTUB1 and catalyses its methylation at lysine 122; this modification does not affect OTUB1's DUB catalytic activity but impairs its non-canonical ability to bind UBC13, thereby relieving OTUB1's suppressive role on ferroptosis.","method":"In vitro methylation assay, site-directed mutagenesis (K122), Co-IP of OTUB1–UBC13, cell viability and intracellular ROS assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro methylation assay, K122 mutant, Co-IP, functional ROS/ferroptosis readout, single lab","pmids":["36822329"],"is_preprint":false},{"year":2023,"finding":"OTUB1 non-canonically stabilises TGFBI by deubiquitinating it at K22 and K25 residues in a manner independent of OTUB1 catalytic activity; OTUB1-mediated TGFBI stabilisation promotes glycolysis and angiogenesis in infantile hemangioma.","method":"Mass spectrometry for substrate identification, Co-IP, ubiquitination assay with site-specific K22/K25 mutagenesis, catalytic mutant analysis, extracellular acidification rate assays, in vivo IH model","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified substrate, site-specific mutagenesis, catalytic-independent mechanism, functional glycolysis assay, single lab","pmids":["36994729"],"is_preprint":false},{"year":2024,"finding":"OTUB1 deubiquitinates and stabilises CHK1, enhancing DNA damage repair capacity and conferring radioresistance in lung cancer; OTUB1 depletion impairs DNA damage repair and overcomes radioresistance.","method":"Co-immunoprecipitation, Western blotting of CHK1 ubiquitination, OTUB1 knockdown with comet assay and immunofluorescence for DNA damage, RNA sequencing, in vivo xenograft with radiotherapy","journal":"International journal of radiation oncology, biology, physics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, DNA damage functional assays, in vivo validation, single lab","pmids":["38266782"],"is_preprint":false},{"year":2024,"finding":"OTUB1 non-canonically stabilises RACK1 via inhibition of its K48-linked ubiquitination (independent of catalytic activity), activating PI3K/AKT and FAK/ERK signalling in hepatocellular carcinoma; OTUB1 transcription is driven by the transcription factor MAZ at the OTUB1 promoter.","method":"Immunoprecipitation-tandem MS for RACK1 identification, Co-IP, K48-ubiquitination assays, catalytic-independent mechanism validation, ChIP-qPCR and dual-luciferase for MAZ-OTUB1 promoter regulation, in vivo xenograft","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified substrate, Co-IP, ubiquitination assay, in vivo validation, ChIP for upstream regulation, single lab","pmids":["38315284"],"is_preprint":false},{"year":2024,"finding":"OTUB1 non-canonically stabilises YTHDF2 by blocking ubiquitin transfer to it independent of deubiquitinase activity; stabilised YTHDF2 binds and promotes m6A-dependent degradation of PRSS8 mRNA, and PRSS8 loss decreases nuclear β-catenin via E-cadherin, promoting prostate cancer proliferation.","method":"In vitro and in vivo ubiquitination assays, Co-IP, m6A-seq and transcriptomic analysis, PRSS8 mRNA stability assays, mouse xenograft model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay, Co-IP, transcriptomic mechanistic dissection, in vivo validation, single lab","pmids":["38462165"],"is_preprint":false},{"year":2024,"finding":"Legionella pneumophila effectors SidC/SdcA ubiquitinate OTUB1 at multiple lysine residues, promoting its association with the Legionella-containing vacuole and interaction with DEPTOR, thereby suppressing MTORC1 signalling and promoting host autophagy; SidE family effectors also induce phosphoribosyl-linked ubiquitination of OTUB1 at Ser16/Ser18 to block its DUB activity.","method":"Biochemical ubiquitination assays with purified effectors, Co-IP of OTUB1-DEPTOR, mTORC1 activity readouts, autophagy assays, BMDM infection model","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reconstituted ubiquitination assays, Co-IP, functional mTORC1 and autophagy readouts in infected cells, single lab","pmids":["38818749"],"is_preprint":false},{"year":2014,"finding":"HSCARG promotes OTUB1 recruitment to TRAF3, facilitating TRAF3 deubiquitination by OTUB1 and suppressing RIG-I-like receptor signalling; HSCARG knockout attenuates TRAF3 deubiquitination by OTUB1, and OTUB1 knockdown abolishes the HSCARG-mediated suppression of IFN-β transcription.","method":"Co-immunoprecipitation, ubiquitination assays of TRAF3, HSCARG KO and OTUB1 KD with IFN-β reporter assays, epistasis experiments","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis between HSCARG and OTUB1 by double KO/KD, Co-IP, functional IFN readout, single lab","pmids":["24763515"],"is_preprint":false},{"year":2017,"finding":"Otub1 stabilises MDMX via non-canonical inhibition of MDM2-mediated MDMX ubiquitination (independently of deubiquitinase activity); Otub1-stabilised MDMX localises to mitochondria, enhances p53 phosphorylation at S46, and promotes mitochondria-mediated apoptosis.","method":"Co-IP, in vitro ubiquitination assay, catalytic mutant analysis, subcellular fractionation, p53-S46 phosphorylation assay, apoptosis assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay, catalytic-independent mechanism, mitochondrial localization by fractionation linked to apoptotic function, single lab","pmids":["28035068"],"is_preprint":false},{"year":2021,"finding":"OTUB1 suppresses K63-linked ubiquitination of TRAF6 via direct binding, thereby inhibiting the TRAF6-ASK1 signalling axis and protecting against NASH-associated hepatic steatosis, inflammation, and fibrosis.","method":"Immunoprecipitation-mass spectrometry for TRAF6 identification, Co-IP, K63-ubiquitination assay of TRAF6, hepatocyte-specific Otub1 overexpression mouse model (HFHC diet), Otub1 KO mice","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction, Co-IP, K63 ubiquitination mechanistic assay, in vivo mouse model, single lab","pmids":["34591986"],"is_preprint":false},{"year":2022,"finding":"ERK/RSK-mediated phosphorylation of YB-1 at Ser102 in hyperglycaemia disrupts its interaction with OTUB1, leading to YB-1 ubiquitination and proteasomal degradation; when the ERK pathway is inhibited, YB-1–OTUB1 interaction is stabilised, preserving YB-1 levels and ameliorating diabetic cardiomyopathy.","method":"Co-immunoprecipitation, ubiquitination assay of YB-1, phospho-specific YB-1(S102) analysis, ERK inhibitor (U0126) in STZ-diabetic mice, echocardiography","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, phosphorylation-dependent interaction, in vivo mouse model with functional cardiac readout, single lab","pmids":["35490780"],"is_preprint":false},{"year":2023,"finding":"OTUB1 non-canonically stabilises CCN6 by inhibiting K48-linked ubiquitination via interaction through its linker domain; OTUB1 deletion reduces CCN6 abundance and increases breast cancer cell migration and proliferation, which is rescued by CCN6 supplementation.","method":"DUB screening assay, Co-IP, K48-ubiquitination assay, OTUB1 deletion mutants (linker domain), breast cancer cell functional assays, allograft model","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DUB screen, Co-IP, K48 ubiquitination assay, domain mapping, in vivo allograft, single lab","pmids":["37608493"],"is_preprint":false},{"year":2025,"finding":"OTUB1 is a novel deubiquitinating enzyme for ERRα; OTUB1 C91 residue is critical for ERRα deubiquitination. The natural product Ailanthone (AIL) inhibits OTUB1 activity by interacting with C91, disrupts the OTUB1–ERRα interaction, and promotes ERRα ubiquitination and proteasomal degradation.","method":"Competitive activity-based protein profiling (ABPP) for target identification, Co-IP, luciferase reporter assay, C91A mutant analysis, surface plasmon resonance, in vivo TNBC mouse model","journal":"Journal of advanced research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ABPP target identification, C91 mutagenesis, Co-IP, functional reporter assay, in vivo validation, single lab","pmids":["39864589"],"is_preprint":false}],"current_model":"OTUB1 is a highly versatile OTU-family deubiquitinase that stabilises a broad spectrum of substrates (including SLC7A11, PD-L1, FOXM1, SMAD2/3, p53, c-IAP1, RAS, YAP, MYC, CHK1, and many others) through two mechanistically distinct modes: canonical K48-linked polyubiquitin chain cleavage, and a non-canonical, catalysis-independent mechanism in which it binds charged E2~ubiquitin thiolester intermediates (UBC13, UBE2D/UBE2E family, UBCH5) and prevents ubiquitin transfer—an activity allosterically stimulated by free ubiquitin and regulated by post-translational modifications of OTUB1 itself including CK2-mediated phosphorylation at Ser16 (which drives nuclear translocalisation), Src/SRMS phosphorylation at Tyr26, SET7-mediated methylation at Lys122 (which impairs E2 binding), monoubiquitination at Lys59/109, persulfidation at Cys91, S-nitrosylation, and FIH-mediated asparagine hydroxylation/covalent complexation."},"narrative":{"mechanistic_narrative":"OTUB1 is an OTU-family deubiquitinase that controls the stability of a broad set of regulatory proteins through two mechanistically distinct activities: canonical cleavage of Lys48-linked polyubiquitin chains, and a catalysis-independent suppression of ubiquitin transfer by E2 conjugating enzymes [PMID:20725033, PMID:22325355, PMID:23955022]. The non-canonical mode was defined at DNA double-strand breaks, where OTUB1 binds and inhibits the E2 UBC13 (UBE2N) to suppress RNF168-dependent K63-linked ubiquitination, blocking 53BP1 focus formation independently of catalytic activity, and extends to UBE2D and UBE2E E2 subfamilies [PMID:20725033]. Crystallographic and biochemical work established the molecular basis: free ubiquitin binds a distal allosteric site to fold an N-terminal ubiquitin-binding helix, increasing OTUB1 affinity for the donor ubiquitin of the charged E2~Ub thioester, occluding the RING-E3 binding surface on UBC13 and blocking acceptor attack, while the canonical K48 distal ubiquitin site is co-opted to engage the thioester [PMID:22367539, PMID:22325355, PMID:22679021]; reciprocally, E2 enzymes stimulate OTUB1's intrinsic K48 isopeptidase activity through the same helix-folding event, creating bidirectional cross-regulation governed by the ratio of charged to free ubiquitin [PMID:23955022]. Through these activities OTUB1 stabilizes numerous substrates by either editing K48 chains or shielding them from E2-mediated ubiquitination, including phospho-SMAD2/3 in TGFβ signaling [PMID:24071738], c-IAP1 to sustain NF-κB/MAPK signaling and prevent necroptosis [PMID:23524849, PMID:33712742], FOXM1 [PMID:26148240], RAS [PMID:26881969], MYC [PMID:35296795], YAP [PMID:36271031], PD-L1 [PMID:33328570], and the cystine antiporter SLC7A11 and GPX4 to restrain ferroptosis [PMID:30709928, PMID:36369321]. p53 pathway control is exerted indirectly: OTUB1 suppresses MDM2-mediated ubiquitination of p53 and MDMX via non-canonical E2 (UbcH5) inhibition, an activity that itself requires OTUB1 monoubiquitination at Lys59/Lys109 [PMID:24403071, PMID:28035068]. OTUB1 also tunes innate and adaptive immunity, deubiquitinating TRAF3/TRAF6 to dampen interferon and TLR responses [PMID:19996094, PMID:24763515, PMID:32024978], setting an IL-15/AKT activation threshold in CD8+ T and NK cells [PMID:31182807], and stabilizing p100 and SOCS1 to constrain NF-κB and JAK/STAT signaling, with conditional knockouts producing autoimmunity and severe experimental autoimmune encephalomyelitis [PMID:31086255, PMID:30944096]. OTUB1 activity is itself extensively regulated by post-translational modification: CK2 phosphorylation at Ser16 drives nuclear accumulation and DNA-repair function [PMID:25872870], Src/SRMS phosphorylation at Tyr26 enables Raptor binding [PMID:35927303], SET7 methylation at Lys122 impairs UBC13 engagement [PMID:36822329], Cys91 persulfidation enhances E2 binding to stabilize SLC7A11 [PMID:37703360], and FIH-mediated asparagine hydroxylation/covalent complexation links OTUB1 to oxygen sensing [PMID:26752685, PMID:31299612]. Otub1 is essential for development, as knockout mice are embryonic lethal [PMID:30282802].","teleology":[{"year":2009,"claim":"Established OTUB1 as a deubiquitinase acting on innate immune signaling adaptors, the first functional context placing it in interferon regulation.","evidence":"Co-IP and IFN-β reporter/antiviral assays with TRAF3 and TRAF6 in cells","pmids":["19996094"],"confidence":"Medium","gaps":["Did not resolve catalytic vs. non-catalytic mechanism","Single lab without structural validation"]},{"year":2009,"claim":"Showed OTUB1 directly deubiquitinates a transcription factor (ERα), introducing the theme of substrate stabilization on chromatin.","evidence":"Mass spec of ERα complex, in vitro DUB assay, ChIP and reporter assays","pmids":["19383985"],"confidence":"Medium","gaps":["Ubiquitin chain linkage specificity not defined","Single lab"]},{"year":2010,"claim":"Discovered the catalysis-independent mode of action—OTUB1 inhibits K63 chain synthesis by binding the E2 UBC13 rather than cleaving chains—redefining how a DUB can restrict ubiquitination.","evidence":"Co-IP, in vitro ubiquitination, catalytic-mutant analysis, and DSB repair readouts","pmids":["20725033"],"confidence":"High","gaps":["Structural basis of E2 inhibition not yet resolved at this stage","Regulation of the switch between canonical and non-canonical modes unknown"]},{"year":2012,"claim":"Provided the structural mechanism: free ubiquitin allosterically activates OTUB1, folding an N-terminal helix that captures the E2~Ub thioester and occludes the RING-E3 site, explaining non-canonical E2 inhibition.","evidence":"X-ray crystallography of OTUB1–UBC13 and OTUB1–Ub aldehyde–UBC13~Ub conjugates, SPR, structure-guided mutagenesis (three independent studies)","pmids":["22367539","22325355","22679021"],"confidence":"High","gaps":["In vivo regulation of allosteric ubiquitin pool not addressed","Which substrates depend on this mode in cells left open"]},{"year":2013,"claim":"Demonstrated bidirectional cross-regulation—E2 enzymes stimulate OTUB1 K48 cleavage via the same helix-folding event—integrating its two activities into one allosteric framework.","evidence":"Reconstituted DUB activity assays and crystallography of human and worm OTUB1/UBCH5B with mutagenesis","pmids":["23955022"],"confidence":"High","gaps":["Cellular thresholds of charged/uncharged E2 not quantified","Substrate-specific consequences not mapped"]},{"year":2013,"claim":"Connected the non-canonical mechanism to physiological signaling outputs by showing OTUB1 sustains phospho-SMAD2/3 and stabilizes c-IAP1, controlling TGFβ-driven migration and TWEAK/NF-κB survival signaling.","evidence":"Signal-dependent Co-IP, catalytic mutants, proteasome-inhibitor rescue, zebrafish c-IAP knockdown, functional migration/death assays","pmids":["24071738","23524849"],"confidence":"High","gaps":["Recruitment determinants to each signaling complex not fully defined"]},{"year":2014,"claim":"Revealed auto-regulation of OTUB1—its own monoubiquitination at Lys59/109 is required to suppress UbcH5 and stabilize p53—and that knockout is embryonically lethal, marking developmental essentiality.","evidence":"In vitro monoubiquitination/ubiquitination assays with lysine-free mutant, CHX chase, apoptosis assays; HSCARG-dependent TRAF3 recruitment; later KO mouse","pmids":["24403071","24763515"],"confidence":"High","gaps":["How monoubiquitination remodels OTUB1 structurally not resolved"]},{"year":2016,"claim":"Extended substrate range to oncogenic RAS and identified oxygen-dependent FIH hydroxylation at Asn22 as a regulator, linking OTUB1 to metabolism and hypoxia.","evidence":"In vitro ubiquitination/imaging/xenografts for RAS; in vitro hydroxylation, N22A interactome and metabolic assays for FIH","pmids":["26881969","26752685"],"confidence":"Medium","gaps":["Physiological oxygen tension dependence in vivo not established","FIH-OTUB1 stoichiometry unresolved at this stage"]},{"year":2018,"claim":"Showed OTUB1 stabilizes the E2 enzyme UBE2E1 itself and the mTOR inhibitor DEPTOR, positioning OTUB1 as a regulator of E2 abundance and mTORC1/autophagy, and confirmed embryonic lethality of Otub1 knockout.","evidence":"In vitro autoubiquitination assays, mouse and human KO cells, Asp88/Cys91 mutant dissection, mTORC1 and autophagy readouts","pmids":["30282802","29382726"],"confidence":"High","gaps":["Non-canonical Asp88-dependent catalysis mechanism incompletely defined"]},{"year":2019,"claim":"Established in vivo immunoregulatory roles via conditional knockouts: OTUB1 sets an IL-15/AKT activation threshold and stabilizes p100, with deficiency causing enhanced anti-tumor immunity or lupus-like autoimmunity.","evidence":"Conditional Otub1 KO mice, membrane fractionation, AKT/NF-κB ubiquitination assays, immune functional and tumor-challenge models","pmids":["31182807","31086255"],"confidence":"High","gaps":["Tissue-specific substrate selection mechanisms not fully defined"]},{"year":2020,"claim":"Broadened OTUB1 into cancer immune evasion and context-dependent E2 control: it stabilizes PD-L1 to suppress CD8+ cytotoxicity, yet in dendritic cells stabilizes UBC13 to promote NF-κB, and relocalizes to mitochondria to activate RIG-I.","evidence":"K48-specific DUB assays, OTUB1 KO/CRISPR cells, DC-specific KO mice, cell-free RIG-I reconstitution, immune functional and infection models","pmids":["33328570","32024978","32023470"],"confidence":"High","gaps":["What dictates whether OTUB1 stabilizes vs. inhibits a given E2 in a given cell remains unresolved"]},{"year":2021,"claim":"Defined OTUB1 as a node in tissue protection and metabolism, stabilizing SOCS1, c-IAP1 (anti-necroptosis), MYC, YAP, and SLC7A11, linking it to JAK/STAT control, glycolysis, Hippo signaling, and ferroptosis.","evidence":"Astrocyte- and hepatocyte-specific conditional KO mice, K48-ubiquitination assays, RIPK1/RIPK3/MLKL necrosome readouts, DUB screens, site-specific lysine mutagenesis","pmids":["30944096","33712742","35296795","36271031","30709928"],"confidence":"High","gaps":["Substrate prioritization across these pathways in a single cell type not addressed"]},{"year":2022,"claim":"Mapped post-translational regulation of OTUB1—Tyr26 phosphorylation enabling Raptor binding and Cys91 persulfidation enhancing E2 binding to stabilize SLC7A11—connecting redox and kinase inputs to OTUB1 output.","evidence":"Co-IP with Y26/Cys91 variants, in vitro kinase and persulfidation site-mapping, GLRX KO, ubiquitination and metabolic readouts; plus HIF-1α and GPX4 stabilization studies","pmids":["35927303","37703360","35732631","36369321"],"confidence":"Medium","gaps":["Crosstalk among the many OTUB1 PTMs not integrated","Most are single-lab findings"]},{"year":2023,"claim":"Showed SET7 methylation at Lys122 selectively impairs the non-canonical UBC13-binding activity without affecting catalysis, providing a switch that derepresses ferroptosis, and extended substrate range to TGFBI, CCN6, and ERRα.","evidence":"In vitro methylation/K122 mutant, Co-IP, ROS/ferroptosis assays; MS substrate identification with site-specific mutagenesis and in vivo models","pmids":["36822329","36994729","37608493","39864589"],"confidence":"Medium","gaps":["Whether methylation and other PTMs act combinatorially is untested","Single-lab substrate validations"]},{"year":2024,"claim":"Continued substrate expansion (CHK1, RACK1, YTHDF2) and revealed pathogen hijacking, with Legionella effectors ubiquitinating OTUB1 to redirect it to the bacterial vacuole and block its DUB activity.","evidence":"Co-IP, K48-ubiquitination assays, transcriptomic/m6A analysis, DNA-damage assays, reconstituted effector ubiquitination and infection models","pmids":["38266782","38315284","38462165","38818749"],"confidence":"Medium","gaps":["Physiological relevance of effector-driven OTUB1 modification beyond infection unknown","Single-lab substrate findings"]},{"year":null,"claim":"How OTUB1 selects between canonical K48 cleavage and non-canonical E2 suppression for any given substrate in a given cellular context—and how its numerous PTMs are coordinated to set that choice—remains the central open question.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating PTM state, free-ubiquitin levels, and substrate selection","Most substrate relationships rest on single-lab studies"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,7,11,14,22,29,30]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,2,4,22]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,9,16]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,10,23]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[23,42]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[12,19]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,3,10,26,37]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,19,20,22,24,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,12,33,38]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,9,16]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,28,17,31,42]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[30,32,13]}],"complexes":[],"partners":["UBC13/UBE2N","UBE2D/UBCH5","UBE2E1","TRAF3","TRAF6","C-IAP1","SLC7A11","DEPTOR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96FW1","full_name":"Ubiquitin thioesterase OTUB1","aliases":["Deubiquitinating enzyme OTUB1","OTU domain-containing ubiquitin aldehyde-binding protein 1","Otubain-1","hOTU1","Ubiquitin-specific-processing protease OTUB1"],"length_aa":271,"mass_kda":31.3,"function":"Hydrolase that can specifically remove 'Lys-48'-linked conjugated ubiquitin from proteins and plays an important regulatory role at the level of protein turnover by preventing degradation (PubMed:12401499, PubMed:12704427, PubMed:14661020, PubMed:23827681). Regulator of T-cell anergy, a phenomenon that occurs when T-cells are rendered unresponsive to antigen rechallenge and no longer respond to their cognate antigen (PubMed:14661020). Acts via its interaction with RNF128/GRAIL, a crucial inductor of CD4 T-cell anergy (PubMed:14661020). Isoform 1 destabilizes RNF128, leading to prevent anergy (PubMed:14661020). In contrast, isoform 2 stabilizes RNF128 and promotes anergy (PubMed:14661020). Surprisingly, it regulates RNF128-mediated ubiquitination, but does not deubiquitinate polyubiquitinated RNF128 (PubMed:14661020). Deubiquitinates estrogen receptor alpha (ESR1) (PubMed:19383985). Mediates deubiquitination of 'Lys-48'-linked polyubiquitin chains, but not 'Lys-63'-linked polyubiquitin chains (PubMed:18954305, PubMed:19211026, PubMed:23827681). Not able to cleave di-ubiquitin (PubMed:18954305, PubMed:23827681). Also capable of removing NEDD8 from NEDD8 conjugates, but with a much lower preference compared to 'Lys-48'-linked ubiquitin (PubMed:18954305, PubMed:23827681) Plays a key non-catalytic role in DNA repair regulation by inhibiting activity of RNF168, an E3 ubiquitin-protein ligase that promotes accumulation of 'Lys-63'-linked histone H2A and H2AX at DNA damage sites (PubMed:20725033, PubMed:22325355). Inhibits RNF168 independently of ubiquitin thioesterase activity by binding and inhibiting UBE2N/UBC13, the E2 partner of RNF168, thereby limiting spreading of 'Lys-63'-linked histone H2A and H2AX marks (PubMed:20725033, PubMed:22325355). Inhibition occurs by binding to free ubiquitin: free ubiquitin acts as an allosteric regulator that increases affinity for UBE2N/UBC13 and disrupts interaction with UBE2V1 (PubMed:20725033, PubMed:22325355). The OTUB1-UBE2N/UBC13-free ubiquitin complex adopts a configuration that mimics a cleaved 'Lys48'-linked di-ubiquitin chain (PubMed:20725033, PubMed:22325355). Acts as a regulator of mTORC1 and mTORC2 complexes (PubMed:29382726, PubMed:35927303). When phosphorylated at Tyr-26, acts as an activator of the mTORC1 complex by mediating deubiquitination of RPTOR via a non-catalytic process: acts by binding and inhibiting the activity of the ubiquitin-conjugating enzyme E2 (UBE2D1/UBCH5A, UBE2W/UBC16 and UBE2N/UBC13), thereby preventing ubiquitination of RPTOR (PubMed:35927303). Can also act as an inhibitor of the mTORC1 and mTORC2 complexes in response to amino acids by mediating non-catalytic deubiquitination of DEPTOR (PubMed:29382726)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96FW1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OTUB1","classification":"Not Classified","n_dependent_lines":69,"n_total_lines":1208,"dependency_fraction":0.057119205298013245},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/OTUB1","total_profiled":1310},"omim":[{"mim_id":"620004","title":"NMRA-LIKE REDOX SENSOR 1; NMRAL1","url":"https://www.omim.org/entry/620004"},{"mim_id":"615712","title":"OTU DEUBIQUITINASE WITH LINEAR LINKAGE SPECIFICITY; OTULIN","url":"https://www.omim.org/entry/615712"},{"mim_id":"612688","title":"RING FINGER PROTEIN 168; RNF168","url":"https://www.omim.org/entry/612688"},{"mim_id":"608337","title":"OTU DOMAIN-CONTAINING UBIQUITIN ALDEHYDE-BINDING PROTEIN 1; OTUB1","url":"https://www.omim.org/entry/608337"},{"mim_id":"603679","title":"UBIQUITIN-CONJUGATING ENZYME E2 N; UBE2N","url":"https://www.omim.org/entry/603679"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Cytoplasmic bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/OTUB1"},"hgnc":{"alias_symbol":["FLJ20113","FLJ40710"],"prev_symbol":[]},"alphafold":{"accession":"Q96FW1","domains":[{"cath_id":"3.30.200.60","chopping":"53-83_231-271","consensus_level":"medium","plddt":94.3953,"start":53,"end":271},{"cath_id":"1.20.1300.20","chopping":"85-229","consensus_level":"medium","plddt":97.177,"start":85,"end":229}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96FW1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96FW1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96FW1-F1-predicted_aligned_error_v6.png","plddt_mean":91.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OTUB1","jax_strain_url":"https://www.jax.org/strain/search?query=OTUB1"},"sequence":{"accession":"Q96FW1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96FW1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96FW1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96FW1"}},"corpus_meta":[{"pmid":"30709928","id":"PMC_30709928","title":"The 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OTUB1 also binds E2s of the UBE2D and UBE2E subfamilies through the same non-canonical mechanism.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assays, siRNA knockdown with DSB repair readouts, catalytic mutant analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, in vitro assays with catalytic-dead mutant, replicated across multiple E2 families, published in Nature\",\n      \"pmids\": [\"20725033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structures of OTUB1–UBC13 and OTUB1 bound to ubiquitin aldehyde plus a chemical UBC13~Ub conjugate reveal that free ubiquitin allosterically binds a distal site on OTUB1, triggering conformational changes that form an N-terminal ubiquitin-binding helix, increase affinity for donor ubiquitin in the E2~Ub thiolester, occlude the RING E3 binding site on UBC13, and block acceptor-ubiquitin attack on the thiolester—thereby inhibiting K63-linked ubiquitin chain synthesis.\",\n      \"method\": \"X-ray crystallography, biochemical ubiquitination assays, structure-guided mutagenesis, surface plasmon resonance\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus reconstituted biochemistry and mutagenesis in a single rigorous study\",\n      \"pmids\": [\"22367539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"OTUB1 co-opts its canonical Lys48-linked distal ubiquitin-binding site to recognize charged E2~Ub thioester intermediates; free ubiquitin binding at this site promotes formation of the inhibited E2 complex, with the donor-ubiquitin Lys48 positioned near the OTUB1 catalytic site in a configuration mimicking K48-linked chain cleavage products.\",\n      \"method\": \"X-ray crystallography, biochemical E2 inhibition assays, mutagenesis, structural analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation, independent confirmation of the same mechanism as PMID:22367539\",\n      \"pmids\": [\"22325355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of human OTUB1 in complex with UBC13 and MMS2 at 3.15 Å resolution reveals atomic-level contacts required for inhibition of K63-linked ubiquitin chain formation; structure-based OTUB1 mutants fail to inhibit K63 chain formation in vitro and histone ubiquitination and 53BP1 foci in vivo.\",\n      \"method\": \"X-ray crystallography, surface plasmon resonance, structure-guided mutagenesis, in vitro ubiquitination assays, cell-based 53BP1 foci assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical and cellular validation, multiple orthogonal methods\",\n      \"pmids\": [\"22679021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"E2 ubiquitin-conjugating enzymes (UBC13, UBCH5) stimulate OTUB1 catalytic cleavage of K48-linked polyubiquitin by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix; this stimulation is regulated by the ratio of charged to uncharged E2 and by concentrations of K48 polyubiquitin and free ubiquitin, establishing bidirectional cross-regulation between OTUB1 and its E2 partners.\",\n      \"method\": \"Biochemical DUB activity assays, X-ray crystallography of human and worm OTUB1/UBCH5B, mutagenesis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus reconstituted biochemistry across two species with mutagenesis\",\n      \"pmids\": [\"23955022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"OTUB1 interacts with TRAF3 and TRAF6 by co-immunoprecipitation and mediates their deubiquitination, thereby negatively regulating virus-triggered type I interferon induction and IRF3/NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown with reporter assays for IFN-β transcription, antiviral response assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with functional knockdown/overexpression data, single lab, two orthogonal readouts\",\n      \"pmids\": [\"19996094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"OTUB1 directly deubiquitinates estrogen receptor alpha (ERα) in vitro and in cells; it interacts with ERα (identified by mass spectrometry of an ERα complex) and negatively regulates ERα-mediated transcription while stabilizing ERα protein on chromatin.\",\n      \"method\": \"Mass spectrometry of ERα complex, in vitro deubiquitination assay, Co-IP, transcriptional reporter assays, ChIP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro DUB assay plus co-IP and functional reporter assays, single lab\",\n      \"pmids\": [\"19383985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"OTUB1 is recruited to the active phospho-SMAD2/3 complex upon TGFβ stimulation; it inhibits ubiquitylation and proteasomal degradation of phospho-SMAD2/3 via non-catalytic E2 inhibition (independent of its isopeptidase activity), thereby sustaining TGFβ-mediated gene transcription and cell migration. Depletion of OTUB1 causes rapid loss of phospho-SMAD2/3 rescued by the proteasomal inhibitor bortezomib.\",\n      \"method\": \"Co-immunoprecipitation (signal-dependent), siRNA knockdown, catalytic mutant analysis, proteasome inhibitor rescue, TGFβ reporter assays, migration assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, catalytic-dead mutant, proteasome inhibitor rescue, functional migration assay; multiple orthogonal methods in one study\",\n      \"pmids\": [\"24071738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"OTUB1 associates with c-IAP1 within the TWEAK receptor signalling complex, disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo, stabilises c-IAP1, and thereby sustains TWEAK-induced canonical NF-κB and MAPK signalling while preventing caspase activation and cell death.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo deubiquitination assays, siRNA knockdown with caspase/NF-κB readouts, zebrafish c-IAP knockdown model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro DUB assay, reciprocal Co-IP, in vivo zebrafish validation, multiple functional readouts\",\n      \"pmids\": [\"23524849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Otub1 suppresses MDM2-mediated p53 ubiquitination and stabilises p53 independently of its deubiquitinase activity by non-canonically inhibiting the E2 enzyme UbcH5; monoubiquitination of Otub1 (primarily at Lys59 and Lys109 by UbcH5) is required for Otub1 to suppress UbcH5 and stabilise p53, as a lysine-free Otub1 mutant (K0) fails to achieve these functions.\",\n      \"method\": \"In vitro ubiquitination/monoubiquitination assays, Co-IP, cycloheximide chase, apoptosis assays, cell proliferation assays, UbcH5 binding studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutants, multiple functional cellular readouts, mechanistic dissection of auto-regulation\",\n      \"pmids\": [\"24403071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Casein kinase 2 (CK2) phosphorylates OTUB1 at Ser16, promoting its nuclear accumulation; pharmacological CK2 inhibition or genetic ablation blocks Ser16 phosphorylation and causes nuclear exclusion of OTUB1. Ser16-phosphorylated OTUB1 is detected exclusively in the nucleus and promotes 53BP1 DNA repair foci formation after ionising radiation.\",\n      \"method\": \"In vitro CK2 phosphorylation assay, phospho-specific antibody, CK2 inhibitor/KO, subcellular fractionation/immunofluorescence, 53BP1 foci assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay, genetic and pharmacological CK2 ablation, direct localization linked to functional DNA repair outcome\",\n      \"pmids\": [\"25872870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"OTUB1 inhibits ubiquitination and proteasomal degradation of FOXM1 through its catalytic (C91-dependent) deubiquitinase activity; the C91S catalytic mutant fails to stabilise FOXM1, increase its half-life, or enhance proliferative and epirubicin-resistance phenotypes.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown and overexpression, cycloheximide chase, catalytic mutant (C91S), K48-ubiquitin linkage analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, catalytic mutant, cycloheximide chase, multiple functional assays in one study\",\n      \"pmids\": [\"26148240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"OTUB1 inhibits RAS mono- and di-ubiquitination independently of its catalytic activity, resulting in sequestration of RAS on the plasma membrane and enhanced RAS activation and ERK1/2 signalling, thereby promoting tumorigenesis in wild-type RAS cells.\",\n      \"method\": \"In vitro ubiquitination assays, catalytic mutant analysis, confocal microscopy for RAS membrane localisation, RAS-GTP pull-down, tumour xenograft assays\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assays with catalytic mutant, RAS membrane localisation by imaging, functional in vivo xenograft data, single lab\",\n      \"pmids\": [\"26881969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FIH (factor inhibiting HIF) hydroxylates OTUB1 at asparagine 22; mutation of N22 profoundly alters OTUB1 protein interaction network and impairs cellular metabolic processes, identifying OTUB1 as a non-HIF substrate whose activity is regulated by oxygen-dependent hydroxylation.\",\n      \"method\": \"In vitro hydroxylation assays, N22A mutant co-immunoprecipitation proteomics, cellular metabolic assays (oxygen consumption, glycolysis)\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro hydroxylation assay plus interactome and metabolic readouts with N22A mutant, single lab\",\n      \"pmids\": [\"26752685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUB1 directly interacts with DEPTOR via its N-terminal domain, deubiquitinates DEPTOR in a Cys91-independent but Asp88-dependent (non-canonical) manner, stabilises DEPTOR, and thereby suppresses amino-acid-induced mTORC1 activation, cellular autophagy, and cell proliferation/size.\",\n      \"method\": \"In vitro deubiquitination assay, co-immunoprecipitation, catalytic-dead and D88A mutant analysis, mTORC1 activity assays (S6K phosphorylation), autophagy assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro DUB assay, D88A and C91S mutants dissect mechanism, multiple mTOR pathway readouts, single lab\",\n      \"pmids\": [\"29382726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUB1 stabilises Snail by preventing its ubiquitination and proteasomal degradation, facilitating epithelial-mesenchymal transition and metastasis of esophageal squamous cell carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA/overexpression, ubiquitination assays, invasion/migration assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination assay and functional invasion readout, single lab\",\n      \"pmids\": [\"29559747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUB1 non-canonically stabilises the E2 enzyme UBE2E1 by suppressing its autoubiquitination in vitro and in cells, preventing proteasomal degradation of UBE2E1; this function is independent of OTUB1 catalytic activity but requires its ability to bind UBE2E1. Otub1 knockout mice exhibit late-stage embryonic lethality.\",\n      \"method\": \"In vitro autoubiquitination assay, OTUB1 KO cell lines (mouse and human), cycloheximide chase, catalytic mutant and binding-defective mutant analysis, mouse knockout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution, genetic KO in two species, catalytic vs. binding mutant dissection, replicated in mouse and human cells\",\n      \"pmids\": [\"30282802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"OTUB1 directly interacts with SLC7A11 (xCT), a key component of the cystine-glutamate antiporter, and stabilises SLC7A11 protein. CD44 overexpression enhances the OTUB1–SLC7A11 interaction, while CD44 depletion partially abrogates it. OTUB1 knockdown destabilises SLC7A11 and promotes ferroptosis in cancer xenografts.\",\n      \"method\": \"Biochemical purification/mass spectrometry to identify OTUB1 as SLC7A11-binding partner, Co-IP, siRNA knockdown, xenograft tumour growth assays, ferroptosis markers\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mass-spec identification, reciprocal Co-IP, in vivo xenograft validation, CD44 epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"30709928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FIH forms an oxygen-sensitive, likely covalent (co-translational amide) bond with OTUB1 within cells, generating a heterotrimeric complex of two FIH and one OTUB1; this complexation regulates OTUB1 deubiquitinase activity in an oxygen-dependent manner, providing a mechanism for hypoxia adaptation.\",\n      \"method\": \"Biochemical co-precipitation, mass spectrometry analysis of bond, co-expression in mammalian and bacterial cells, DUB activity assays, oxygen-dependency experiments\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods, novel covalent bond proposed with supporting MS data, single lab\",\n      \"pmids\": [\"31299612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"IL-15 induces membrane recruitment of Otub1, which then non-canonically inhibits ubiquitin-dependent AKT activation, setting a threshold for CD8+ T cell and NK cell activation; Otub1 deficiency causes AKT hyperactivation, enhanced T cell metabolic reprogramming, and augmented anti-cancer immunity.\",\n      \"method\": \"Conditional Otub1 KO mice, IL-15 stimulation assays, membrane fractionation, ubiquitination assays of AKT, T cell and NK cell functional assays, tumour challenge models\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo, membrane recruitment localization, ubiquitination mechanistic readout, multiple immune cell functional assays\",\n      \"pmids\": [\"31182807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Otub1 stabilises p100 (NF-κB precursor) by preventing its ubiquitination and degradation; Otub1 deficiency causes both increased signal-induced p100 processing (non-canonical NF-κB) and steady-state p100 degradation (leading to aberrant canonical NF-κB activation), resulting in B-cell hyperplasia and lupus-like autoimmunity.\",\n      \"method\": \"B cell-conditional Otub1 KO mice, ubiquitination assays, NF-κB pathway activation readouts, immunophenotyping, autoimmunity assessment\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo, ubiquitination mechanistic assays, multiple NF-κB pathway readouts and autoimmune phenotype\",\n      \"pmids\": [\"31086255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The ubiquitin-like modifier FAT10 non-covalently interacts with OTUB1 and stimulates its K48-linked DUB activity; FAT10 also strengthens OTUB1's non-catalytic suppression of K63 polyubiquitylation on TRAF3 and increases OTUB1 interaction with UBCH5B, while covalent FAT10ylation leads to OTUB1 proteasomal degradation.\",\n      \"method\": \"In vitro DUB activity assays, Co-IP, ubiquitination assays of TRAF3, FAT10 covalent vs. non-covalent interaction analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro DUB assays, Co-IP, functional ubiquitination assays for TRAF3, single lab\",\n      \"pmids\": [\"30718280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUB1 removes K48-linked ubiquitin chains from PD-L1 via its catalytic deubiquitinase activity, blocking ERAD-dependent PD-L1 degradation, stabilising PD-L1 at the tumour cell surface, and suppressing CD8+ T cell-mediated cytotoxicity.\",\n      \"method\": \"Co-immunoprecipitation, K48-ubiquitin chain-specific deubiquitination assays, OTUB1 KO/knockdown, PD-1 binding assay, PBMC cytotoxicity assay, in vivo tumour model with CD8+ T cell infiltration\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — DUB assay, K48-specific ubiquitin analysis, functional immune assays in vitro and in vivo, multiple orthogonal methods\",\n      \"pmids\": [\"33328570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUB1 activates RIG-I antiviral signalling via a dual mechanism: hydrolysis of K48-linked polyubiquitin and formation of an E2-repressive complex with UBCH5c. Upon IAV infection, OTUB1 relocalises from the nucleus to mitochondrial membranes together with RIG-I. Influenza A NS1 protein triggers proteasomal degradation of OTUB1 to antagonise this signalling.\",\n      \"method\": \"Cell-free reconstitution system with [35S]IRF3, purified RIG-I, mitochondrial membranes, and OTUB1 variants; immunofluorescence for relocalization; OTUB1 CRISPR/knockdown with IFN readouts; NS1 degradation assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cell-free reconstitution system, localization experiment, functional antiviral assays, NS1 mechanistic study; single lab\",\n      \"pmids\": [\"32023470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUB1 promotes NF-κB activity in dendritic cells through K48-linked deubiquitination and stabilisation of the E2 enzyme UBC13, which in turn increases K63-linked ubiquitination of IRAK1 and TRAF6, augmenting cytokine (IL-6, IL-12, TNF) production upon TLR stimulation.\",\n      \"method\": \"DC-specific Otub1 KO mice, ubiquitination assays for UBC13/IRAK1/TRAF6, cytokine measurement, T. gondii infection model\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo, mechanistic ubiquitination assays for multiple pathway components, in vivo infection model with functional outcome\",\n      \"pmids\": [\"32024978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Otub1 directly interacts with c-Maf and abrogates K48-linked polyubiquitination of c-Maf, preventing its degradation and enhancing its transcriptional activity; this deubiquitination depends on Otub1's Lys71 and N terminus but is independent of UBE2O.\",\n      \"method\": \"Mass spectrometry identification of Otub1-c-Maf interaction, Co-IP, K48-ubiquitination assays, deletion/point mutant analysis, myeloma cell survival assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction, Co-IP, ubiquitination assays with structure-function mutants, single lab\",\n      \"pmids\": [\"32842143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 inhibits MSH2 ubiquitination by blocking E2 ubiquitin transfer activity (non-canonical mechanism), maintaining MSH2 protein stability; OTUB1 depletion promotes MSH2 ubiquitination, increases mutation frequency, and causes resistance to genotoxic agents.\",\n      \"method\": \"Functional ubiquitination and deubiquitination assays, OTUB1 knockdown, mutation frequency assays, drug sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and cellular ubiquitination assays, functional mutation/drug resistance readouts, single lab\",\n      \"pmids\": [\"33640455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 stabilises SOCS1 via K48-linked deubiquitination, thereby inhibiting IFN-γ-induced JAK2/STAT1 signalling hyperactivation in astrocytes. Astrocyte-specific Otub1 KO mice develop more severe EAE due to astrocyte hyperactivation.\",\n      \"method\": \"Astrocyte-specific conditional Otub1 KO mice, K48-ubiquitination assays for SOCS1, JAK/STAT signalling readouts, EAE model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo, mechanistic ubiquitination assays, JAK/STAT pathway readouts, EAE functional phenotype\",\n      \"pmids\": [\"30944096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 prevents hepatocyte necroptosis by stabilising c-IAP1 through K48-deubiquitination, thereby maintaining K63-linked ubiquitination of RIPK1 and preventing RIPK1/RIPK3 necrosome formation and MLKL phosphorylation. OTUB1 deletion in liver parenchymal cells causes lethal necroptosis upon bacterial infection or TNF challenge.\",\n      \"method\": \"Hepatocyte-specific Otub1 KO mice (OTUB1LPC-KO), human HepG2 OTUB1-KO cells, K48-ubiquitination assay for c-IAP1, RIPK1 K63-ubiquitination, RIPK3/MLKL phosphorylation, necrostatin-1s rescue, MLKL KO rescue\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — hepatocyte-specific KO in vivo, human cell KO, mechanistic ubiquitination cascade dissected, genetic rescue with MLKL KO and necrostatin-1s\",\n      \"pmids\": [\"33712742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 directly deubiquitinates YAP protein via its OTU domain at multiple lysine sites (K90, K280, K343, K494, K497), stabilising YAP and promoting Hippo pathway suppression in gastric cancer.\",\n      \"method\": \"DUB siRNA library screen, Co-IP, in vitro and in vivo deubiquitination assays, site-directed mutagenesis of YAP lysines\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DUB screen followed by Co-IP and ubiquitination assays with defined lysine sites, single lab\",\n      \"pmids\": [\"36271031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 deubiquitinates and stabilises MYC at the K323 site, preventing its proteasomal degradation; stabilised MYC promotes HK2 expression and aerobic glycolysis in breast cancer. OTUB1-mediated MYC stability is confirmed in OTUB1-knockout mice.\",\n      \"method\": \"Co-IP, in vitro deubiquitination assay, site-directed mutagenesis (K323), OTUB1-KO mice, glycolysis assays, in vivo tumorigenesis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro DUB assay, site-specific mutagenesis, OTUB1-KO mouse validation, functional metabolic readout\",\n      \"pmids\": [\"35296795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CST1 recruits OTUB1 to GPX4, relieving GPX4 ubiquitination and improving its protein stability; this reduces intracellular ROS and inhibits ferroptosis in gastric cancer cells. The CST1–OTUB1–GPX4 interaction was confirmed by Co-IP combined with mass spectrometry.\",\n      \"method\": \"Co-immunoprecipitation combined with mass spectrometry, ubiquitination assay, siRNA knockdown, ROS assays, in vivo metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-confirmed Co-IP, ubiquitination assay, functional ferroptosis and metastasis readouts, single lab\",\n      \"pmids\": [\"36369321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUB1 stabilises HIF-1α under hypoxia via non-canonical inhibition of K48-linked polyubiquitination (independent of PHDs/VHL/FIH); OTUB1 depletion reduces endogenous HIF-1α protein and suppresses hypoxia-induced glycolytic reprogramming.\",\n      \"method\": \"Co-IP, K48-ubiquitination assay, OTUB1 knockdown under hypoxia, HIF-1α protein stability assays, glycolysis metabolic assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional hypoxia/metabolic readouts, single lab\",\n      \"pmids\": [\"35732631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUB1 non-canonically stabilises Raptor (mTORC1 component) by preventing its ubiquitination; phosphorylation of OTUB1 at Tyr26 by Src and SRMS kinases is required for the OTUB1–Raptor interaction, and dephosphorylation of OTUB1 at Y26 destabilises Raptor and sensitises cancer cells to anti-cancer drugs via mitochondrial ROS-mediated dysfunction.\",\n      \"method\": \"Biochemical interaction assays, Co-IP with Y26 mutant, in vitro kinase assays (Src/SRMS), ubiquitination assays of Raptor, drug sensitivity assays, mitochondrial ROS measurement\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro kinase assay, Y26 mutant dissects function, functional drug-sensitisation assay, single lab\",\n      \"pmids\": [\"35927303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Endogenous H2S (produced by CTH) persulfidates OTUB1 at cysteine 91, promoting OTUB1 interaction with the E2 UBCH5A and thereby decreasing ubiquitination and proteasomal degradation of SLC7A11/xCT, increasing cellular glutathione; GLRX reverses this by deglutathionylation (at C23 or C204) of OTUB1.\",\n      \"method\": \"S-glutathionylation/persulfidation site-mapping, Co-IP of OTUB1-UBCH5A, ubiquitination and stability assays of SLC7A11, GLRX knockout/overexpression, GSH measurement\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific PTM mapping, Co-IP, ubiquitination assay, GLRX KO functional validation, single lab\",\n      \"pmids\": [\"37703360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SET7 lysine monomethylase directly interacts with OTUB1 and catalyses its methylation at lysine 122; this modification does not affect OTUB1's DUB catalytic activity but impairs its non-canonical ability to bind UBC13, thereby relieving OTUB1's suppressive role on ferroptosis.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis (K122), Co-IP of OTUB1–UBC13, cell viability and intracellular ROS assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro methylation assay, K122 mutant, Co-IP, functional ROS/ferroptosis readout, single lab\",\n      \"pmids\": [\"36822329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUB1 non-canonically stabilises TGFBI by deubiquitinating it at K22 and K25 residues in a manner independent of OTUB1 catalytic activity; OTUB1-mediated TGFBI stabilisation promotes glycolysis and angiogenesis in infantile hemangioma.\",\n      \"method\": \"Mass spectrometry for substrate identification, Co-IP, ubiquitination assay with site-specific K22/K25 mutagenesis, catalytic mutant analysis, extracellular acidification rate assays, in vivo IH model\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified substrate, site-specific mutagenesis, catalytic-independent mechanism, functional glycolysis assay, single lab\",\n      \"pmids\": [\"36994729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUB1 deubiquitinates and stabilises CHK1, enhancing DNA damage repair capacity and conferring radioresistance in lung cancer; OTUB1 depletion impairs DNA damage repair and overcomes radioresistance.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting of CHK1 ubiquitination, OTUB1 knockdown with comet assay and immunofluorescence for DNA damage, RNA sequencing, in vivo xenograft with radiotherapy\",\n      \"journal\": \"International journal of radiation oncology, biology, physics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, DNA damage functional assays, in vivo validation, single lab\",\n      \"pmids\": [\"38266782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUB1 non-canonically stabilises RACK1 via inhibition of its K48-linked ubiquitination (independent of catalytic activity), activating PI3K/AKT and FAK/ERK signalling in hepatocellular carcinoma; OTUB1 transcription is driven by the transcription factor MAZ at the OTUB1 promoter.\",\n      \"method\": \"Immunoprecipitation-tandem MS for RACK1 identification, Co-IP, K48-ubiquitination assays, catalytic-independent mechanism validation, ChIP-qPCR and dual-luciferase for MAZ-OTUB1 promoter regulation, in vivo xenograft\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified substrate, Co-IP, ubiquitination assay, in vivo validation, ChIP for upstream regulation, single lab\",\n      \"pmids\": [\"38315284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUB1 non-canonically stabilises YTHDF2 by blocking ubiquitin transfer to it independent of deubiquitinase activity; stabilised YTHDF2 binds and promotes m6A-dependent degradation of PRSS8 mRNA, and PRSS8 loss decreases nuclear β-catenin via E-cadherin, promoting prostate cancer proliferation.\",\n      \"method\": \"In vitro and in vivo ubiquitination assays, Co-IP, m6A-seq and transcriptomic analysis, PRSS8 mRNA stability assays, mouse xenograft model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay, Co-IP, transcriptomic mechanistic dissection, in vivo validation, single lab\",\n      \"pmids\": [\"38462165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Legionella pneumophila effectors SidC/SdcA ubiquitinate OTUB1 at multiple lysine residues, promoting its association with the Legionella-containing vacuole and interaction with DEPTOR, thereby suppressing MTORC1 signalling and promoting host autophagy; SidE family effectors also induce phosphoribosyl-linked ubiquitination of OTUB1 at Ser16/Ser18 to block its DUB activity.\",\n      \"method\": \"Biochemical ubiquitination assays with purified effectors, Co-IP of OTUB1-DEPTOR, mTORC1 activity readouts, autophagy assays, BMDM infection model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstituted ubiquitination assays, Co-IP, functional mTORC1 and autophagy readouts in infected cells, single lab\",\n      \"pmids\": [\"38818749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HSCARG promotes OTUB1 recruitment to TRAF3, facilitating TRAF3 deubiquitination by OTUB1 and suppressing RIG-I-like receptor signalling; HSCARG knockout attenuates TRAF3 deubiquitination by OTUB1, and OTUB1 knockdown abolishes the HSCARG-mediated suppression of IFN-β transcription.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays of TRAF3, HSCARG KO and OTUB1 KD with IFN-β reporter assays, epistasis experiments\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis between HSCARG and OTUB1 by double KO/KD, Co-IP, functional IFN readout, single lab\",\n      \"pmids\": [\"24763515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Otub1 stabilises MDMX via non-canonical inhibition of MDM2-mediated MDMX ubiquitination (independently of deubiquitinase activity); Otub1-stabilised MDMX localises to mitochondria, enhances p53 phosphorylation at S46, and promotes mitochondria-mediated apoptosis.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, catalytic mutant analysis, subcellular fractionation, p53-S46 phosphorylation assay, apoptosis assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay, catalytic-independent mechanism, mitochondrial localization by fractionation linked to apoptotic function, single lab\",\n      \"pmids\": [\"28035068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 suppresses K63-linked ubiquitination of TRAF6 via direct binding, thereby inhibiting the TRAF6-ASK1 signalling axis and protecting against NASH-associated hepatic steatosis, inflammation, and fibrosis.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry for TRAF6 identification, Co-IP, K63-ubiquitination assay of TRAF6, hepatocyte-specific Otub1 overexpression mouse model (HFHC diet), Otub1 KO mice\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction, Co-IP, K63 ubiquitination mechanistic assay, in vivo mouse model, single lab\",\n      \"pmids\": [\"34591986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERK/RSK-mediated phosphorylation of YB-1 at Ser102 in hyperglycaemia disrupts its interaction with OTUB1, leading to YB-1 ubiquitination and proteasomal degradation; when the ERK pathway is inhibited, YB-1–OTUB1 interaction is stabilised, preserving YB-1 levels and ameliorating diabetic cardiomyopathy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay of YB-1, phospho-specific YB-1(S102) analysis, ERK inhibitor (U0126) in STZ-diabetic mice, echocardiography\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, phosphorylation-dependent interaction, in vivo mouse model with functional cardiac readout, single lab\",\n      \"pmids\": [\"35490780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUB1 non-canonically stabilises CCN6 by inhibiting K48-linked ubiquitination via interaction through its linker domain; OTUB1 deletion reduces CCN6 abundance and increases breast cancer cell migration and proliferation, which is rescued by CCN6 supplementation.\",\n      \"method\": \"DUB screening assay, Co-IP, K48-ubiquitination assay, OTUB1 deletion mutants (linker domain), breast cancer cell functional assays, allograft model\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DUB screen, Co-IP, K48 ubiquitination assay, domain mapping, in vivo allograft, single lab\",\n      \"pmids\": [\"37608493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUB1 is a novel deubiquitinating enzyme for ERRα; OTUB1 C91 residue is critical for ERRα deubiquitination. The natural product Ailanthone (AIL) inhibits OTUB1 activity by interacting with C91, disrupts the OTUB1–ERRα interaction, and promotes ERRα ubiquitination and proteasomal degradation.\",\n      \"method\": \"Competitive activity-based protein profiling (ABPP) for target identification, Co-IP, luciferase reporter assay, C91A mutant analysis, surface plasmon resonance, in vivo TNBC mouse model\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ABPP target identification, C91 mutagenesis, Co-IP, functional reporter assay, in vivo validation, single lab\",\n      \"pmids\": [\"39864589\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OTUB1 is a highly versatile OTU-family deubiquitinase that stabilises a broad spectrum of substrates (including SLC7A11, PD-L1, FOXM1, SMAD2/3, p53, c-IAP1, RAS, YAP, MYC, CHK1, and many others) through two mechanistically distinct modes: canonical K48-linked polyubiquitin chain cleavage, and a non-canonical, catalysis-independent mechanism in which it binds charged E2~ubiquitin thiolester intermediates (UBC13, UBE2D/UBE2E family, UBCH5) and prevents ubiquitin transfer—an activity allosterically stimulated by free ubiquitin and regulated by post-translational modifications of OTUB1 itself including CK2-mediated phosphorylation at Ser16 (which drives nuclear translocalisation), Src/SRMS phosphorylation at Tyr26, SET7-mediated methylation at Lys122 (which impairs E2 binding), monoubiquitination at Lys59/109, persulfidation at Cys91, S-nitrosylation, and FIH-mediated asparagine hydroxylation/covalent complexation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"OTUB1 is an OTU-family deubiquitinase that controls the stability of a broad set of regulatory proteins through two mechanistically distinct activities: canonical cleavage of Lys48-linked polyubiquitin chains, and a catalysis-independent suppression of ubiquitin transfer by E2 conjugating enzymes [#0, #2, #4]. The non-canonical mode was defined at DNA double-strand breaks, where OTUB1 binds and inhibits the E2 UBC13 (UBE2N) to suppress RNF168-dependent K63-linked ubiquitination, blocking 53BP1 focus formation independently of catalytic activity, and extends to UBE2D and UBE2E E2 subfamilies [#0]. Crystallographic and biochemical work established the molecular basis: free ubiquitin binds a distal allosteric site to fold an N-terminal ubiquitin-binding helix, increasing OTUB1 affinity for the donor ubiquitin of the charged E2~Ub thioester, occluding the RING-E3 binding surface on UBC13 and blocking acceptor attack, while the canonical K48 distal ubiquitin site is co-opted to engage the thioester [#1, #2, #3]; reciprocally, E2 enzymes stimulate OTUB1's intrinsic K48 isopeptidase activity through the same helix-folding event, creating bidirectional cross-regulation governed by the ratio of charged to free ubiquitin [#4]. Through these activities OTUB1 stabilizes numerous substrates by either editing K48 chains or shielding them from E2-mediated ubiquitination, including phospho-SMAD2/3 in TGF\\u03b2 signaling [#7], c-IAP1 to sustain NF-\\u03baB/MAPK signaling and prevent necroptosis [#8, #28], FOXM1 [#11], RAS [#12], MYC [#30], YAP [#29], PD-L1 [#22], and the cystine antiporter SLC7A11 and GPX4 to restrain ferroptosis [#17, #31]. p53 pathway control is exerted indirectly: OTUB1 suppresses MDM2-mediated ubiquitination of p53 and MDMX via non-canonical E2 (UbcH5) inhibition, an activity that itself requires OTUB1 monoubiquitination at Lys59/Lys109 [#9, #42]. OTUB1 also tunes innate and adaptive immunity, deubiquitinating TRAF3/TRAF6 to dampen interferon and TLR responses [#5, #41, #24], setting an IL-15/AKT activation threshold in CD8+ T and NK cells [#19], and stabilizing p100 and SOCS1 to constrain NF-\\u03baB and JAK/STAT signaling, with conditional knockouts producing autoimmunity and severe experimental autoimmune encephalomyelitis [#20, #27]. OTUB1 activity is itself extensively regulated by post-translational modification: CK2 phosphorylation at Ser16 drives nuclear accumulation and DNA-repair function [#10], Src/SRMS phosphorylation at Tyr26 enables Raptor binding [#33], SET7 methylation at Lys122 impairs UBC13 engagement [#35], Cys91 persulfidation enhances E2 binding to stabilize SLC7A11 [#34], and FIH-mediated asparagine hydroxylation/covalent complexation links OTUB1 to oxygen sensing [#13, #18]. Otub1 is essential for development, as knockout mice are embryonic lethal [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established OTUB1 as a deubiquitinase acting on innate immune signaling adaptors, the first functional context placing it in interferon regulation.\",\n      \"evidence\": \"Co-IP and IFN-\\u03b2 reporter/antiviral assays with TRAF3 and TRAF6 in cells\",\n      \"pmids\": [\"19996094\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve catalytic vs. non-catalytic mechanism\", \"Single lab without structural validation\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed OTUB1 directly deubiquitinates a transcription factor (ER\\u03b1), introducing the theme of substrate stabilization on chromatin.\",\n      \"evidence\": \"Mass spec of ER\\u03b1 complex, in vitro DUB assay, ChIP and reporter assays\",\n      \"pmids\": [\"19383985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain linkage specificity not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovered the catalysis-independent mode of action\\u2014OTUB1 inhibits K63 chain synthesis by binding the E2 UBC13 rather than cleaving chains\\u2014redefining how a DUB can restrict ubiquitination.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, catalytic-mutant analysis, and DSB repair readouts\",\n      \"pmids\": [\"20725033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of E2 inhibition not yet resolved at this stage\", \"Regulation of the switch between canonical and non-canonical modes unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided the structural mechanism: free ubiquitin allosterically activates OTUB1, folding an N-terminal helix that captures the E2~Ub thioester and occludes the RING-E3 site, explaining non-canonical E2 inhibition.\",\n      \"evidence\": \"X-ray crystallography of OTUB1\\u2013UBC13 and OTUB1\\u2013Ub aldehyde\\u2013UBC13~Ub conjugates, SPR, structure-guided mutagenesis (three independent studies)\",\n      \"pmids\": [\"22367539\", \"22325355\", \"22679021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulation of allosteric ubiquitin pool not addressed\", \"Which substrates depend on this mode in cells left open\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated bidirectional cross-regulation\\u2014E2 enzymes stimulate OTUB1 K48 cleavage via the same helix-folding event\\u2014integrating its two activities into one allosteric framework.\",\n      \"evidence\": \"Reconstituted DUB activity assays and crystallography of human and worm OTUB1/UBCH5B with mutagenesis\",\n      \"pmids\": [\"23955022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular thresholds of charged/uncharged E2 not quantified\", \"Substrate-specific consequences not mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected the non-canonical mechanism to physiological signaling outputs by showing OTUB1 sustains phospho-SMAD2/3 and stabilizes c-IAP1, controlling TGF\\u03b2-driven migration and TWEAK/NF-\\u03baB survival signaling.\",\n      \"evidence\": \"Signal-dependent Co-IP, catalytic mutants, proteasome-inhibitor rescue, zebrafish c-IAP knockdown, functional migration/death assays\",\n      \"pmids\": [\"24071738\", \"23524849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recruitment determinants to each signaling complex not fully defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed auto-regulation of OTUB1\\u2014its own monoubiquitination at Lys59/109 is required to suppress UbcH5 and stabilize p53\\u2014and that knockout is embryonically lethal, marking developmental essentiality.\",\n      \"evidence\": \"In vitro monoubiquitination/ubiquitination assays with lysine-free mutant, CHX chase, apoptosis assays; HSCARG-dependent TRAF3 recruitment; later KO mouse\",\n      \"pmids\": [\"24403071\", \"24763515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How monoubiquitination remodels OTUB1 structurally not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended substrate range to oncogenic RAS and identified oxygen-dependent FIH hydroxylation at Asn22 as a regulator, linking OTUB1 to metabolism and hypoxia.\",\n      \"evidence\": \"In vitro ubiquitination/imaging/xenografts for RAS; in vitro hydroxylation, N22A interactome and metabolic assays for FIH\",\n      \"pmids\": [\"26881969\", \"26752685\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological oxygen tension dependence in vivo not established\", \"FIH-OTUB1 stoichiometry unresolved at this stage\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed OTUB1 stabilizes the E2 enzyme UBE2E1 itself and the mTOR inhibitor DEPTOR, positioning OTUB1 as a regulator of E2 abundance and mTORC1/autophagy, and confirmed embryonic lethality of Otub1 knockout.\",\n      \"evidence\": \"In vitro autoubiquitination assays, mouse and human KO cells, Asp88/Cys91 mutant dissection, mTORC1 and autophagy readouts\",\n      \"pmids\": [\"30282802\", \"29382726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Non-canonical Asp88-dependent catalysis mechanism incompletely defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established in vivo immunoregulatory roles via conditional knockouts: OTUB1 sets an IL-15/AKT activation threshold and stabilizes p100, with deficiency causing enhanced anti-tumor immunity or lupus-like autoimmunity.\",\n      \"evidence\": \"Conditional Otub1 KO mice, membrane fractionation, AKT/NF-\\u03baB ubiquitination assays, immune functional and tumor-challenge models\",\n      \"pmids\": [\"31182807\", \"31086255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific substrate selection mechanisms not fully defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened OTUB1 into cancer immune evasion and context-dependent E2 control: it stabilizes PD-L1 to suppress CD8+ cytotoxicity, yet in dendritic cells stabilizes UBC13 to promote NF-\\u03baB, and relocalizes to mitochondria to activate RIG-I.\",\n      \"evidence\": \"K48-specific DUB assays, OTUB1 KO/CRISPR cells, DC-specific KO mice, cell-free RIG-I reconstitution, immune functional and infection models\",\n      \"pmids\": [\"33328570\", \"32024978\", \"32023470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What dictates whether OTUB1 stabilizes vs. inhibits a given E2 in a given cell remains unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined OTUB1 as a node in tissue protection and metabolism, stabilizing SOCS1, c-IAP1 (anti-necroptosis), MYC, YAP, and SLC7A11, linking it to JAK/STAT control, glycolysis, Hippo signaling, and ferroptosis.\",\n      \"evidence\": \"Astrocyte- and hepatocyte-specific conditional KO mice, K48-ubiquitination assays, RIPK1/RIPK3/MLKL necrosome readouts, DUB screens, site-specific lysine mutagenesis\",\n      \"pmids\": [\"30944096\", \"33712742\", \"35296795\", \"36271031\", \"30709928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate prioritization across these pathways in a single cell type not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped post-translational regulation of OTUB1\\u2014Tyr26 phosphorylation enabling Raptor binding and Cys91 persulfidation enhancing E2 binding to stabilize SLC7A11\\u2014connecting redox and kinase inputs to OTUB1 output.\",\n      \"evidence\": \"Co-IP with Y26/Cys91 variants, in vitro kinase and persulfidation site-mapping, GLRX KO, ubiquitination and metabolic readouts; plus HIF-1\\u03b1 and GPX4 stabilization studies\",\n      \"pmids\": [\"35927303\", \"37703360\", \"35732631\", \"36369321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Crosstalk among the many OTUB1 PTMs not integrated\", \"Most are single-lab findings\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed SET7 methylation at Lys122 selectively impairs the non-canonical UBC13-binding activity without affecting catalysis, providing a switch that derepresses ferroptosis, and extended substrate range to TGFBI, CCN6, and ERR\\u03b1.\",\n      \"evidence\": \"In vitro methylation/K122 mutant, Co-IP, ROS/ferroptosis assays; MS substrate identification with site-specific mutagenesis and in vivo models\",\n      \"pmids\": [\"36822329\", \"36994729\", \"37608493\", \"39864589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether methylation and other PTMs act combinatorially is untested\", \"Single-lab substrate validations\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Continued substrate expansion (CHK1, RACK1, YTHDF2) and revealed pathogen hijacking, with Legionella effectors ubiquitinating OTUB1 to redirect it to the bacterial vacuole and block its DUB activity.\",\n      \"evidence\": \"Co-IP, K48-ubiquitination assays, transcriptomic/m6A analysis, DNA-damage assays, reconstituted effector ubiquitination and infection models\",\n      \"pmids\": [\"38266782\", \"38315284\", \"38462165\", \"38818749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of effector-driven OTUB1 modification beyond infection unknown\", \"Single-lab substrate findings\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How OTUB1 selects between canonical K48 cleavage and non-canonical E2 suppression for any given substrate in a given cellular context\\u2014and how its numerous PTMs are coordinated to set that choice\\u2014remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating PTM state, free-ubiquitin levels, and substrate selection\", \"Most substrate relationships rest on single-lab studies\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 7, 11, 14, 22, 29, 30]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 2, 4, 22]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 9, 16]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10, 23]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [23, 42]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [12, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 3, 10, 26, 37]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 19, 20, 22, 24, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 12, 33, 38]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 9, 16]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 28, 17, 31, 42]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [30, 32, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBC13/UBE2N\", \"UBE2D/UBCH5\", \"UBE2E1\", \"TRAF3\", \"TRAF6\", \"c-IAP1\", \"SLC7A11\", \"DEPTOR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}