{"gene":"TRIM35","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":2014,"finding":"TRIM35 interacts with pyruvate kinase isoform M2 (PKM2) via its coiled-coil domain, and suppresses PKM2 Y105 phosphorylation, thereby reducing the Warburg effect and tumorigenicity in hepatocellular carcinoma cells.","method":"Mass spectrometry, Co-IP, coiled-coil domain deletion mutants, in vitro phosphorylation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (MS, Co-IP, domain mutagenesis, functional readout) in single study","pmids":["25263439"],"is_preprint":false},{"year":2015,"finding":"TRIM35 negatively regulates TLR7/9-mediated type I interferon production by interacting with IRF7 and promoting its K48-linked ubiquitination and proteasomal degradation, acting as a negative feedback regulator.","method":"Co-IP, ubiquitination assay, proteasome inhibitor experiments, knockdown/overexpression with IFN-β reporter","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus ubiquitination assay with defined linkage and functional readout","pmids":["25907537"],"is_preprint":false},{"year":2003,"finding":"HLS5 (mouse ortholog of TRIM35) localizes to cytoplasmic granules and punctate nuclear bodies, and enforced expression inhibits cell growth, clonogenicity, and tumorigenicity in HeLa cells.","method":"Subcellular localization by microscopy, colony formation assay, xenograft tumor model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct localization experiment with functional consequence, but limited mechanistic detail","pmids":["14662771"],"is_preprint":false},{"year":2007,"finding":"HLS5 (mouse ortholog of TRIM35) relocates from cytoplasmic granules to the nucleus under GATA-1 influence, associates with FOG-1 and GATA-1, suppresses GATA-1-mediated transactivation, and reduces GATA-1 binding to DNA, thereby impeding erythroid maturation.","method":"Fluorescence microscopy (localization), Co-IP, chromatin/DNA binding assays, reporter transactivation assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, reporter assay, localization) with defined functional phenotype","pmids":["18063753"],"is_preprint":false},{"year":2022,"finding":"TRIM35 acts as an E3 ubiquitin ligase that ubiquitinates PKM2, regulating the transition between PKM2 tetramer and dimer forms, thereby suppressing the Warburg effect in breast cancer cells.","method":"Ubiquitination assay, Co-IP, overexpression/knockdown with metabolic readouts (glucose uptake, lactate), xenograft model","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assay with functional metabolic readout, single lab","pmids":["36196894"],"is_preprint":false},{"year":2022,"finding":"TRIM35 functions as an E3 ubiquitin ligase to ubiquitinate and degrade PDK1, resulting in inactivation of AKT signaling and promotion of apoptosis in breast cancer cells.","method":"Ubiquitination assay, co-IP, overexpression/knockdown with apoptosis and proliferation readouts, xenograft model","journal":"Neoplasma","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assay with defined substrate and functional pathway placement, single lab","pmids":["35081724"],"is_preprint":false},{"year":2022,"finding":"TRIM35 (as an E3 ubiquitin ligase) promotes K48-linked ubiquitination and proteasomal degradation of nuclear PKM2 (S37P-PKM2) in cardiomyocytes, leading to destabilization of GATA4/6 and increased P53, resulting in heart failure. Cardiomyocyte-specific TRIM35 overexpression in mice recapitulates dilated cardiac dysfunction.","method":"Cardiomyocyte-specific transgenic/knockout mouse models, Co-IP, ubiquitination assay, immunofluorescence, echocardiography","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1-2 — in vivo mouse models with multiple molecular readouts (Co-IP, ubiquitination), mechanistic rescue experiments, translational patient data","pmids":["36322626"],"is_preprint":false},{"year":2021,"finding":"TRIM35 functions as an E3 ubiquitin ligase to mediate ubiquitination and proteasomal degradation of CLOCK, a circadian regulator, thereby suppressing DLBCL cell proliferation and modulating NK cell infiltration in the tumor microenvironment.","method":"Co-IP, ubiquitination assay, overexpression with proliferation and immune infiltration readouts","journal":"Journal of immunology research","confidence":"Medium","confidence_rationale":"Tier 2-3 — ubiquitination assay with defined substrate and in vivo immune phenotype, single lab","pmids":["34124276"],"is_preprint":false},{"year":2022,"finding":"TRIM35 interacts with TIGAR and promotes its polyubiquitination and proteasomal degradation; TRIM35 knockdown alleviates renal ischemia-reperfusion injury by inhibiting oxidative stress and enhancing mitochondrial fusion.","method":"Co-IP, ubiquitination assay, knockdown with mitochondrial fusion and oxidative stress readouts","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assay with defined functional readout, single lab","pmids":["35421414"],"is_preprint":false},{"year":2023,"finding":"TRIM35 mediates K63-linked ubiquitination of LSD1 at lysine 422, repressing LSD1 demethylase activity; suppressed LSD1 facilitates ERGIC1 transcription, autophagy inhibition, IFNGR1 stabilization, and enhanced IFN-γ signaling, increasing MHC class I expression and immune surveillance in NSCLC.","method":"Co-IP, ubiquitination assay with linkage specificity, site-directed mutagenesis (K422), demethylase activity assay, gene expression and flow cytometry","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — site-specific ubiquitination with enzymatic activity assay, multiple downstream pathway validations, mechanistic cascade defined","pmids":["37979167"],"is_preprint":false},{"year":2023,"finding":"TRIM35 interacts with adenovirus E1A protein and promotes its K48-linked ubiquitination and degradation, with K253 and K285 of E1A identified as key ubiquitination sites, thereby inhibiting adenovirus replication.","method":"Co-IP, ubiquitination assay, site-directed mutagenesis of E1A (K253A, K285A), viral replication assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1-2 — Co-IP, ubiquitination with defined sites by mutagenesis, functional antiviral readout","pmids":["37578239"],"is_preprint":false},{"year":2021,"finding":"Porcine TRIM35 directly interacts with TRAF3 and catalyzes its K63-linked polyubiquitination, promoting IFN-β production; the RING and PRY/SPRY domains are essential for E3 ligase activity.","method":"Co-IP, K63-linked ubiquitination assay, domain deletion mutants, IFN-β reporter assay","journal":"Developmental and comparative immunology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus ubiquitination assay with domain mapping, porcine ortholog consistent with mammalian gene","pmids":["34626690"],"is_preprint":false},{"year":2024,"finding":"TRIM35 negatively regulates the cGAS-STING signaling pathway by directly interacting with STING in the cytoplasm and removing K63-linked ubiquitin from STING through its RING domain (C36 and C44 sites), impairing STING interaction with TBK1/IKKε and attenuating IFN-β production.","method":"Co-IP, colocalization, ubiquitination/deubiquitination assay, RING domain mutagenesis (C36A, C44A), TBK1/IKKε interaction assays","journal":"Inflammation","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic dissection with domain mutagenesis, ubiquitin linkage specificity, and defined molecular interaction disrupted","pmids":["39088122"],"is_preprint":false},{"year":2024,"finding":"TRIM35 directly monoubiquitinates lysine-120 (K120) on histone H2B in postnatal mature cardiomyocytes, promoting chromatin remodeling and accessibility of P53 to its transcriptional promoter targets, leading to increased P53 transcriptional activity and heart failure.","method":"ChIP-seq, RNA-seq, in vitro ubiquitination assay, TRIM35 transgenic mouse model, patient dilated cardiomyopathy samples","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitination with specific residue identified, ChIP-seq for chromatin remodeling, in vivo transgenic validation, and translational patient data","pmids":["38860363"],"is_preprint":false},{"year":2024,"finding":"TRIM35 interacts with and ubiquitinates the amino acid transporter SLC7A5, up-regulating it to enhance amino acid transport and activate mTORC1 signaling in cardiac fibroblasts, promoting fibroblast proliferation, migration, and differentiation, and inducing cardiac remodeling.","method":"LC-MS/MS, Co-IP, ubiquitination assay, adenoviral/AAV overexpression, fibroblast-specific Trim35 knockout mice (TAC model), mTORC1 inhibitor (Rapamycin) rescue","journal":"Cell communication and signaling","confidence":"High","confidence_rationale":"Tier 1-2 — proteomic identification of substrate, Co-IP, ubiquitination assay, in vivo conditional KO with functional rescue","pmids":["39304904"],"is_preprint":false},{"year":2023,"finding":"TRIM35 interacts with IRF5 and promotes its ubiquitination and degradation, thereby suppressing IRF5-driven glycolysis (via LDHA) in hepatocellular carcinoma.","method":"Co-IP, ubiquitination assay, knockdown with metabolite and glycolysis readouts, IHC in patient samples","journal":"Journal of digestive diseases","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP and ubiquitination assay with defined functional consequence, single lab","pmids":["37594849"],"is_preprint":false},{"year":2025,"finding":"TRIM35 directly binds genomic promoters as a DNA-binding protein, interacts with histone H3, catalyzes its non-proteolytic ubiquitination, which recruits p300 for H3K27 acetylation, activating transcription of HSPA6 and suppressing breast cancer progression.","method":"ChIP-PCR, Co-IP, ubiquitination assay, p300 recruitment assay, H3K27 acetylation ChIP, overexpression functional assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (ChIP, Co-IP, ubiquitination, epigenetic readout) but single lab and novel claim","pmids":["41136372"],"is_preprint":false},{"year":2025,"finding":"Endothelial TRIM35 inhibits MMP10 expression and secretion by promoting K63-linked ubiquitination of RelB and maintaining its nuclear localization, suppressing noncanonical NF-κB-driven MMP10 transcription and thereby inhibiting vascular graft calcification.","method":"Single-cell RNA-seq, conditional endothelial TRIM35 knockout mice (arterial isograft model), Co-IP, ubiquitination assay, nuclear fractionation, in vitro coculture calcification assay","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1-2 — in vivo conditional KO with defined phenotypic rescue, Co-IP plus ubiquitination assay with linkage specificity and nuclear localization functional consequence","pmids":["39865905"],"is_preprint":false},{"year":2025,"finding":"TRIM35 interacts with Wnt3a (via ubiquitination) and regulates glucose metabolism in pulmonary artery endothelial cells; tsRNA-3040b suppresses TRIM35 transcription by promoting R-loop formation at its genomic locus, leading to disordered glycolysis and PAECs proliferation.","method":"ChIP-PCR, Dot-blot (R-loop detection), Co-IP, endogenous co-precipitation, molecular docking, enzyme activity assays for glycolytic enzymes","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with ubiquitination substrate, R-loop validated by orthogonal methods, but mechanistic chain has multiple steps in single lab","pmids":["41351040"],"is_preprint":false},{"year":2025,"finding":"TRIM35 ubiquitinates and promotes degradation of EIF3E, modulating the CDK4/Cyclin D1 signaling pathway to suppress proliferation, invasion, and migration of endometrial cancer cells.","method":"Co-IP, ubiquitination assay, overexpression/knockdown with proliferation, invasion, and cell cycle readouts; EIF3E rescue experiment","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination with defined pathway readout and rescue, single lab","pmids":["41429342"],"is_preprint":false}],"current_model":"TRIM35 is a RING-domain E3 ubiquitin ligase (and deubiquitinase) that regulates multiple substrates—including PKM2, IRF7, IRF5, LSD1, TRAF3, STING, TIGAR, CLOCK, PDK1, SLC7A5, RelB, E1A, EIF3E, H2B-K120, and histone H3—through K48-linked proteasomal degradation, K63-linked non-degradative ubiquitination, or monoubiquitination, thereby controlling cancer metabolism (Warburg effect), innate immune/interferon signaling, cardiac remodeling, and epigenetic gene regulation."},"narrative":{"teleology":[{"year":2003,"claim":"The initial characterization of HLS5/TRIM35 established it as a cytoplasmic- and nuclear-body-localized protein with growth-suppressive and anti-tumorigenic activity, setting the stage for identifying its molecular substrates.","evidence":"Microscopy and colony formation/xenograft assays in HeLa cells","pmids":["14662771"],"confidence":"Medium","gaps":["No molecular substrate or E3 ligase activity identified","Mechanism of growth suppression unknown"]},{"year":2007,"claim":"Discovery that HLS5/TRIM35 dynamically relocalizes from cytoplasmic granules to the nucleus under GATA-1 influence and suppresses GATA-1 transactivation by reducing its DNA binding revealed TRIM35 as a context-dependent transcriptional modulator in erythroid differentiation.","evidence":"Co-IP of HLS5 with GATA-1/FOG-1, reporter transactivation assays, DNA-binding assays in erythroid cells","pmids":["18063753"],"confidence":"High","gaps":["Whether TRIM35 ubiquitinates GATA-1 or FOG-1 was not tested","Physiological role in erythropoiesis in vivo not established"]},{"year":2014,"claim":"Identification of PKM2 as a direct TRIM35-interacting partner via its coiled-coil domain established the first mechanistic link between TRIM35 and cancer metabolism, showing that TRIM35 suppresses PKM2 Y105 phosphorylation and the Warburg effect in hepatocellular carcinoma.","evidence":"Mass spectrometry, Co-IP, coiled-coil domain deletion mutants, in vitro phosphorylation assays in HCC cells","pmids":["25263439"],"confidence":"High","gaps":["Whether TRIM35 directly ubiquitinates PKM2 was not yet demonstrated","Upstream regulation of TRIM35 expression unknown"]},{"year":2015,"claim":"Demonstration that TRIM35 catalyzes K48-linked ubiquitination of IRF7 for proteasomal degradation defined TRIM35 as a bona fide E3 ubiquitin ligase and revealed its role as a negative feedback regulator of TLR7/9-mediated type I interferon production.","evidence":"Co-IP, K48-linkage-specific ubiquitination assay, proteasome inhibitor rescue, IFN-β reporter in TLR-stimulated cells","pmids":["25907537"],"confidence":"High","gaps":["In vivo relevance for antiviral immunity not shown","Structural basis for IRF7 recognition unknown"]},{"year":2021,"claim":"Extension to innate immune signaling via porcine TRAF3 (K63-linked ubiquitination promoting IFN-β) and to cancer biology via CLOCK degradation in DLBCL showed TRIM35 can employ both K48 and K63 linkages on distinct substrates to achieve opposing outcomes on immune and proliferative pathways.","evidence":"Co-IP, linkage-specific ubiquitination assays, domain deletion mutants (RING, PRY/SPRY), IFN-β reporter, immune infiltration analysis","pmids":["34626690","34124276"],"confidence":"Medium","gaps":["TRAF3 work in porcine system not validated in human cells","CLOCK ubiquitination linkage type not specified","In vivo immune phenotype for TRAF3 not tested"]},{"year":2022,"claim":"A suite of studies identified additional TRIM35 degradation substrates—PKM2 (now shown to be directly ubiquitinated), PDK1, and TIGAR—demonstrating TRIM35's broad role in controlling cancer metabolism, AKT signaling, and mitochondrial homeostasis through K48-linked proteasomal pathways.","evidence":"Ubiquitination assays, Co-IP, metabolic readouts, apoptosis assays, xenograft models (breast cancer), renal ischemia-reperfusion injury models","pmids":["36196894","35081724","35421414"],"confidence":"Medium","gaps":["Each substrate studied by a single lab without independent replication","Structural basis for multi-substrate recognition unresolved","TIGAR ubiquitination linkage type not specified"]},{"year":2022,"claim":"In vivo demonstration that TRIM35 ubiquitinates nuclear PKM2 (S37-phosphorylated form) at K48 linkage in cardiomyocytes, destabilizing GATA4/6 and activating P53 to cause dilated cardiomyopathy, established TRIM35 as a pathogenic driver in heart failure using cardiomyocyte-specific transgenic/knockout mouse models and patient samples.","evidence":"Cardiomyocyte-specific TRIM35 overexpression and KO mice, echocardiography, Co-IP, ubiquitination assay, patient DCM samples","pmids":["36322626"],"confidence":"High","gaps":["Whether cardiac TRIM35 upregulation is cause or consequence in human DCM requires longitudinal data","Therapeutic targetability not explored"]},{"year":2023,"claim":"Discovery that TRIM35 catalyzes K63-linked ubiquitination of LSD1 at K422 to repress its demethylase activity, leading to ERGIC1-dependent autophagy inhibition and IFNGR1 stabilization that enhances MHC-I-mediated immune surveillance, connected TRIM35's epigenetic and immune functions in a single mechanistic cascade in NSCLC.","evidence":"Site-directed mutagenesis (K422), linkage-specific ubiquitination, LSD1 demethylase activity assay, flow cytometry for MHC-I","pmids":["37979167"],"confidence":"High","gaps":["In vivo anti-tumor immune efficacy not tested with TRIM35 modulation","Whether LSD1 K63-ubiquitination is reversible by a specific DUB unknown"]},{"year":2023,"claim":"Identification of IRF5 as a TRIM35 substrate linked ubiquitin-mediated IRF5 degradation to suppression of LDHA-driven glycolysis in HCC, extending TRIM35's metabolic regulatory reach to IRF family members beyond IRF7.","evidence":"Co-IP, ubiquitination assay, glycolysis metabolite readouts, patient IHC in HCC","pmids":["37594849"],"confidence":"Medium","gaps":["Single lab, not independently replicated","IRF5 ubiquitination linkage and site not mapped"]},{"year":2023,"claim":"TRIM35 was shown to target adenovirus E1A oncoprotein at K253 and K285 for K48-linked ubiquitination and degradation, establishing a direct antiviral effector function.","evidence":"Co-IP, ubiquitination with E1A lysine mutants (K253A, K285A), viral replication assays","pmids":["37578239"],"confidence":"High","gaps":["Whether TRIM35 is IFN-induced during adenovirus infection not determined","Breadth of antiviral activity beyond adenovirus untested"]},{"year":2024,"claim":"TRIM35 was found to directly monoubiquitinate histone H2B at K120 in cardiomyocytes, opening chromatin for P53 transcriptional activation and contributing to heart failure, establishing TRIM35 as a histone-modifying E3 ligase with epigenetic function in vivo.","evidence":"In vitro ubiquitination assay (H2BK120), ChIP-seq, RNA-seq, TRIM35 transgenic mice, patient DCM samples","pmids":["38860363"],"confidence":"High","gaps":["Whether TRIM35 acts as a general H2BK120 ubiquitin ligase outside the heart is unknown","Relationship to canonical H2BK120 writers (RNF20/40) not addressed"]},{"year":2024,"claim":"Discovery that TRIM35 removes K63-linked ubiquitin from STING via its RING domain (C36/C44), disrupting STING–TBK1/IKKε interaction and attenuating cGAS-STING-mediated IFN-β production, revealed an unexpected deubiquitinase-like activity for this RING E3 ligase.","evidence":"Co-IP, colocalization, deubiquitination assay, RING domain mutagenesis (C36A, C44A), TBK1/IKKε interaction mapping","pmids":["39088122"],"confidence":"High","gaps":["Deubiquitinase mechanism for a RING E3 ligase is biochemically unusual and requires reconstitution with purified components","Whether this is direct catalytic DUB activity or facilitated by a DUB partner is unresolved"]},{"year":2024,"claim":"In cardiac fibroblasts, TRIM35 ubiquitinates the amino acid transporter SLC7A5, upregulating it to activate mTORC1 signaling and promote fibroblast-driven cardiac remodeling, extending TRIM35's cardiac pathology from cardiomyocytes to fibroblasts.","evidence":"LC-MS/MS substrate identification, Co-IP, fibroblast-specific Trim35 KO mice under TAC, rapamycin rescue","pmids":["39304904"],"confidence":"High","gaps":["How TRIM35-mediated ubiquitination upregulates (rather than degrades) SLC7A5 is mechanistically unclear","Whether non-degradative ubiquitination stabilizes or traffics SLC7A5 not resolved"]},{"year":2025,"claim":"TRIM35 was shown to ubiquitinate histone H3 non-proteolytically, recruiting p300 for H3K27 acetylation and activating HSPA6 transcription, and separately to ubiquitinate RelB via K63 linkage to maintain its nuclear retention and suppress NF-κB-driven vascular calcification, consolidating TRIM35's role as an epigenetic and transcriptional regulator beyond degradation.","evidence":"ChIP-PCR, Co-IP, H3K27ac ChIP, p300 recruitment assay (breast cancer); conditional endothelial TRIM35 KO mice in arterial isograft model, nuclear fractionation (vascular calcification)","pmids":["41136372","39865905"],"confidence":"Medium","gaps":["H3 ubiquitination site not mapped to a specific lysine","H3 ubiquitination claim from single lab awaits independent replication","How TRIM35 directly binds DNA promoters despite lacking a canonical DNA-binding domain is unexplained"]},{"year":null,"claim":"Key unresolved questions include the structural basis for TRIM35's remarkably broad substrate repertoire, how it switches between K48-linked degradative, K63-linked non-degradative, monoubiquitinating, and apparent deubiquitinating activities, and whether TRIM35 itself is regulated by post-translational modification or cofactor association in a tissue-specific manner.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure available","Mechanism of activity-type switching (K48 vs K63 vs monoUb vs deubiquitination) unknown","Upstream regulatory inputs controlling TRIM35 expression and activity not systematically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,4,5,6,7,8,9,10,11,12,13,14,15,16,17,19]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,4,5,6,7,9,10,11,13,14,17,19]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[13,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3,16]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3,6,13,16,17]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,12]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,9,11,12]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,4,6,15,18]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4,5,6,7,9,10,13,14,19]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[13,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,13,14]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3,16,17]}],"complexes":[],"partners":["PKM2","IRF7","LSD1","STING","TRAF3","SLC7A5","RELB","GATA1"],"other_free_text":[]},"mechanistic_narrative":"TRIM35 is a RING-domain E3 ubiquitin ligase that functions as a broad regulator of cancer metabolism, innate immune signaling, cardiac remodeling, and epigenetic transcription through ubiquitin-dependent control of diverse substrates. It catalyzes K48-linked polyubiquitination and proteasomal degradation of metabolic regulators (PKM2, IRF5, PDK1, EIF3E, CLOCK, TIGAR) and immune signaling factors (IRF7, adenovirus E1A), suppressing glycolysis/Warburg effect and modulating interferon responses [PMID:25263439, PMID:36196894, PMID:25907537, PMID:37578239, PMID:35081724, PMID:37594849]. TRIM35 also mediates K63-linked non-degradative ubiquitination of LSD1 (repressing its demethylase activity to enhance IFN-γ signaling), TRAF3 (promoting IFN-β production), and RelB (maintaining nuclear retention to suppress NF-κB–driven MMP10 transcription), and removes K63-linked ubiquitin from STING to attenuate cGAS-STING signaling [PMID:37979167, PMID:34626690, PMID:39865905, PMID:39088122]. In cardiomyocytes, TRIM35 monoubiquitinates histone H2B at K120 to remodel chromatin and activate P53-dependent transcription, and ubiquitinates nuclear PKM2 to destabilize GATA4/6, with cardiomyocyte-specific overexpression sufficient to induce dilated cardiomyopathy in mice [PMID:38860363, PMID:36322626]."},"prefetch_data":{"uniprot":{"accession":"Q9UPQ4","full_name":"E3 ubiquitin-protein ligase TRIM35","aliases":["Hemopoietic lineage switch protein 5"],"length_aa":493,"mass_kda":56.5,"function":"E3 ubiquitin-protein ligase that participates in multiple biological processes including cell death, glucose metabolism, and in particular, the innate immune response. Mediates 'Lys-63'-linked polyubiquitination of TRAF3 thereby promoting type I interferon production via RIG-I signaling pathway (PubMed:32562145). Can also catalyze 'Lys-48'-linked polyubiquitination and proteasomal degradation of viral proteins such as influenza virus PB2 (PubMed:32562145). Acts as a negative feedback regulator of TLR7- and TLR9-triggered signaling. Mechanistically, promotes the 'Lys-48'-linked ubiquitination of IRF7 and induces its degradation via a proteasome-dependent pathway (PubMed:25907537). Reduces FGFR1-dependent tyrosine phosphorylation of PKM, inhibiting PKM-dependent lactate production, glucose metabolism, and cell growth (PubMed:25263439)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UPQ4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRIM35","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/TRIM35","total_profiled":1310},"omim":[{"mim_id":"617007","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 35; TRIM35","url":"https://www.omim.org/entry/617007"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TRIM35"},"hgnc":{"alias_symbol":["KIAA1098","MAIR","HLS5"],"prev_symbol":[]},"alphafold":{"accession":"Q9UPQ4","domains":[{"cath_id":"3.30.40.10","chopping":"15-90","consensus_level":"medium","plddt":88.5426,"start":15,"end":90},{"cath_id":"-","chopping":"183-297","consensus_level":"high","plddt":95.3306,"start":183,"end":297},{"cath_id":"2.60.120.920","chopping":"305-375_390-479","consensus_level":"high","plddt":89.0218,"start":305,"end":479}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPQ4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPQ4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UPQ4-F1-predicted_aligned_error_v6.png","plddt_mean":88.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRIM35","jax_strain_url":"https://www.jax.org/strain/search?query=TRIM35"},"sequence":{"accession":"Q9UPQ4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UPQ4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UPQ4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UPQ4"}},"corpus_meta":[{"pmid":"12874256","id":"PMC_12874256","title":"Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.","date":"2003","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/12874256","citation_count":84,"is_preprint":false},{"pmid":"25263439","id":"PMC_25263439","title":"TRIM35 Interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinoma.","date":"2014","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/25263439","citation_count":75,"is_preprint":false},{"pmid":"25576919","id":"PMC_25576919","title":"Co-expression of PKM2 and TRIM35 predicts survival and recurrence in hepatocellular carcinoma.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/25576919","citation_count":64,"is_preprint":false},{"pmid":"28394882","id":"PMC_28394882","title":"miR-4417 Targets Tripartite Motif-Containing 35 (TRIM35) and Regulates Pyruvate Kinase Muscle 2 (PKM2) Phosphorylation to Promote Proliferation and Suppress Apoptosis in Hepatocellular Carcinoma Cells.","date":"2017","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/28394882","citation_count":33,"is_preprint":false},{"pmid":"25907537","id":"PMC_25907537","title":"TRIM35 negatively regulates TLR7- and TLR9-mediated type I interferon production by targeting IRF7.","date":"2015","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/25907537","citation_count":31,"is_preprint":false},{"pmid":"18097021","id":"PMC_18097021","title":"Caspase-independent cell death by CD300LF (MAIR-V), an inhibitory immunoglobulin-like receptor on myeloid cells.","date":"2008","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18097021","citation_count":28,"is_preprint":false},{"pmid":"14662771","id":"PMC_14662771","title":"HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14662771","citation_count":28,"is_preprint":false},{"pmid":"36196894","id":"PMC_36196894","title":"TRIM35 ubiquitination regulates the expression of PKM2 tetramer and dimer and affects the malignant behaviour of breast cancer by regulating the Warburg effect.","date":"2022","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36196894","citation_count":26,"is_preprint":false},{"pmid":"17202337","id":"PMC_17202337","title":"Dual assemblies of an activating immune receptor, MAIR-II, with ITAM-bearing adapters DAP12 and FcRgamma chain on peritoneal macrophages.","date":"2007","source":"Journal of immunology (Baltimore, Md. : 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regulates the Warburg effect of hypoxic HCC cells through miR-103a-3p/TRIM35.","date":"2024","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38642184","citation_count":1,"is_preprint":false},{"pmid":"39865905","id":"PMC_39865905","title":"Endothelial TRIM35-Regulated MMP10 Release Exacerbates Calcification of Vascular Grafts.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39865905","citation_count":1,"is_preprint":false},{"pmid":"28397257","id":"PMC_28397257","title":"Retracted: Mice lacking the Raf-1 kinase inhibitor protein exhibit exaggerated hypoxia-induced pulmonary hypertension, by I Morecroft, B Doyle, M Nilsen, W Kolch, K Mair and MR MacLean. British Journal of Pharmacology, volume 163(5): 948-963, published in June 2011; DOI 10.1111/j.1476-5381.2011.01305.x.","date":"2017","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28397257","citation_count":1,"is_preprint":false},{"pmid":"39088122","id":"PMC_39088122","title":"TRIM35 Negatively Regulates the cGAS-STING-Mediated Signaling Pathway by Attenuating K63-Linked Ubiquitination of STING.","date":"2024","source":"Inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/39088122","citation_count":0,"is_preprint":false},{"pmid":"41351040","id":"PMC_41351040","title":"tsRNA-3040b accumulates R-loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation.","date":"2025","source":"Cellular & molecular biology letters","url":"https://pubmed.ncbi.nlm.nih.gov/41351040","citation_count":0,"is_preprint":false},{"pmid":"41429342","id":"PMC_41429342","title":"TRIM35 inhibits endometrial cancer progression via ubiquitination and degradation of EIF3E.","date":"2025","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/41429342","citation_count":0,"is_preprint":false},{"pmid":"41136372","id":"PMC_41136372","title":"TRIM35, a novel DNA-binding protein, epigenetically modifies H3 to promote HSPA6 transcription and suppress breast cancer progression.","date":"2025","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/41136372","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19396,"output_tokens":4433,"usd":0.062342},"stage2":{"model":"claude-opus-4-6","input_tokens":7962,"output_tokens":4239,"usd":0.218677},"total_usd":0.281019,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"TRIM35 interacts with pyruvate kinase isoform M2 (PKM2) via its coiled-coil domain, and suppresses PKM2 Y105 phosphorylation, thereby reducing the Warburg effect and tumorigenicity in hepatocellular carcinoma cells.\",\n      \"method\": \"Mass spectrometry, Co-IP, coiled-coil domain deletion mutants, in vitro phosphorylation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (MS, Co-IP, domain mutagenesis, functional readout) in single study\",\n      \"pmids\": [\"25263439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRIM35 negatively regulates TLR7/9-mediated type I interferon production by interacting with IRF7 and promoting its K48-linked ubiquitination and proteasomal degradation, acting as a negative feedback regulator.\",\n      \"method\": \"Co-IP, ubiquitination assay, proteasome inhibitor experiments, knockdown/overexpression with IFN-β reporter\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus ubiquitination assay with defined linkage and functional readout\",\n      \"pmids\": [\"25907537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"HLS5 (mouse ortholog of TRIM35) localizes to cytoplasmic granules and punctate nuclear bodies, and enforced expression inhibits cell growth, clonogenicity, and tumorigenicity in HeLa cells.\",\n      \"method\": \"Subcellular localization by microscopy, colony formation assay, xenograft tumor model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct localization experiment with functional consequence, but limited mechanistic detail\",\n      \"pmids\": [\"14662771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HLS5 (mouse ortholog of TRIM35) relocates from cytoplasmic granules to the nucleus under GATA-1 influence, associates with FOG-1 and GATA-1, suppresses GATA-1-mediated transactivation, and reduces GATA-1 binding to DNA, thereby impeding erythroid maturation.\",\n      \"method\": \"Fluorescence microscopy (localization), Co-IP, chromatin/DNA binding assays, reporter transactivation assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, reporter assay, localization) with defined functional phenotype\",\n      \"pmids\": [\"18063753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM35 acts as an E3 ubiquitin ligase that ubiquitinates PKM2, regulating the transition between PKM2 tetramer and dimer forms, thereby suppressing the Warburg effect in breast cancer cells.\",\n      \"method\": \"Ubiquitination assay, Co-IP, overexpression/knockdown with metabolic readouts (glucose uptake, lactate), xenograft model\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay with functional metabolic readout, single lab\",\n      \"pmids\": [\"36196894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM35 functions as an E3 ubiquitin ligase to ubiquitinate and degrade PDK1, resulting in inactivation of AKT signaling and promotion of apoptosis in breast cancer cells.\",\n      \"method\": \"Ubiquitination assay, co-IP, overexpression/knockdown with apoptosis and proliferation readouts, xenograft model\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay with defined substrate and functional pathway placement, single lab\",\n      \"pmids\": [\"35081724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM35 (as an E3 ubiquitin ligase) promotes K48-linked ubiquitination and proteasomal degradation of nuclear PKM2 (S37P-PKM2) in cardiomyocytes, leading to destabilization of GATA4/6 and increased P53, resulting in heart failure. Cardiomyocyte-specific TRIM35 overexpression in mice recapitulates dilated cardiac dysfunction.\",\n      \"method\": \"Cardiomyocyte-specific transgenic/knockout mouse models, Co-IP, ubiquitination assay, immunofluorescence, echocardiography\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vivo mouse models with multiple molecular readouts (Co-IP, ubiquitination), mechanistic rescue experiments, translational patient data\",\n      \"pmids\": [\"36322626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM35 functions as an E3 ubiquitin ligase to mediate ubiquitination and proteasomal degradation of CLOCK, a circadian regulator, thereby suppressing DLBCL cell proliferation and modulating NK cell infiltration in the tumor microenvironment.\",\n      \"method\": \"Co-IP, ubiquitination assay, overexpression with proliferation and immune infiltration readouts\",\n      \"journal\": \"Journal of immunology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ubiquitination assay with defined substrate and in vivo immune phenotype, single lab\",\n      \"pmids\": [\"34124276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM35 interacts with TIGAR and promotes its polyubiquitination and proteasomal degradation; TRIM35 knockdown alleviates renal ischemia-reperfusion injury by inhibiting oxidative stress and enhancing mitochondrial fusion.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown with mitochondrial fusion and oxidative stress readouts\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assay with defined functional readout, single lab\",\n      \"pmids\": [\"35421414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM35 mediates K63-linked ubiquitination of LSD1 at lysine 422, repressing LSD1 demethylase activity; suppressed LSD1 facilitates ERGIC1 transcription, autophagy inhibition, IFNGR1 stabilization, and enhanced IFN-γ signaling, increasing MHC class I expression and immune surveillance in NSCLC.\",\n      \"method\": \"Co-IP, ubiquitination assay with linkage specificity, site-directed mutagenesis (K422), demethylase activity assay, gene expression and flow cytometry\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — site-specific ubiquitination with enzymatic activity assay, multiple downstream pathway validations, mechanistic cascade defined\",\n      \"pmids\": [\"37979167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM35 interacts with adenovirus E1A protein and promotes its K48-linked ubiquitination and degradation, with K253 and K285 of E1A identified as key ubiquitination sites, thereby inhibiting adenovirus replication.\",\n      \"method\": \"Co-IP, ubiquitination assay, site-directed mutagenesis of E1A (K253A, K285A), viral replication assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — Co-IP, ubiquitination with defined sites by mutagenesis, functional antiviral readout\",\n      \"pmids\": [\"37578239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Porcine TRIM35 directly interacts with TRAF3 and catalyzes its K63-linked polyubiquitination, promoting IFN-β production; the RING and PRY/SPRY domains are essential for E3 ligase activity.\",\n      \"method\": \"Co-IP, K63-linked ubiquitination assay, domain deletion mutants, IFN-β reporter assay\",\n      \"journal\": \"Developmental and comparative immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ubiquitination assay with domain mapping, porcine ortholog consistent with mammalian gene\",\n      \"pmids\": [\"34626690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM35 negatively regulates the cGAS-STING signaling pathway by directly interacting with STING in the cytoplasm and removing K63-linked ubiquitin from STING through its RING domain (C36 and C44 sites), impairing STING interaction with TBK1/IKKε and attenuating IFN-β production.\",\n      \"method\": \"Co-IP, colocalization, ubiquitination/deubiquitination assay, RING domain mutagenesis (C36A, C44A), TBK1/IKKε interaction assays\",\n      \"journal\": \"Inflammation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic dissection with domain mutagenesis, ubiquitin linkage specificity, and defined molecular interaction disrupted\",\n      \"pmids\": [\"39088122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM35 directly monoubiquitinates lysine-120 (K120) on histone H2B in postnatal mature cardiomyocytes, promoting chromatin remodeling and accessibility of P53 to its transcriptional promoter targets, leading to increased P53 transcriptional activity and heart failure.\",\n      \"method\": \"ChIP-seq, RNA-seq, in vitro ubiquitination assay, TRIM35 transgenic mouse model, patient dilated cardiomyopathy samples\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitination with specific residue identified, ChIP-seq for chromatin remodeling, in vivo transgenic validation, and translational patient data\",\n      \"pmids\": [\"38860363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM35 interacts with and ubiquitinates the amino acid transporter SLC7A5, up-regulating it to enhance amino acid transport and activate mTORC1 signaling in cardiac fibroblasts, promoting fibroblast proliferation, migration, and differentiation, and inducing cardiac remodeling.\",\n      \"method\": \"LC-MS/MS, Co-IP, ubiquitination assay, adenoviral/AAV overexpression, fibroblast-specific Trim35 knockout mice (TAC model), mTORC1 inhibitor (Rapamycin) rescue\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — proteomic identification of substrate, Co-IP, ubiquitination assay, in vivo conditional KO with functional rescue\",\n      \"pmids\": [\"39304904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM35 interacts with IRF5 and promotes its ubiquitination and degradation, thereby suppressing IRF5-driven glycolysis (via LDHA) in hepatocellular carcinoma.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown with metabolite and glycolysis readouts, IHC in patient samples\",\n      \"journal\": \"Journal of digestive diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and ubiquitination assay with defined functional consequence, single lab\",\n      \"pmids\": [\"37594849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM35 directly binds genomic promoters as a DNA-binding protein, interacts with histone H3, catalyzes its non-proteolytic ubiquitination, which recruits p300 for H3K27 acetylation, activating transcription of HSPA6 and suppressing breast cancer progression.\",\n      \"method\": \"ChIP-PCR, Co-IP, ubiquitination assay, p300 recruitment assay, H3K27 acetylation ChIP, overexpression functional assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, Co-IP, ubiquitination, epigenetic readout) but single lab and novel claim\",\n      \"pmids\": [\"41136372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Endothelial TRIM35 inhibits MMP10 expression and secretion by promoting K63-linked ubiquitination of RelB and maintaining its nuclear localization, suppressing noncanonical NF-κB-driven MMP10 transcription and thereby inhibiting vascular graft calcification.\",\n      \"method\": \"Single-cell RNA-seq, conditional endothelial TRIM35 knockout mice (arterial isograft model), Co-IP, ubiquitination assay, nuclear fractionation, in vitro coculture calcification assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vivo conditional KO with defined phenotypic rescue, Co-IP plus ubiquitination assay with linkage specificity and nuclear localization functional consequence\",\n      \"pmids\": [\"39865905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM35 interacts with Wnt3a (via ubiquitination) and regulates glucose metabolism in pulmonary artery endothelial cells; tsRNA-3040b suppresses TRIM35 transcription by promoting R-loop formation at its genomic locus, leading to disordered glycolysis and PAECs proliferation.\",\n      \"method\": \"ChIP-PCR, Dot-blot (R-loop detection), Co-IP, endogenous co-precipitation, molecular docking, enzyme activity assays for glycolytic enzymes\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with ubiquitination substrate, R-loop validated by orthogonal methods, but mechanistic chain has multiple steps in single lab\",\n      \"pmids\": [\"41351040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM35 ubiquitinates and promotes degradation of EIF3E, modulating the CDK4/Cyclin D1 signaling pathway to suppress proliferation, invasion, and migration of endometrial cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, overexpression/knockdown with proliferation, invasion, and cell cycle readouts; EIF3E rescue experiment\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination with defined pathway readout and rescue, single lab\",\n      \"pmids\": [\"41429342\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRIM35 is a RING-domain E3 ubiquitin ligase (and deubiquitinase) that regulates multiple substrates—including PKM2, IRF7, IRF5, LSD1, TRAF3, STING, TIGAR, CLOCK, PDK1, SLC7A5, RelB, E1A, EIF3E, H2B-K120, and histone H3—through K48-linked proteasomal degradation, K63-linked non-degradative ubiquitination, or monoubiquitination, thereby controlling cancer metabolism (Warburg effect), innate immune/interferon signaling, cardiac remodeling, and epigenetic gene regulation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TRIM35 is a RING-domain E3 ubiquitin ligase that functions as a broad regulator of cancer metabolism, innate immune signaling, cardiac remodeling, and epigenetic transcription through ubiquitin-dependent control of diverse substrates. It catalyzes K48-linked polyubiquitination and proteasomal degradation of metabolic regulators (PKM2, IRF5, PDK1, EIF3E, CLOCK, TIGAR) and immune signaling factors (IRF7, adenovirus E1A), suppressing glycolysis/Warburg effect and modulating interferon responses [PMID:25263439, PMID:36196894, PMID:25907537, PMID:37578239, PMID:35081724, PMID:37594849]. TRIM35 also mediates K63-linked non-degradative ubiquitination of LSD1 (repressing its demethylase activity to enhance IFN-γ signaling), TRAF3 (promoting IFN-β production), and RelB (maintaining nuclear retention to suppress NF-κB–driven MMP10 transcription), and removes K63-linked ubiquitin from STING to attenuate cGAS-STING signaling [PMID:37979167, PMID:34626690, PMID:39865905, PMID:39088122]. In cardiomyocytes, TRIM35 monoubiquitinates histone H2B at K120 to remodel chromatin and activate P53-dependent transcription, and ubiquitinates nuclear PKM2 to destabilize GATA4/6, with cardiomyocyte-specific overexpression sufficient to induce dilated cardiomyopathy in mice [PMID:38860363, PMID:36322626].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"The initial characterization of HLS5/TRIM35 established it as a cytoplasmic- and nuclear-body-localized protein with growth-suppressive and anti-tumorigenic activity, setting the stage for identifying its molecular substrates.\",\n      \"evidence\": \"Microscopy and colony formation/xenograft assays in HeLa cells\",\n      \"pmids\": [\"14662771\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular substrate or E3 ligase activity identified\", \"Mechanism of growth suppression unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovery that HLS5/TRIM35 dynamically relocalizes from cytoplasmic granules to the nucleus under GATA-1 influence and suppresses GATA-1 transactivation by reducing its DNA binding revealed TRIM35 as a context-dependent transcriptional modulator in erythroid differentiation.\",\n      \"evidence\": \"Co-IP of HLS5 with GATA-1/FOG-1, reporter transactivation assays, DNA-binding assays in erythroid cells\",\n      \"pmids\": [\"18063753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM35 ubiquitinates GATA-1 or FOG-1 was not tested\", \"Physiological role in erythropoiesis in vivo not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of PKM2 as a direct TRIM35-interacting partner via its coiled-coil domain established the first mechanistic link between TRIM35 and cancer metabolism, showing that TRIM35 suppresses PKM2 Y105 phosphorylation and the Warburg effect in hepatocellular carcinoma.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, coiled-coil domain deletion mutants, in vitro phosphorylation assays in HCC cells\",\n      \"pmids\": [\"25263439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM35 directly ubiquitinates PKM2 was not yet demonstrated\", \"Upstream regulation of TRIM35 expression unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstration that TRIM35 catalyzes K48-linked ubiquitination of IRF7 for proteasomal degradation defined TRIM35 as a bona fide E3 ubiquitin ligase and revealed its role as a negative feedback regulator of TLR7/9-mediated type I interferon production.\",\n      \"evidence\": \"Co-IP, K48-linkage-specific ubiquitination assay, proteasome inhibitor rescue, IFN-β reporter in TLR-stimulated cells\",\n      \"pmids\": [\"25907537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance for antiviral immunity not shown\", \"Structural basis for IRF7 recognition unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extension to innate immune signaling via porcine TRAF3 (K63-linked ubiquitination promoting IFN-β) and to cancer biology via CLOCK degradation in DLBCL showed TRIM35 can employ both K48 and K63 linkages on distinct substrates to achieve opposing outcomes on immune and proliferative pathways.\",\n      \"evidence\": \"Co-IP, linkage-specific ubiquitination assays, domain deletion mutants (RING, PRY/SPRY), IFN-β reporter, immune infiltration analysis\",\n      \"pmids\": [\"34626690\", \"34124276\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRAF3 work in porcine system not validated in human cells\", \"CLOCK ubiquitination linkage type not specified\", \"In vivo immune phenotype for TRAF3 not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A suite of studies identified additional TRIM35 degradation substrates—PKM2 (now shown to be directly ubiquitinated), PDK1, and TIGAR—demonstrating TRIM35's broad role in controlling cancer metabolism, AKT signaling, and mitochondrial homeostasis through K48-linked proteasomal pathways.\",\n      \"evidence\": \"Ubiquitination assays, Co-IP, metabolic readouts, apoptosis assays, xenograft models (breast cancer), renal ischemia-reperfusion injury models\",\n      \"pmids\": [\"36196894\", \"35081724\", \"35421414\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each substrate studied by a single lab without independent replication\", \"Structural basis for multi-substrate recognition unresolved\", \"TIGAR ubiquitination linkage type not specified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"In vivo demonstration that TRIM35 ubiquitinates nuclear PKM2 (S37-phosphorylated form) at K48 linkage in cardiomyocytes, destabilizing GATA4/6 and activating P53 to cause dilated cardiomyopathy, established TRIM35 as a pathogenic driver in heart failure using cardiomyocyte-specific transgenic/knockout mouse models and patient samples.\",\n      \"evidence\": \"Cardiomyocyte-specific TRIM35 overexpression and KO mice, echocardiography, Co-IP, ubiquitination assay, patient DCM samples\",\n      \"pmids\": [\"36322626\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cardiac TRIM35 upregulation is cause or consequence in human DCM requires longitudinal data\", \"Therapeutic targetability not explored\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that TRIM35 catalyzes K63-linked ubiquitination of LSD1 at K422 to repress its demethylase activity, leading to ERGIC1-dependent autophagy inhibition and IFNGR1 stabilization that enhances MHC-I-mediated immune surveillance, connected TRIM35's epigenetic and immune functions in a single mechanistic cascade in NSCLC.\",\n      \"evidence\": \"Site-directed mutagenesis (K422), linkage-specific ubiquitination, LSD1 demethylase activity assay, flow cytometry for MHC-I\",\n      \"pmids\": [\"37979167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo anti-tumor immune efficacy not tested with TRIM35 modulation\", \"Whether LSD1 K63-ubiquitination is reversible by a specific DUB unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of IRF5 as a TRIM35 substrate linked ubiquitin-mediated IRF5 degradation to suppression of LDHA-driven glycolysis in HCC, extending TRIM35's metabolic regulatory reach to IRF family members beyond IRF7.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, glycolysis metabolite readouts, patient IHC in HCC\",\n      \"pmids\": [\"37594849\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, not independently replicated\", \"IRF5 ubiquitination linkage and site not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"TRIM35 was shown to target adenovirus E1A oncoprotein at K253 and K285 for K48-linked ubiquitination and degradation, establishing a direct antiviral effector function.\",\n      \"evidence\": \"Co-IP, ubiquitination with E1A lysine mutants (K253A, K285A), viral replication assays\",\n      \"pmids\": [\"37578239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM35 is IFN-induced during adenovirus infection not determined\", \"Breadth of antiviral activity beyond adenovirus untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"TRIM35 was found to directly monoubiquitinate histone H2B at K120 in cardiomyocytes, opening chromatin for P53 transcriptional activation and contributing to heart failure, establishing TRIM35 as a histone-modifying E3 ligase with epigenetic function in vivo.\",\n      \"evidence\": \"In vitro ubiquitination assay (H2BK120), ChIP-seq, RNA-seq, TRIM35 transgenic mice, patient DCM samples\",\n      \"pmids\": [\"38860363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM35 acts as a general H2BK120 ubiquitin ligase outside the heart is unknown\", \"Relationship to canonical H2BK120 writers (RNF20/40) not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that TRIM35 removes K63-linked ubiquitin from STING via its RING domain (C36/C44), disrupting STING–TBK1/IKKε interaction and attenuating cGAS-STING-mediated IFN-β production, revealed an unexpected deubiquitinase-like activity for this RING E3 ligase.\",\n      \"evidence\": \"Co-IP, colocalization, deubiquitination assay, RING domain mutagenesis (C36A, C44A), TBK1/IKKε interaction mapping\",\n      \"pmids\": [\"39088122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deubiquitinase mechanism for a RING E3 ligase is biochemically unusual and requires reconstitution with purified components\", \"Whether this is direct catalytic DUB activity or facilitated by a DUB partner is unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In cardiac fibroblasts, TRIM35 ubiquitinates the amino acid transporter SLC7A5, upregulating it to activate mTORC1 signaling and promote fibroblast-driven cardiac remodeling, extending TRIM35's cardiac pathology from cardiomyocytes to fibroblasts.\",\n      \"evidence\": \"LC-MS/MS substrate identification, Co-IP, fibroblast-specific Trim35 KO mice under TAC, rapamycin rescue\",\n      \"pmids\": [\"39304904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TRIM35-mediated ubiquitination upregulates (rather than degrades) SLC7A5 is mechanistically unclear\", \"Whether non-degradative ubiquitination stabilizes or traffics SLC7A5 not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"TRIM35 was shown to ubiquitinate histone H3 non-proteolytically, recruiting p300 for H3K27 acetylation and activating HSPA6 transcription, and separately to ubiquitinate RelB via K63 linkage to maintain its nuclear retention and suppress NF-κB-driven vascular calcification, consolidating TRIM35's role as an epigenetic and transcriptional regulator beyond degradation.\",\n      \"evidence\": \"ChIP-PCR, Co-IP, H3K27ac ChIP, p300 recruitment assay (breast cancer); conditional endothelial TRIM35 KO mice in arterial isograft model, nuclear fractionation (vascular calcification)\",\n      \"pmids\": [\"41136372\", \"39865905\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"H3 ubiquitination site not mapped to a specific lysine\", \"H3 ubiquitination claim from single lab awaits independent replication\", \"How TRIM35 directly binds DNA promoters despite lacking a canonical DNA-binding domain is unexplained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for TRIM35's remarkably broad substrate repertoire, how it switches between K48-linked degradative, K63-linked non-degradative, monoubiquitinating, and apparent deubiquitinating activities, and whether TRIM35 itself is regulated by post-translational modification or cofactor association in a tissue-specific manner.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal or cryo-EM structure available\", \"Mechanism of activity-type switching (K48 vs K63 vs monoUb vs deubiquitination) unknown\", \"Upstream regulatory inputs controlling TRIM35 expression and activity not systematically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 4, 5, 6, 7, 9, 10, 11, 13, 14, 17, 19]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [13, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3, 16]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3, 6, 13, 16, 17]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 9, 11, 12]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 4, 6, 15, 18]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4, 5, 6, 7, 9, 10, 13, 14, 19]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [13, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 13, 14]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 16, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PKM2\",\n      \"IRF7\",\n      \"LSD1\",\n      \"STING\",\n      \"TRAF3\",\n      \"SLC7A5\",\n      \"RelB\",\n      \"GATA1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}