{"gene":"USP49","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2013,"finding":"USP49 is a histone H2B-specific deubiquitinase that forms a complex with RuvB-like1 (RVB1) and SUG1, deubiquitinates histone H2B in vitro and in vivo, and is required for efficient cotranscriptional splicing of a large set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) at affected exons and upstream/downstream intronic splicing elements, and altered U1A and U2B association with chromatin and nascent pre-mRNA. Elevated uH2B also increased nucleosome stability at these exons.","method":"Biochemical purification, in vitro deubiquitinase assay, Co-IP (complex with RVB1/SUG1), RNA-seq (>9000 isoform changes), ChIP, siRNA knockdown","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic activity demonstrated, complex purified and validated, multiple orthogonal methods (biochemistry, ChIP, RNA-seq)","pmids":["23824326"],"is_preprint":false},{"year":2017,"finding":"USP49 deubiquitinates and stabilizes FKBP51, which in turn enhances PHLPP-mediated dephosphorylation of AKT at Ser473, thereby negatively regulating AKT activation. USP49 inhibited pancreatic cancer cell proliferation and enhanced gemcitabine sensitivity in a FKBP51-AKT-dependent manner.","method":"Co-IP, in vivo ubiquitination assay, knockdown/overexpression with AKT phosphorylation readout, epistasis rescue experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assays, pathway epistasis with multiple orthogonal methods in a single focused study","pmids":["28363942"],"is_preprint":false},{"year":2018,"finding":"USP49 interacts with the N-terminus of p53, deubiquitinates p53 (suppressing multiple forms of its ubiquitination), and stabilizes p53 protein. USP49 expression is transcriptionally upregulated by p53 in response to DNA damage, forming a positive feedback loop. USP49 knockout mice are more susceptible to AOM/DSS-induced colon tumors.","method":"DUB library screen, Co-IP, in vivo ubiquitination assay, KO mouse model, etoposide sensitivity assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, KO mouse; single lab with multiple methods","pmids":["29748582"],"is_preprint":false},{"year":2018,"finding":"USP49 directly interacts with DUSP1 and deubiquitinates it, thereby stabilizing DUSP1 protein. Stabilized DUSP1 dephosphorylates JNK1/2, and this USP49-DUSP1-JNK1/2 axis protects cardiomyocytes from ischemia-reperfusion-induced apoptosis.","method":"Co-immunoprecipitation, ubiquitination analysis, overexpression/knockdown with JNK phosphorylation and apoptosis readouts, in vivo rat I/R model (TUNEL)","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay, single lab, two orthogonal methods","pmids":["30246457"],"is_preprint":false},{"year":2019,"finding":"USP49 interacts with MITA/STING and removes K63-linked ubiquitin chains from MITA after HSV-1 infection, inhibiting MITA aggregation and subsequent recruitment of TBK1 to the signaling complex, thereby attenuating type I interferon and proinflammatory cytokine production. Usp49-/- mice exhibit resistance to lethal HSV-1 infection.","method":"Co-IP, in vivo K63-linked deubiquitination assay, MITA aggregation assay, TBK1 recruitment assay, siRNA/CRISPR KO, Usp49-/- mouse model","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, linkage-specific ubiquitination assay, genetic KO in cells and mice, multiple orthogonal methods","pmids":["30943264"],"is_preprint":false},{"year":2019,"finding":"USP49 directly interacts with APOBEC3G (A3G) and removes ubiquitin from it, stabilizing A3G protein and enhancing its anti-HIV-1 activity. USP49 also counteracts a Vif- and cullin-ring-independent pathway of A3G degradation.","method":"Co-IP, in vitro deubiquitination assay, overexpression/knockdown with A3G stability and HIV replication readouts","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and deubiquitination assay, single lab, two orthogonal methods","pmids":["31397674"],"is_preprint":false},{"year":2022,"finding":"Fbxo45, an F-box protein forming an atypical SCF E3 ligase complex, directly binds USP49 through its SPRY domain and mediates K48-linked ubiquitination and proteasomal degradation of USP49. This Fbxo45-mediated USP49 degradation is enhanced by NEK6 kinase. Loss of Fbxo45 stabilizes USP49, inhibiting pancreatic cancer cell viability and motility.","method":"Co-IP, ubiquitination assay, cycloheximide chase, NEK6 kinase assay, xenograft mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with domain mapping, single lab, multiple methods","pmids":["35279684"],"is_preprint":false},{"year":2022,"finding":"USP49 is transcriptionally activated by c-MYC and deubiquitinates BAG2, stabilizing it to promote CRC cell survival and chemoresistance.","method":"Promoter analysis (c-MYC binding), Co-IP, ubiquitination assay, knockdown/overexpression with proliferation and chemoresistance readouts","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay, single lab, two orthogonal methods","pmids":["35367823"],"is_preprint":false},{"year":2022,"finding":"USP49 deubiquitinates γH2AX (ubiquitylated at K15); a catalytic-dead USP49 mutant interacts with and colocalizes with γH2AX. Overexpressed USP49 suppressed γH2AX ubiquitylation, inhibited 53BP1 foci formation at DSBs, and increased cell sensitivity to DSB-inducing drugs. Endogenous USP49 is degraded via the proteasome upon DSB induction.","method":"Overexpression and catalytic mutant analysis, Co-IP, immunofluorescence colocalization, ubiquitination assay, 53BP1 foci formation assay, proteasome inhibitor treatment","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic mutant validation plus multiple cellular assays, single lab","pmids":["35598681"],"is_preprint":false},{"year":2022,"finding":"USP49 directly binds the MDM2 gene promoter (by ChIP) and deubiquitinates H2B at this locus; USP49 knockout increases H2Bub enrichment at the MDM2 gene, elevating MDM2 transcription and suppressing p53 target gene expression, thereby promoting HCT116 cell proliferation.","method":"ChIP, H2Bub ChIP, RT-PCR for p53 target genes, KO and overexpression","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP linking USP49 H2B-DUB activity to MDM2 promoter regulation, single lab, two orthogonal methods","pmids":["35072515"],"is_preprint":false},{"year":2021,"finding":"USP49 is a GRβ-binding protein and the interaction depends on GRβ ubiquitination status. USP49 knockdown in glioblastoma cells increased GRβ ubiquitination and amplified GRβ oncogenic effects, indicating USP49 modulates GRβ stability through deubiquitination.","method":"Co-IP, ubiquitination assay, K733R GRβ mutant, siRNA knockdown with proliferation/invasion/apoptosis readouts","journal":"Molecular cancer research : MCR","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and ubiquitination assay, no direct in vitro deubiquitination of GRβ demonstrated","pmids":["34610959"],"is_preprint":false},{"year":2023,"finding":"USP49 loss impairs cancer cell proliferation, increases aneuploidy, and allows cells to override the spindle assembly checkpoint (SAC) arrest induced by nocodazole. New binding partners of USP49 identified include ribophorin 1, USP44, and different centrins.","method":"siRNA/shRNA knockdown, nocodazole SAC assay, aneuploidy measurement, MS-based interactome (binding partners)","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional SAC assay with defined phenotype plus MS-identified binding partners, single lab","pmids":["36702832"],"is_preprint":false},{"year":2024,"finding":"USP49 stabilizes SIRT1 by inhibiting its ubiquitination and degradation, thereby promoting autophagy and carboplatin resistance in retinoblastoma cells. IGF2BP3-mediated m6A modification upregulates USP49 expression in an m6A-dependent manner.","method":"Co-IP, ubiquitination assay, autophagy flux assay, IGF2BP3 m6A-dependent regulation assay, rescue experiments, xenograft model","journal":"The Kaohsiung journal of medical sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without direct in vitro deubiquitination reconstitution","pmids":["39497328"],"is_preprint":false},{"year":2024,"finding":"USP49 binds IκBα and mediates its deubiquitination, leading to IκBα stabilization, cytoplasmic retention of NF-κB, and suppression of NF-κB activity. miR-361-3p inhibits USP49 expression by binding its 3' UTR, relieving IκBα deubiquitination and thus activating NF-κB to promote inflammation and apoptosis in sepsis-induced myocardial injury.","method":"Co-immunoprecipitation, western blot, dual-luciferase reporter (miR-361-3p/Usp49 3'UTR), CLP rat model, LPS cell model","journal":"Toxicology research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP-based binding and deubiquitination inferred, single lab, no direct in vitro deubiquitination assay","pmids":["39568464"],"is_preprint":false},{"year":2025,"finding":"USP49 interacts with HDAC3 (validated by IP-mass spectrometry and Co-IP); USP49 downregulation augments HDAC3 ubiquitination, reducing HDAC3 protein stability. In the context of cold atmospheric plasma treatment, reduced USP49 destabilizes HDAC3, altering H3K18 lactylation and p53-driven ferroptosis in endometrial cancer.","method":"Immunoprecipitation-mass spectrometry, Co-IP, immunofluorescence co-localization, molecular docking, ubiquitination assay, xenograft model","journal":"Journal of translational medicine","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — IP-MS and Co-IP support interaction, but deubiquitination of HDAC3 by USP49 is inferred rather than directly reconstituted","pmids":["40234906"],"is_preprint":false},{"year":2025,"finding":"ATM promotes liquid-liquid phase separation (LLPS) of USP49 and its recruitment to DNA double-strand breaks upon ionizing radiation. USP49 deubiquitinates RPA70 and recruits it along with Rad51 to DSBs, promoting homologous recombination repair and radioresistance in ESCC. p53 further augments USP49 transcription upon IR.","method":"LLPS assay, IR-induced DSB foci (immunofluorescence), Co-IP, ubiquitination assay, HR repair assay, knockdown/overexpression in vivo xenograft","journal":"International journal of biological macromolecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; LLPS and HR claims require further validation","pmids":["40460957"],"is_preprint":false},{"year":2025,"finding":"USP49 interacts with PKMYT1 and limits its ubiquitination and proteasomal degradation, thereby stabilizing PKMYT1 and promoting G2/M cell cycle progression and malignant phenotypes in triple-negative breast cancer cells.","method":"Co-IP, ubiquitination assay, knockdown/overexpression with cell cycle and viability readouts, PKMYT1 rescue experiments","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without in vitro reconstitution","pmids":["41443044"],"is_preprint":false},{"year":2025,"finding":"USP49 interacts with and deubiquitinates GRPR (gastrin-releasing peptide receptor), stabilizing it and promoting PI3K/AKT-driven tumor survival in medullary thyroid carcinoma. The natural flavonoid vitexin inhibits USP49, thereby destabilizing GRPR.","method":"Co-IP, ubiquitination assay, vitexin treatment with GRPR stability and AKT signaling readouts, xenograft mouse model","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay, no direct in vitro deubiquitination reconstitution","pmids":["41349242"],"is_preprint":false},{"year":2025,"finding":"FBXO2 E3 ubiquitin ligase directly binds USP49 and targets it for ubiquitin-mediated proteasomal degradation, reducing USP49 stability and function in hepatocellular carcinoma cells.","method":"Co-IP, in vivo ubiquitination assay, cycloheximide chase, siRNA knockdown rescue experiments, xenograft model","journal":"Frontiers in immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay","pmids":["41035649"],"is_preprint":false}],"current_model":"USP49 is a deubiquitinase (DUB) with multiple verified substrates: it specifically deubiquitinates histone H2B (forming a complex with RVB1 and SUG1) to regulate cotranscriptional pre-mRNA splicing and MDM2-p53 axis transcription; it stabilizes FKBP51 to negatively regulate AKT via PHLPP-mediated dephosphorylation; it removes K63-linked ubiquitin from MITA/STING to attenuate innate antiviral signaling; it deubiquitinates and stabilizes p53 forming a positive feedback loop in the DNA damage response; it deubiquitinates γH2AX to regulate 53BP1 recruitment and DSB repair pathway choice; and it stabilizes additional substrates including DUSP1, A3G, BAG2, SIRT1, GRPR, PKMYT1, RPA70, and HDAC3 in various cellular contexts. USP49 itself is targeted for proteasomal degradation by Fbxo45/NEK6 and FBXO2 E3 ligase complexes, and undergoes ATM-promoted liquid-liquid phase separation at DSBs."},"narrative":{"mechanistic_narrative":"USP49 is a deubiquitinating enzyme that acts both at chromatin and on individual protein substrates to control gene expression, genome integrity, and signaling [PMID:23824326, PMID:28363942]. Its founding activity is the specific removal of monoubiquitin from histone H2B as part of a complex with RVB1 and SUG1, which controls cotranscriptional splicing of a large set of exons and modulates nucleosome stability [PMID:23824326]. This chromatin-directed H2B deubiquitination extends to gene-specific regulation: USP49 binds the MDM2 promoter and removes H2Bub there, restraining MDM2 transcription and thereby de-repressing p53 target genes [PMID:35072515]. Beyond chromatin, USP49 stabilizes diverse substrates by reversing their ubiquitination — it deubiquitinates and stabilizes FKBP51 to promote PHLPP-mediated dephosphorylation of AKT-Ser473 [PMID:28363942], removes K63-linked ubiquitin from MITA/STING to block TBK1 recruitment and dampen type I interferon responses [PMID:30943264], and stabilizes APOBEC3G to enhance its anti-HIV-1 activity [PMID:31397674]. USP49 also engages the DNA damage response, interacting with and deubiquitinating p53 in a p53-induced positive feedback loop, with Usp49-knockout mice showing increased susceptibility to colon tumors [PMID:29748582]. Functionally, loss of USP49 impairs proliferation, increases aneuploidy, and permits override of the nocodazole-induced spindle assembly checkpoint [PMID:36702832]. USP49 protein level is itself controlled by ubiquitin-proteasome turnover through the Fbxo45/NEK6 axis [PMID:35279684].","teleology":[{"year":2013,"claim":"Established USP49 as a histone H2B-specific deubiquitinase and linked this chromatin activity to a cellular process — cotranscriptional pre-mRNA splicing.","evidence":"Biochemical purification, in vitro DUB assay, Co-IP defining an RVB1/SUG1 complex, and ChIP/RNA-seq after knockdown","pmids":["23824326"],"confidence":"High","gaps":["Structural basis of H2B recognition not resolved","How splicing factor association is mechanistically coupled to uH2B removal not fully defined"]},{"year":2017,"claim":"Extended USP49 activity beyond chromatin to a signaling substrate, showing it stabilizes FKBP51 to suppress AKT activation.","evidence":"Reciprocal Co-IP, in vivo ubiquitination assay, and pathway epistasis with AKT-Ser473 readout in pancreatic cancer cells","pmids":["28363942"],"confidence":"High","gaps":["Whether USP49 directly deubiquitinates FKBP51 in vitro not shown","Ubiquitin linkage specificity on FKBP51 not defined"]},{"year":2018,"claim":"Placed USP49 in the DNA damage response as a p53 stabilizer within a feed-forward loop, with tumor-suppressive consequences in vivo.","evidence":"DUB library screen, Co-IP, in vivo ubiquitination assay, and AOM/DSS tumorigenesis in Usp49-knockout mice","pmids":["29748582"],"confidence":"Medium","gaps":["Direct in vitro deubiquitination of p53 not reconstituted","Relationship to chromatin-based MDM2 regulation not integrated at this stage"]},{"year":2018,"claim":"Showed substrate stabilization as a recurring mode, with USP49 protecting DUSP1 to restrain JNK signaling and apoptosis in cardiomyocytes.","evidence":"Co-IP, ubiquitination analysis, and JNK/apoptosis readouts with an in vivo rat I/R model","pmids":["30246457"],"confidence":"Medium","gaps":["No in vitro deubiquitination reconstitution","Tissue specificity of the DUSP1 interaction not established"]},{"year":2019,"claim":"Defined a linkage-specific role in innate immunity, with USP49 stripping K63 chains from MITA/STING to limit antiviral signaling.","evidence":"Reciprocal Co-IP, K63-linkage deubiquitination assay, MITA aggregation and TBK1 recruitment assays, and Usp49-/- mice resistant to HSV-1","pmids":["30943264"],"confidence":"High","gaps":["Whether the same complex partners (RVB1/SUG1) participate not addressed","Structural mechanism of MITA aggregation control unknown"]},{"year":2019,"claim":"Showed USP49 can promote antiviral restriction by stabilizing APOBEC3G against a Vif/cullin-independent degradation route.","evidence":"Co-IP, in vitro deubiquitination assay, and A3G stability/HIV replication readouts","pmids":["31397674"],"confidence":"Medium","gaps":["Identity of the Vif-independent degradation pathway not defined","Physiological context in primary cells not tested"]},{"year":2022,"claim":"Connected USP49 chromatin H2B-DUB activity to gene-specific transcriptional control of the p53 axis via the MDM2 promoter.","evidence":"ChIP and H2Bub ChIP at MDM2, RT-PCR of p53 targets, and KO/overexpression in HCT116","pmids":["35072515"],"confidence":"Medium","gaps":["How USP49 is recruited to specific promoters unknown","Generality across other promoters not mapped"]},{"year":2022,"claim":"Identified the mechanism controlling USP49 abundance, showing an Fbxo45 SCF complex enhanced by NEK6 drives its K48-linked proteasomal degradation.","evidence":"Co-IP with SPRY-domain mapping, ubiquitination assay, cycloheximide chase, NEK6 kinase assay, and xenograft","pmids":["35279684"],"confidence":"Medium","gaps":["Signals controlling Fbxo45 engagement of USP49 unknown","Whether degradation is context- or substrate-dependent unresolved"]},{"year":2022,"claim":"Linked USP49 to DSB repair pathway choice by showing it deubiquitinates K15-ubiquitylated gamma-H2AX and limits 53BP1 recruitment.","evidence":"Catalytic-dead mutant interaction/colocalization, ubiquitination assay, 53BP1 foci assay, and proteasome inhibitor analysis","pmids":["35598681"],"confidence":"Medium","gaps":["Endogenous timing of gamma-H2AX deubiquitination during repair not resolved","Balance with the p53-stabilizing role not reconciled"]},{"year":2022,"claim":"Showed USP49 stabilizes BAG2 downstream of c-MYC transcriptional activation to promote colorectal cancer survival and chemoresistance.","evidence":"Promoter analysis of c-MYC binding, Co-IP, ubiquitination assay, and proliferation/chemoresistance readouts","pmids":["35367823"],"confidence":"Medium","gaps":["No in vitro deubiquitination reconstitution","Direct versus indirect stabilization of BAG2 not separated"]},{"year":2023,"claim":"Defined a cell-division phenotype for USP49 loss — proliferation defects, aneuploidy, and SAC override — and expanded its interactome.","evidence":"siRNA/shRNA knockdown, nocodazole SAC and aneuploidy assays, and MS-based interactome (ribophorin 1, USP44, centrins)","pmids":["36702832"],"confidence":"Medium","gaps":["Substrate mediating the SAC phenotype not identified","Functional significance of new interactors not validated"]},{"year":2025,"claim":"Proposed a DSB-localized condensate model in which ATM-driven LLPS recruits USP49 to deubiquitinate RPA70 and promote homologous recombination.","evidence":"LLPS assay, IR-induced foci, Co-IP, ubiquitination assay, and HR repair readouts in ESCC with xenograft","pmids":["40460957"],"confidence":"Low","gaps":["LLPS and HR claims await independent validation","Reconciliation with the earlier 53BP1-restraining DSB role unresolved"]},{"year":null,"claim":"How USP49 selects among its many candidate substrates across chromatin, signaling, immunity, and DNA repair contexts — and which interactions are direct, in vitro-reconstituted deubiquitination events versus inferred stabilizations — remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Substrate-targeting determinants and recruitment mechanisms undefined","Many proposed substrates rest on Co-IP plus cellular ubiquitination assays without enzymatic reconstitution","No structural model of USP49 with any substrate"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4,5,8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,9]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,6]}],"complexes":["USP49-RVB1-SUG1 H2B-deubiquitinase complex"],"partners":["RVB1","SUG1","FKBP51","STING1","APOBEC3G","TP53","MDM2","FBXO45"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q70CQ1","full_name":"Ubiquitin carboxyl-terminal hydrolase 49","aliases":["Deubiquitinating enzyme 49","Ubiquitin thioesterase 49","Ubiquitin-specific-processing protease 49"],"length_aa":688,"mass_kda":79.2,"function":"Deubiquitinase that plays a role in several cellular processes including transcriptional regulation, cell cycle progression or innate immunity. Specifically deubiquitinates histone H2B at 'Lys-120' (H2BK120Ub), a specific tag for epigenetic transcriptional activation and acts as a regulator of mRNA splicing (PubMed:23824326). Antagonizes DNA double-strand break-induced ubiquitination of H2AX thereby participating in the maintenance of genome integrity (PubMed:35598681). Plays a role in the negative regulation of cell proliferation through the AKT pathway by deubiquitinating FKBP51, leading to enhanced AKT1 dephosphorylation by PHLPP1 (PubMed:28363942). Also regulates the mitotic spindle checkpoint and prevents aneuploidy (PubMed:36702832). Negatively regulates cellular antiviral responses via deconjugating 'Lys-63'-linked ubiquitination of STING1 (PubMed:30943264). Significantly enhances the anti-HIV-1 activity of APOBEC3G. Mechanistically, stabilizes APOBEC3G by counteracting HIV-1 Vif-mediated APOBEC3G ubiquitination (PubMed:31397674)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q70CQ1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP49","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP49","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"retina","ntpm":8.3}],"url":"https://www.proteinatlas.org/search/USP49"},"hgnc":{"alias_symbol":["MGC20741"],"prev_symbol":[]},"alphafold":{"accession":"Q70CQ1","domains":[{"cath_id":"3.30.40.10","chopping":"2-106","consensus_level":"medium","plddt":82.6316,"start":2,"end":106}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q70CQ1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q70CQ1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q70CQ1-F1-predicted_aligned_error_v6.png","plddt_mean":71.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP49","jax_strain_url":"https://www.jax.org/strain/search?query=USP49"},"sequence":{"accession":"Q70CQ1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q70CQ1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q70CQ1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q70CQ1"}},"corpus_meta":[{"pmid":"28363942","id":"PMC_28363942","title":"USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51-AKT signaling.","date":"2017","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/28363942","citation_count":91,"is_preprint":false},{"pmid":"23824326","id":"PMC_23824326","title":"USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing.","date":"2013","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/23824326","citation_count":85,"is_preprint":false},{"pmid":"35279684","id":"PMC_35279684","title":"Fbxo45 facilitates pancreatic carcinoma progression by targeting USP49 for ubiquitination and degradation.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35279684","citation_count":57,"is_preprint":false},{"pmid":"30943264","id":"PMC_30943264","title":"USP49 negatively regulates cellular antiviral responses via deconjugating K63-linked ubiquitination of MITA.","date":"2019","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/30943264","citation_count":46,"is_preprint":false},{"pmid":"29748582","id":"PMC_29748582","title":"USP49 participates in the DNA damage response by forming a positive feedback loop with p53.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29748582","citation_count":40,"is_preprint":false},{"pmid":"31397674","id":"PMC_31397674","title":"USP49 potently stabilizes APOBEC3G protein by removing ubiquitin and inhibits HIV-1 replication.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31397674","citation_count":27,"is_preprint":false},{"pmid":"30246457","id":"PMC_30246457","title":"USP49 inhibits ischemia-reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1-JNK1/2 signaling.","date":"2018","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/30246457","citation_count":25,"is_preprint":false},{"pmid":"35367823","id":"PMC_35367823","title":"c-MYC-USP49-BAG2 axis promotes proliferation and chemoresistance of colorectal cancer cells in vitro.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35367823","citation_count":25,"is_preprint":false},{"pmid":"32691951","id":"PMC_32691951","title":"The long non-coding RNA HLNC1 potentiates hepatocellular carcinoma progression via interaction with USP49.","date":"2020","source":"Journal of clinical laboratory analysis","url":"https://pubmed.ncbi.nlm.nih.gov/32691951","citation_count":13,"is_preprint":false},{"pmid":"40234906","id":"PMC_40234906","title":"Cold atmospheric plasma drives USP49/HDAC3 axis mediated ferroptosis as a novel therapeutic strategy in endometrial cancer via reinforcing lactylation dependent p53 expression.","date":"2025","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40234906","citation_count":11,"is_preprint":false},{"pmid":"34610959","id":"PMC_34610959","title":"Oncogenic Activity of Glucocorticoid Receptor β Is Controlled by Ubiquitination-Dependent Interaction with USP49 in Glioblastoma Cells.","date":"2021","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/34610959","citation_count":11,"is_preprint":false},{"pmid":"35598681","id":"PMC_35598681","title":"USP49 is a novel deubiquitylating enzyme for γ H2AX in DNA double-strand break repair.","date":"2022","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/35598681","citation_count":9,"is_preprint":false},{"pmid":"35072515","id":"PMC_35072515","title":"USP49-Mediated Histone H2B Deubiquitination Regulates HCT116 Cell Proliferation through MDM2-p53 Axis.","date":"2022","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/35072515","citation_count":6,"is_preprint":false},{"pmid":"39497328","id":"PMC_39497328","title":"IGF2BP3-dependent N6-methyladenosine modification of USP49 promotes carboplatin resistance in retinoblastoma by enhancing autophagy via regulating the stabilization of SIRT1.","date":"2024","source":"The Kaohsiung journal of medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39497328","citation_count":4,"is_preprint":false},{"pmid":"40460957","id":"PMC_40460957","title":"USP49 undergoes liquid-liquid phase separation and stabilizes RPA70 to induce radioresistance through homologous recombination repair in esophageal squamous cell carcinoma.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40460957","citation_count":3,"is_preprint":false},{"pmid":"39568464","id":"PMC_39568464","title":"miR-361-3p overexpression promotes apoptosis and inflammation by regulating the USP49/IκBα/NF-κB pathway to aggravate sepsis-induced myocardial injury.","date":"2024","source":"Toxicology research","url":"https://pubmed.ncbi.nlm.nih.gov/39568464","citation_count":3,"is_preprint":false},{"pmid":"41035649","id":"PMC_41035649","title":"FBXO2 promotes hepatocellular carcinoma progression and sorafenib resistance by targeting USP49 for proteasomal degradation.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41035649","citation_count":2,"is_preprint":false},{"pmid":"36702832","id":"PMC_36702832","title":"USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36702832","citation_count":1,"is_preprint":false},{"pmid":"41443044","id":"PMC_41443044","title":"USP49 promotes the malignancy of triple-negative breast cancer cells by regulating PKMYT1 ubiquitination and stability.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/41443044","citation_count":0,"is_preprint":false},{"pmid":"41349242","id":"PMC_41349242","title":"Vitexin targets USP49-GRPR deubiquitination axis in medullary thyroid carcinoma therapy.","date":"2025","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/41349242","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12107,"output_tokens":4873,"usd":0.054708,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12920,"output_tokens":3905,"usd":0.081113,"stage2_stop_reason":"end_turn"},"total_usd":0.135821,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"USP49 is a histone H2B-specific deubiquitinase that forms a complex with RuvB-like1 (RVB1) and SUG1, deubiquitinates histone H2B in vitro and in vivo, and is required for efficient cotranscriptional splicing of a large set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) at affected exons and upstream/downstream intronic splicing elements, and altered U1A and U2B association with chromatin and nascent pre-mRNA. Elevated uH2B also increased nucleosome stability at these exons.\",\n      \"method\": \"Biochemical purification, in vitro deubiquitinase assay, Co-IP (complex with RVB1/SUG1), RNA-seq (>9000 isoform changes), ChIP, siRNA knockdown\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic activity demonstrated, complex purified and validated, multiple orthogonal methods (biochemistry, ChIP, RNA-seq)\",\n      \"pmids\": [\"23824326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP49 deubiquitinates and stabilizes FKBP51, which in turn enhances PHLPP-mediated dephosphorylation of AKT at Ser473, thereby negatively regulating AKT activation. USP49 inhibited pancreatic cancer cell proliferation and enhanced gemcitabine sensitivity in a FKBP51-AKT-dependent manner.\",\n      \"method\": \"Co-IP, in vivo ubiquitination assay, knockdown/overexpression with AKT phosphorylation readout, epistasis rescue experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assays, pathway epistasis with multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"28363942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP49 interacts with the N-terminus of p53, deubiquitinates p53 (suppressing multiple forms of its ubiquitination), and stabilizes p53 protein. USP49 expression is transcriptionally upregulated by p53 in response to DNA damage, forming a positive feedback loop. USP49 knockout mice are more susceptible to AOM/DSS-induced colon tumors.\",\n      \"method\": \"DUB library screen, Co-IP, in vivo ubiquitination assay, KO mouse model, etoposide sensitivity assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, KO mouse; single lab with multiple methods\",\n      \"pmids\": [\"29748582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP49 directly interacts with DUSP1 and deubiquitinates it, thereby stabilizing DUSP1 protein. Stabilized DUSP1 dephosphorylates JNK1/2, and this USP49-DUSP1-JNK1/2 axis protects cardiomyocytes from ischemia-reperfusion-induced apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination analysis, overexpression/knockdown with JNK phosphorylation and apoptosis readouts, in vivo rat I/R model (TUNEL)\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"30246457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP49 interacts with MITA/STING and removes K63-linked ubiquitin chains from MITA after HSV-1 infection, inhibiting MITA aggregation and subsequent recruitment of TBK1 to the signaling complex, thereby attenuating type I interferon and proinflammatory cytokine production. Usp49-/- mice exhibit resistance to lethal HSV-1 infection.\",\n      \"method\": \"Co-IP, in vivo K63-linked deubiquitination assay, MITA aggregation assay, TBK1 recruitment assay, siRNA/CRISPR KO, Usp49-/- mouse model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, linkage-specific ubiquitination assay, genetic KO in cells and mice, multiple orthogonal methods\",\n      \"pmids\": [\"30943264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP49 directly interacts with APOBEC3G (A3G) and removes ubiquitin from it, stabilizing A3G protein and enhancing its anti-HIV-1 activity. USP49 also counteracts a Vif- and cullin-ring-independent pathway of A3G degradation.\",\n      \"method\": \"Co-IP, in vitro deubiquitination assay, overexpression/knockdown with A3G stability and HIV replication readouts\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and deubiquitination assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"31397674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Fbxo45, an F-box protein forming an atypical SCF E3 ligase complex, directly binds USP49 through its SPRY domain and mediates K48-linked ubiquitination and proteasomal degradation of USP49. This Fbxo45-mediated USP49 degradation is enhanced by NEK6 kinase. Loss of Fbxo45 stabilizes USP49, inhibiting pancreatic cancer cell viability and motility.\",\n      \"method\": \"Co-IP, ubiquitination assay, cycloheximide chase, NEK6 kinase assay, xenograft mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with domain mapping, single lab, multiple methods\",\n      \"pmids\": [\"35279684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP49 is transcriptionally activated by c-MYC and deubiquitinates BAG2, stabilizing it to promote CRC cell survival and chemoresistance.\",\n      \"method\": \"Promoter analysis (c-MYC binding), Co-IP, ubiquitination assay, knockdown/overexpression with proliferation and chemoresistance readouts\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"35367823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP49 deubiquitinates γH2AX (ubiquitylated at K15); a catalytic-dead USP49 mutant interacts with and colocalizes with γH2AX. Overexpressed USP49 suppressed γH2AX ubiquitylation, inhibited 53BP1 foci formation at DSBs, and increased cell sensitivity to DSB-inducing drugs. Endogenous USP49 is degraded via the proteasome upon DSB induction.\",\n      \"method\": \"Overexpression and catalytic mutant analysis, Co-IP, immunofluorescence colocalization, ubiquitination assay, 53BP1 foci formation assay, proteasome inhibitor treatment\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant validation plus multiple cellular assays, single lab\",\n      \"pmids\": [\"35598681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP49 directly binds the MDM2 gene promoter (by ChIP) and deubiquitinates H2B at this locus; USP49 knockout increases H2Bub enrichment at the MDM2 gene, elevating MDM2 transcription and suppressing p53 target gene expression, thereby promoting HCT116 cell proliferation.\",\n      \"method\": \"ChIP, H2Bub ChIP, RT-PCR for p53 target genes, KO and overexpression\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP linking USP49 H2B-DUB activity to MDM2 promoter regulation, single lab, two orthogonal methods\",\n      \"pmids\": [\"35072515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP49 is a GRβ-binding protein and the interaction depends on GRβ ubiquitination status. USP49 knockdown in glioblastoma cells increased GRβ ubiquitination and amplified GRβ oncogenic effects, indicating USP49 modulates GRβ stability through deubiquitination.\",\n      \"method\": \"Co-IP, ubiquitination assay, K733R GRβ mutant, siRNA knockdown with proliferation/invasion/apoptosis readouts\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and ubiquitination assay, no direct in vitro deubiquitination of GRβ demonstrated\",\n      \"pmids\": [\"34610959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP49 loss impairs cancer cell proliferation, increases aneuploidy, and allows cells to override the spindle assembly checkpoint (SAC) arrest induced by nocodazole. New binding partners of USP49 identified include ribophorin 1, USP44, and different centrins.\",\n      \"method\": \"siRNA/shRNA knockdown, nocodazole SAC assay, aneuploidy measurement, MS-based interactome (binding partners)\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional SAC assay with defined phenotype plus MS-identified binding partners, single lab\",\n      \"pmids\": [\"36702832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP49 stabilizes SIRT1 by inhibiting its ubiquitination and degradation, thereby promoting autophagy and carboplatin resistance in retinoblastoma cells. IGF2BP3-mediated m6A modification upregulates USP49 expression in an m6A-dependent manner.\",\n      \"method\": \"Co-IP, ubiquitination assay, autophagy flux assay, IGF2BP3 m6A-dependent regulation assay, rescue experiments, xenograft model\",\n      \"journal\": \"The Kaohsiung journal of medical sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without direct in vitro deubiquitination reconstitution\",\n      \"pmids\": [\"39497328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP49 binds IκBα and mediates its deubiquitination, leading to IκBα stabilization, cytoplasmic retention of NF-κB, and suppression of NF-κB activity. miR-361-3p inhibits USP49 expression by binding its 3' UTR, relieving IκBα deubiquitination and thus activating NF-κB to promote inflammation and apoptosis in sepsis-induced myocardial injury.\",\n      \"method\": \"Co-immunoprecipitation, western blot, dual-luciferase reporter (miR-361-3p/Usp49 3'UTR), CLP rat model, LPS cell model\",\n      \"journal\": \"Toxicology research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP-based binding and deubiquitination inferred, single lab, no direct in vitro deubiquitination assay\",\n      \"pmids\": [\"39568464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP49 interacts with HDAC3 (validated by IP-mass spectrometry and Co-IP); USP49 downregulation augments HDAC3 ubiquitination, reducing HDAC3 protein stability. In the context of cold atmospheric plasma treatment, reduced USP49 destabilizes HDAC3, altering H3K18 lactylation and p53-driven ferroptosis in endometrial cancer.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry, Co-IP, immunofluorescence co-localization, molecular docking, ubiquitination assay, xenograft model\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — IP-MS and Co-IP support interaction, but deubiquitination of HDAC3 by USP49 is inferred rather than directly reconstituted\",\n      \"pmids\": [\"40234906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATM promotes liquid-liquid phase separation (LLPS) of USP49 and its recruitment to DNA double-strand breaks upon ionizing radiation. USP49 deubiquitinates RPA70 and recruits it along with Rad51 to DSBs, promoting homologous recombination repair and radioresistance in ESCC. p53 further augments USP49 transcription upon IR.\",\n      \"method\": \"LLPS assay, IR-induced DSB foci (immunofluorescence), Co-IP, ubiquitination assay, HR repair assay, knockdown/overexpression in vivo xenograft\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; LLPS and HR claims require further validation\",\n      \"pmids\": [\"40460957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP49 interacts with PKMYT1 and limits its ubiquitination and proteasomal degradation, thereby stabilizing PKMYT1 and promoting G2/M cell cycle progression and malignant phenotypes in triple-negative breast cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown/overexpression with cell cycle and viability readouts, PKMYT1 rescue experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without in vitro reconstitution\",\n      \"pmids\": [\"41443044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP49 interacts with and deubiquitinates GRPR (gastrin-releasing peptide receptor), stabilizing it and promoting PI3K/AKT-driven tumor survival in medullary thyroid carcinoma. The natural flavonoid vitexin inhibits USP49, thereby destabilizing GRPR.\",\n      \"method\": \"Co-IP, ubiquitination assay, vitexin treatment with GRPR stability and AKT signaling readouts, xenograft mouse model\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay, no direct in vitro deubiquitination reconstitution\",\n      \"pmids\": [\"41349242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO2 E3 ubiquitin ligase directly binds USP49 and targets it for ubiquitin-mediated proteasomal degradation, reducing USP49 stability and function in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-IP, in vivo ubiquitination assay, cycloheximide chase, siRNA knockdown rescue experiments, xenograft model\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay\",\n      \"pmids\": [\"41035649\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP49 is a deubiquitinase (DUB) with multiple verified substrates: it specifically deubiquitinates histone H2B (forming a complex with RVB1 and SUG1) to regulate cotranscriptional pre-mRNA splicing and MDM2-p53 axis transcription; it stabilizes FKBP51 to negatively regulate AKT via PHLPP-mediated dephosphorylation; it removes K63-linked ubiquitin from MITA/STING to attenuate innate antiviral signaling; it deubiquitinates and stabilizes p53 forming a positive feedback loop in the DNA damage response; it deubiquitinates γH2AX to regulate 53BP1 recruitment and DSB repair pathway choice; and it stabilizes additional substrates including DUSP1, A3G, BAG2, SIRT1, GRPR, PKMYT1, RPA70, and HDAC3 in various cellular contexts. USP49 itself is targeted for proteasomal degradation by Fbxo45/NEK6 and FBXO2 E3 ligase complexes, and undergoes ATM-promoted liquid-liquid phase separation at DSBs.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP49 is a deubiquitinating enzyme that acts both at chromatin and on individual protein substrates to control gene expression, genome integrity, and signaling [#0, #1]. Its founding activity is the specific removal of monoubiquitin from histone H2B as part of a complex with RVB1 and SUG1, which controls cotranscriptional splicing of a large set of exons and modulates nucleosome stability [#0]. This chromatin-directed H2B deubiquitination extends to gene-specific regulation: USP49 binds the MDM2 promoter and removes H2Bub there, restraining MDM2 transcription and thereby de-repressing p53 target genes [#9]. Beyond chromatin, USP49 stabilizes diverse substrates by reversing their ubiquitination — it deubiquitinates and stabilizes FKBP51 to promote PHLPP-mediated dephosphorylation of AKT-Ser473 [#1], removes K63-linked ubiquitin from MITA/STING to block TBK1 recruitment and dampen type I interferon responses [#4], and stabilizes APOBEC3G to enhance its anti-HIV-1 activity [#5]. USP49 also engages the DNA damage response, interacting with and deubiquitinating p53 in a p53-induced positive feedback loop, with Usp49-knockout mice showing increased susceptibility to colon tumors [#2]. Functionally, loss of USP49 impairs proliferation, increases aneuploidy, and permits override of the nocodazole-induced spindle assembly checkpoint [#11]. USP49 protein level is itself controlled by ubiquitin-proteasome turnover through the Fbxo45/NEK6 axis [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established USP49 as a histone H2B-specific deubiquitinase and linked this chromatin activity to a cellular process — cotranscriptional pre-mRNA splicing.\",\n      \"evidence\": \"Biochemical purification, in vitro DUB assay, Co-IP defining an RVB1/SUG1 complex, and ChIP/RNA-seq after knockdown\",\n      \"pmids\": [\"23824326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of H2B recognition not resolved\", \"How splicing factor association is mechanistically coupled to uH2B removal not fully defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended USP49 activity beyond chromatin to a signaling substrate, showing it stabilizes FKBP51 to suppress AKT activation.\",\n      \"evidence\": \"Reciprocal Co-IP, in vivo ubiquitination assay, and pathway epistasis with AKT-Ser473 readout in pancreatic cancer cells\",\n      \"pmids\": [\"28363942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP49 directly deubiquitinates FKBP51 in vitro not shown\", \"Ubiquitin linkage specificity on FKBP51 not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed USP49 in the DNA damage response as a p53 stabilizer within a feed-forward loop, with tumor-suppressive consequences in vivo.\",\n      \"evidence\": \"DUB library screen, Co-IP, in vivo ubiquitination assay, and AOM/DSS tumorigenesis in Usp49-knockout mice\",\n      \"pmids\": [\"29748582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro deubiquitination of p53 not reconstituted\", \"Relationship to chromatin-based MDM2 regulation not integrated at this stage\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed substrate stabilization as a recurring mode, with USP49 protecting DUSP1 to restrain JNK signaling and apoptosis in cardiomyocytes.\",\n      \"evidence\": \"Co-IP, ubiquitination analysis, and JNK/apoptosis readouts with an in vivo rat I/R model\",\n      \"pmids\": [\"30246457\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro deubiquitination reconstitution\", \"Tissue specificity of the DUSP1 interaction not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a linkage-specific role in innate immunity, with USP49 stripping K63 chains from MITA/STING to limit antiviral signaling.\",\n      \"evidence\": \"Reciprocal Co-IP, K63-linkage deubiquitination assay, MITA aggregation and TBK1 recruitment assays, and Usp49-/- mice resistant to HSV-1\",\n      \"pmids\": [\"30943264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same complex partners (RVB1/SUG1) participate not addressed\", \"Structural mechanism of MITA aggregation control unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed USP49 can promote antiviral restriction by stabilizing APOBEC3G against a Vif/cullin-independent degradation route.\",\n      \"evidence\": \"Co-IP, in vitro deubiquitination assay, and A3G stability/HIV replication readouts\",\n      \"pmids\": [\"31397674\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the Vif-independent degradation pathway not defined\", \"Physiological context in primary cells not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected USP49 chromatin H2B-DUB activity to gene-specific transcriptional control of the p53 axis via the MDM2 promoter.\",\n      \"evidence\": \"ChIP and H2Bub ChIP at MDM2, RT-PCR of p53 targets, and KO/overexpression in HCT116\",\n      \"pmids\": [\"35072515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How USP49 is recruited to specific promoters unknown\", \"Generality across other promoters not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the mechanism controlling USP49 abundance, showing an Fbxo45 SCF complex enhanced by NEK6 drives its K48-linked proteasomal degradation.\",\n      \"evidence\": \"Co-IP with SPRY-domain mapping, ubiquitination assay, cycloheximide chase, NEK6 kinase assay, and xenograft\",\n      \"pmids\": [\"35279684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signals controlling Fbxo45 engagement of USP49 unknown\", \"Whether degradation is context- or substrate-dependent unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked USP49 to DSB repair pathway choice by showing it deubiquitinates K15-ubiquitylated gamma-H2AX and limits 53BP1 recruitment.\",\n      \"evidence\": \"Catalytic-dead mutant interaction/colocalization, ubiquitination assay, 53BP1 foci assay, and proteasome inhibitor analysis\",\n      \"pmids\": [\"35598681\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous timing of gamma-H2AX deubiquitination during repair not resolved\", \"Balance with the p53-stabilizing role not reconciled\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed USP49 stabilizes BAG2 downstream of c-MYC transcriptional activation to promote colorectal cancer survival and chemoresistance.\",\n      \"evidence\": \"Promoter analysis of c-MYC binding, Co-IP, ubiquitination assay, and proliferation/chemoresistance readouts\",\n      \"pmids\": [\"35367823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro deubiquitination reconstitution\", \"Direct versus indirect stabilization of BAG2 not separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined a cell-division phenotype for USP49 loss — proliferation defects, aneuploidy, and SAC override — and expanded its interactome.\",\n      \"evidence\": \"siRNA/shRNA knockdown, nocodazole SAC and aneuploidy assays, and MS-based interactome (ribophorin 1, USP44, centrins)\",\n      \"pmids\": [\"36702832\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate mediating the SAC phenotype not identified\", \"Functional significance of new interactors not validated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed a DSB-localized condensate model in which ATM-driven LLPS recruits USP49 to deubiquitinate RPA70 and promote homologous recombination.\",\n      \"evidence\": \"LLPS assay, IR-induced foci, Co-IP, ubiquitination assay, and HR repair readouts in ESCC with xenograft\",\n      \"pmids\": [\"40460957\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"LLPS and HR claims await independent validation\", \"Reconciliation with the earlier 53BP1-restraining DSB role unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP49 selects among its many candidate substrates across chromatin, signaling, immunity, and DNA repair contexts — and which interactions are direct, in vitro-reconstituted deubiquitination events versus inferred stabilizations — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Substrate-targeting determinants and recruitment mechanisms undefined\", \"Many proposed substrates rest on Co-IP plus cellular ubiquitination assays without enzymatic reconstitution\", \"No structural model of USP49 with any substrate\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4, 5, 8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [\"USP49-RVB1-SUG1 H2B-deubiquitinase complex\"],\n    \"partners\": [\"RVB1\", \"SUG1\", \"FKBP51\", \"STING1\", \"APOBEC3G\", \"TP53\", \"MDM2\", \"FBXO45\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}