{"gene":"TRIM69","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2012,"finding":"Human TRIM69 (hTRIM69) functions as an E3 ubiquitin ligase in an E2 conjugating enzyme-selective fashion; an intact RING finger domain is indispensable for ubiquitination activity in vitro and in vivo. TRIM69 localizes in both cytoplasm and nucleus in a speckled aggregating pattern, also requiring an intact RING finger domain.","method":"In vitro ubiquitination assay, RING domain mutagenesis, proteasome inhibitor treatment, subcellular fractionation/imaging","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus in vivo ubiquitination validation, single lab but multiple orthogonal methods","pmids":["23131556"],"is_preprint":false},{"year":2003,"finding":"RNF36 (TRIM69) localizes to nucleus in a speckled pattern, co-localizes and physically interacts with PML; nuclear localization is regulated by phosphorylation potentially mediated by p38, as p38 inhibitor SB203580 causes cytoplasmic translocation. Overexpression induces apoptosis associated with elevated Bax, caspase-2, and RIP.","method":"Double immunofluorescence staining, co-immunoprecipitation, in vitro phosphorylation assay, p38 inhibitor treatment, DNA fragmentation assay, flow cytometry, TUNEL staining","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus localization experiments and multiple downstream readouts, single lab","pmids":["12837286"],"is_preprint":false},{"year":2018,"finding":"TRIM69 directly interacts with dengue virus NS3 protein, mediates its K48-linked polyubiquitination and proteasomal degradation; the RING domain (E3 ligase activity) is critical for antiviral activity; Lys104 of NS3 is the target ubiquitination site. TRIM69 does not modulate IFN signaling.","method":"Co-immunoprecipitation, ubiquitination assay, RING domain mutants, site-directed mutagenesis of NS3 Lys104, mouse in vivo DENV infection model","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination, mutagenesis of both ligase and substrate, plus in vivo mouse model; replicated mechanistically by independent structural study (PMID:35397701)","pmids":["30142214"],"is_preprint":false},{"year":2019,"finding":"TRIM69 potently inhibits vesicular stomatitis Indiana virus (VSIV) replication; a single amino acid substitution in the VSIV phosphoprotein governs sensitivity to TRIM69, and TRIM69 exhibits signatures of positive selection consistent with an antiviral role.","method":"Arrayed ISG expression screening, viral replication assays, VSIV mutant analysis","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional ISG screen with mechanistic follow-up via single amino acid substitution in viral target, single lab","pmids":["31375575"],"is_preprint":false},{"year":2019,"finding":"TRIM69 inhibits VSIV replication by physically associating with the viral phosphoprotein (P), blocking pioneer transcription of incoming virion RNA and preventing viral mRNA synthesis; higher-order TRIM69 multimerization (not proteasomal degradation of P) is required for antiviral activity.","method":"Loss-of-function screen (IFN-α vs VSIV), co-immunoprecipitation of TRIM69 with VSIV P, viral transcription assays, multimerization-deficient mutant analysis","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function screen plus Co-IP, mechanistic dissection of transcription inhibition, multimerization mutants; independently consistent with PMID:31375575 and PMID:33021497","pmids":["31578292"],"is_preprint":false},{"year":2019,"finding":"TRIM69 interacts with p53, induces p53 ubiquitination, and suppresses UVB-induced apoptosis and ROS production in human lens epithelial cells; Foxo3a acts as a transcription factor driving TRIM69 promoter activity, placing Foxo3a upstream of TRIM69 in the regulatory axis.","method":"Co-immunoprecipitation, ubiquitination assay, TRIM69 overexpression/knockdown, p53 overexpression epistasis, Foxo3a promoter-activity assay","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus epistasis with p53, single lab","pmids":["30844644"],"is_preprint":false},{"year":2019,"finding":"TRIM69 directly interacts with ASK1 in microglial cells and deubiquitinates ASK1, suppressing the MKK4/MKK7/JNK phosphorylation cascade and NF-κB signaling; Trim69 knockout mice show exaggerated HFD-induced hippocampal inflammation and apoptosis.","method":"Co-immunoprecipitation, ubiquitination/deubiquitination assay, Trim69 KO mice (HFD model), microglial cell-specific Trim69 suppression, LPS challenge","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and biochemical deubiquitination assay with KO mouse model, single lab","pmids":["31178136"],"is_preprint":false},{"year":2020,"finding":"The RING domain of TRIM69 mediates higher-order oligomeric assembly (not the B-box domain as seen for other TRIM proteins); crystal structure of the RING domain resolved to 2.1 Å identifies the oligomerization interface and regions outside the four-helix bundle that support assembly.","method":"SEC-MALLS, X-ray crystallography (2.1 Å resolution)","journal":"Acta crystallographica. Section D, Structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus biophysical assembly analysis; mechanistically consistent with functional requirement for multimerization shown in PMID:31578292","pmids":["33021497"],"is_preprint":false},{"year":2022,"finding":"TRIM69 associates directly with microtubules and promotes accumulation of stable microtubules; this microtubule remodeling activity underlies TRIM69's broad antiviral activity (HIV-1, HIV-2, SIVMAC, VSV, SARS-CoV-2) in IFN-stimulated myeloid cells.","method":"Direct microtubule binding assay, stable microtubule quantification, TRIM69 loss-of-function, multiple viral replication assays in myeloid cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct microtubule association assay, multiple orthogonal viral restriction assays, functional link between MT remodeling and antiviral activity established","pmids":["36251989"],"is_preprint":false},{"year":2022,"finding":"Hydrogen-deuterium exchange mass spectrometry mapped the binding interface between TRIM69 and DENV NS2B-NS3Δpro; NS2B-NS3Δpro is sufficient as a TRIM69 ubiquitination substrate (confirmed by ELISA and in vitro assay); target regions on NS3 are conserved across DENV serotypes and other flaviviruses including Zika, West Nile, and Japanese encephalitis virus.","method":"HDXMS (hydrogen-deuterium exchange mass spectrometry), ELISA-based ubiquitination assay, in vitro ubiquitination assay","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — Tier 1 structural mapping (HDXMS) plus in vitro ubiquitination assay, provides molecular resolution of the interaction interface","pmids":["35397701"],"is_preprint":false},{"year":2022,"finding":"lncFAL directly binds FSP1 and competitively abolishes TRIM69-dependent FSP1 polyubiquitination and degradation, thereby reducing ferroptosis vulnerability in hepatocellular carcinoma cells.","method":"RNA pulldown, co-immunoprecipitation, ubiquitination assay, in vitro and in vivo ferroptosis assays","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay identifying FSP1 as TRIM69 substrate, with functional rescue, single lab","pmids":["36423520"],"is_preprint":false},{"year":2023,"finding":"TRIM69 interacts with PRKCD through its B-box domain and catalyzes K48-linked polyubiquitination of PRKCD, promoting its proteasomal degradation; this suppresses BDNF production and inhibits anoikis resistance and metastasis in gastric cancer cells.","method":"Co-immunoprecipitation, mass spectrometry, K48-linked ubiquitination assay, B-box domain mutants, PRKCD/BDNF overexpression epistasis, in vitro and in vivo metastasis assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with MS identification, linkage-specific ubiquitination, domain mutants, epistasis experiments, in vivo validation","pmids":["37864033"],"is_preprint":false},{"year":2023,"finding":"TRIM69 directly binds MST2 and redistributes it to the perinuclear cytoskeleton; TRIM69 promotes MST2 association with PLK1 and stimulates MST2 phosphorylation at S15 (a PLK1 site critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation requiring PRC1 and DYNEIN; TRIM69 ablation causes centrosome scattering and chromosome segregation defects.","method":"Co-immunoprecipitation identifying MST2 as binding partner, phosphorylation assay (pS15-MST2), live imaging, TRIM69 knockdown/ablation with centrosome/chromosome phenotype readouts","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, phosphorylation assay, live imaging, multiple orthogonal phenotypic readouts, mechanistic pathway dissection","pmids":["37739411"],"is_preprint":false},{"year":2025,"finding":"TRIM69 binding to microtubules is mediated by a basic surface in its SPRY domain that interacts with the C-terminal tails of tubulins; this surface is conserved in mammalian TRIM69s and is critical for MT remodeling and antiviral function. TRIM69-driven MT remodeling inhibits docking and nuclear migration of HIV virion cores by promoting stalling of dynein/dynactin motor complexes.","method":"In silico modeling, TIRF microscopy, cell-based HIV infection assays, SPRY domain mutagenesis, dynein/dynactin motor complex analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — structural modeling plus TIRF microscopy plus mutagenesis plus mechanistic cell assays; peer-reviewed publication consistent with preprint","pmids":["41066114"],"is_preprint":false},{"year":2025,"finding":"Microtubule acetylation (via αTAT1) is not required for TRIM69-mediated restriction of HIV-1; TRIM69 restricts a diverse range of viruses independently of microtubule acetylation.","method":"CRISPR/Cas9 knockout of αTAT1, HIV-1 infectivity assay, TRIM69 restriction assay in αTAT1-KO cells","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with functional viral assay; result is negative/dissociative (acetylation not required), single lab","pmids":["39804091"],"is_preprint":false},{"year":2026,"finding":"TRIM69 directly binds STING and promotes its K63-linked ubiquitination, leading to increased STING dimerization and downstream TBK1 activation, thereby potentiating cGAS-STING-mediated type I IFN production during antiviral innate immunity.","method":"Co-immunoprecipitation, K63-linked ubiquitination assay, STING dimerization assay, TBK1 activation readout, IFN-I production assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus downstream signaling readouts, single lab","pmids":["41865946"],"is_preprint":false},{"year":2016,"finding":"Zebrafish trim69 knockdown activates the tp53 apoptosis pathway during development; injection of human TRIM69 mRNA rescues the apoptotic phenotype, demonstrating functional conservation.","method":"Morpholino knockdown in zebrafish, tp53 pathway activation readout, human mRNA rescue experiment","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — zebrafish KD with cross-species mRNA rescue, places TRIM69 upstream of p53 apoptosis pathway","pmids":["27031046"],"is_preprint":false},{"year":2016,"finding":"TRIM69 interacts with c-Jun in human cells; TRIM69 knockdown increases c-Jun expression and overexpression decreases it; double knockdown of c-Jun and trim69 rescues brain development defects in zebrafish, placing TRIM69 upstream of the AP-1 pathway in neurogenesis.","method":"Co-immunoprecipitation (TRIM69–c-Jun), overexpression/knockdown in human cells, zebrafish double knockdown epistasis with brain development readout","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus epistasis in zebrafish model, single lab","pmids":["27050765"],"is_preprint":false},{"year":2025,"finding":"TRIM69 SPRY domain basic surface binds C-terminal tails of tubulins, promotes stable microtubule accumulation, and inhibits dynein/dynactin-dependent nuclear migration of HIV virion cores (preprint version consistent with published PMID:41066114).","method":"In silico modeling, TIRF microscopy, cell-based assays, SPRY domain mutagenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint; findings replicated in peer-reviewed publication PMID:41066114","pmids":["bio_10.1101_2025.02.06.636796"],"is_preprint":true}],"current_model":"TRIM69 is an interferon-stimulated E3 ubiquitin ligase whose RING domain mediates higher-order oligomerization and whose activities span multiple mechanisms: it ubiquitinates and degrades specific viral proteins (dengue NS3 at Lys104, VSIV phosphoprotein sequestered via multimerization) and cellular substrates (p53, PRKCD via K48-linkage, FSP1, and STING via K63-linkage); it directly binds microtubules through a basic surface in its SPRY domain to promote stable microtubule accumulation and inhibit dynein/dynactin-dependent nuclear transport of HIV virion cores; it also regulates centrosome dynamics and chromosome segregation by binding MST2, redistributing it to the perinuclear cytoskeleton, and stimulating PLK1-dependent MST2-S15 phosphorylation required for centrosome disjunction."},"narrative":{"mechanistic_narrative":"TRIM69 is a RING-domain E3 ubiquitin ligase and interferon-stimulated antiviral effector that acts through two distinct modes—substrate ubiquitination and microtubule-based remodeling—to control viral replication, cell death, and mitotic architecture [PMID:23131556, PMID:36251989]. Its RING domain is indispensable for E2-selective ubiquitination activity and also mediates higher-order oligomeric assembly, an unusual property resolved by crystallography to 2.1 Å, that is required both for catalysis and for its non-degradative antiviral function [PMID:23131556, PMID:33021497]. As a ligase, TRIM69 directly binds and ubiquitinates diverse substrates with defined linkage specificities: it K48-polyubiquitinates dengue virus NS3 at Lys104 to drive proteasomal degradation and restrict flavivirus replication [PMID:30142214, PMID:35397701], degrades cellular targets including p53, PRKCD (via its B-box domain, K48-linked), and the ferroptosis suppressor FSP1 [PMID:30844644, PMID:37864033, PMID:36423520], and conversely promotes K63-linked ubiquitination of STING to potentiate STING dimerization, TBK1 activation, and type I interferon production [PMID:41865946]. In a ubiquitination-independent arm, TRIM69 binds vesicular stomatitis virus phosphoprotein through multimerization to block pioneer viral transcription [PMID:31578292], and engages microtubules directly via a conserved basic surface in its SPRY domain that contacts tubulin C-terminal tails, promoting stable microtubule accumulation that stalls dynein/dynactin motors and inhibits nuclear transport of HIV virion cores, underlying broad restriction of HIV, SIV, VSV, and SARS-CoV-2 [PMID:36251989, PMID:41066114]. TRIM69 additionally regulates mitotic fidelity by binding MST2, redistributing it to the perinuclear cytoskeleton and stimulating PLK1-dependent MST2-S15 phosphorylation required for centrosome disjunction, such that its loss causes centrosome scattering and chromosome segregation defects [PMID:37739411].","teleology":[{"year":2003,"claim":"Established TRIM69 (RNF36) as a nuclear-speckle protein that interacts with PML and drives apoptosis, the first hint of its cellular role.","evidence":"Co-immunoprecipitation, immunofluorescence, in vitro phosphorylation, and apoptosis readouts in human cells","pmids":["12837286"],"confidence":"Medium","gaps":["No enzymatic activity assigned at this stage","Functional consequence of PML interaction not mechanistically defined","p38 phosphorylation link inferred from inhibitor treatment only"]},{"year":2012,"claim":"Defined TRIM69 biochemically as a RING-dependent, E2-selective E3 ubiquitin ligase, supplying the catalytic basis for its later substrate-directed functions.","evidence":"In vitro ubiquitination assay with RING mutagenesis plus subcellular imaging","pmids":["23131556"],"confidence":"High","gaps":["No physiological substrate identified","E2 partners not enumerated","Link between speckled localization and ligase activity unresolved"]},{"year":2016,"claim":"Placed TRIM69 upstream of p53-dependent apoptosis and the AP-1/c-Jun pathway in development, with cross-species rescue confirming functional conservation.","evidence":"Zebrafish morpholino knockdown with human mRNA rescue and c-Jun epistasis","pmids":["27031046","27050765"],"confidence":"Medium","gaps":["Direct biochemical link between TRIM69 and tp53/c-Jun in vivo not established","Whether regulation is via ubiquitination unaddressed","Developmental phenotypes not connected to ligase activity"]},{"year":2018,"claim":"Identified the first defined viral substrate, showing TRIM69 K48-ubiquitinates dengue NS3 at Lys104 to restrict infection, demonstrating direct antiviral ligase activity.","evidence":"Co-IP, ubiquitination assays, RING and NS3-Lys104 mutagenesis, and a mouse DENV infection model","pmids":["30142214"],"confidence":"High","gaps":["IFN signaling explicitly not modulated, leaving upstream regulation open","Breadth across flaviviruses not yet tested","Structural basis of NS3 recognition unresolved at this stage"]},{"year":2019,"claim":"Revealed a ubiquitination-independent antiviral mechanism: TRIM69 multimerizes and binds VSIV phosphoprotein to block pioneer viral transcription rather than degrade it.","evidence":"ISG and loss-of-function screens, Co-IP with VSIV P, viral transcription assays, and multimerization-deficient mutants","pmids":["31375575","31578292"],"confidence":"High","gaps":["Structural basis of multimerization not yet defined","How P sequestration blocks transcription mechanistically unclear","Generality of non-degradative mode to other viruses untested"]},{"year":2019,"claim":"Connected TRIM69 to redox and inflammatory signaling by showing p53 ubiquitination (Foxo3a-driven) and ASK1 deubiquitination, expanding its cellular substrate repertoire.","evidence":"Co-IP and ubiquitination/deubiquitination assays plus Trim69 KO mice and epistasis","pmids":["30844644","31178136"],"confidence":"Medium","gaps":["A single E3 ligase exhibiting deubiquitination of ASK1 is mechanistically unusual and not reconciled","Linkage types and direct vs indirect effects not fully resolved","Tissue-specific relevance limited to lens/microglia models"]},{"year":2020,"claim":"Resolved the structural basis of TRIM69 assembly, showing the RING domain—not the B-box—mediates higher-order oligomerization, rationalizing the multimerization requirement for antiviral activity.","evidence":"SEC-MALLS and 2.1 Å X-ray crystallography of the RING domain","pmids":["33021497"],"confidence":"High","gaps":["Full-length assembly architecture not visualized","How oligomerization couples to substrate ubiquitination unresolved","Structure does not address microtubule or P-protein binding"]},{"year":2022,"claim":"Discovered a third mechanism—direct microtubule binding and stabilization—as the basis for TRIM69's broad antiviral restriction in myeloid cells.","evidence":"Direct microtubule binding assays, stable MT quantification, and multiple viral restriction assays (HIV, SIV, VSV, SARS-CoV-2)","pmids":["36251989"],"confidence":"High","gaps":["MT-binding determinant not yet localized to a domain","Relationship between MT remodeling and ligase activity unclear","How stable MTs restrict diverse viruses not mechanistically defined"]},{"year":2022,"claim":"Mapped the DENV NS3 interaction interface at molecular resolution and showed flavivirus conservation, generalizing TRIM69's antiviral substrate recognition.","evidence":"HDX-MS interface mapping and ELISA/in vitro ubiquitination of NS2B-NS3Δpro","pmids":["35397701"],"confidence":"High","gaps":["Functional restriction of Zika/WNV/JEV not directly demonstrated","Co-crystal structure absent","Stoichiometry of ubiquitin transfer unresolved"]},{"year":2022,"claim":"Extended TRIM69's degradative ligase role into cancer cell death control by identifying FSP1 as a substrate whose degradation governs ferroptosis vulnerability.","evidence":"RNA pulldown, Co-IP, ubiquitination assays, and ferroptosis assays with lncFAL competition","pmids":["36423520"],"confidence":"Medium","gaps":["Ubiquitination linkage type and site on FSP1 not defined","Direct vs lncRNA-mediated regulation entangled","Single tumor context (HCC)"]},{"year":2023,"claim":"Defined TRIM69's mitotic function—binding MST2 and stimulating PLK1-dependent S15 phosphorylation for centrosome disjunction—and a B-box-dependent PRKCD-degradation pathway in cancer metastasis.","evidence":"Reciprocal Co-IP, MS, phosphorylation and linkage-specific ubiquitination assays, domain mutants, live imaging, and in vivo metastasis/centrosome readouts","pmids":["37739411","37864033"],"confidence":"High","gaps":["Whether centrosome role requires ligase activity unclear","How MST2 redistribution couples to microtubule binding unresolved","Connection between mitotic and antiviral functions not addressed"]},{"year":2025,"claim":"Pinpointed the SPRY-domain basic surface contacting tubulin C-terminal tails as the molecular basis of microtubule remodeling, mechanistically linking MT stabilization to stalled dynein/dynactin and blocked HIV core nuclear transport.","evidence":"In silico modeling, TIRF microscopy, SPRY mutagenesis, and HIV infection/motor-complex assays; microtubule acetylation shown dispensable by αTAT1 knockout","pmids":["41066114","39804091"],"confidence":"High","gaps":["Co-structure of SPRY–tubulin not solved","How motor stalling is achieved at molecular level incomplete","Generality beyond HIV core transport not fully mapped"]},{"year":2026,"claim":"Showed TRIM69 acts as a positive regulator of innate immune signaling by K63-ubiquitinating STING to enhance dimerization and type I IFN output, distinguishing this from its degradative K48 activities.","evidence":"Co-IP, K63-linkage ubiquitination assay, STING dimerization, TBK1 activation, and IFN-I production readouts","pmids":["41865946"],"confidence":"Medium","gaps":["STING ubiquitination site not mapped","Single lab without reciprocal in vivo validation","Reconciliation with TRIM69's own ISG-stimulated expression unaddressed"]},{"year":null,"claim":"How TRIM69's three modes—linkage-specific ubiquitination, oligomeric viral-protein sequestration, and SPRY-mediated microtubule remodeling—are coordinated and regulated within a single protein remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model linking ligase, multimerization, and MT-binding activities","Determinants of K48- vs K63-linkage substrate choice unknown","In vivo importance of mitotic vs antiviral roles not disentangled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2,11,15]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,5,11,15]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[8,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,12,13]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,4,8,15]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[12]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,5,10,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,11]}],"complexes":[],"partners":["NS3","PML","P53","ASK1","PRKCD","FSP1","MST2","STING"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86WT6","full_name":"E3 ubiquitin-protein ligase TRIM69","aliases":["RFP-like domain-containing protein trimless","RING finger protein 36","RING-type E3 ubiquitin transferase TRIM69","Tripartite motif-containing protein 69"],"length_aa":500,"mass_kda":57.4,"function":"E3 ubiquitin ligase that plays an important role in antiviral immunity by restricting different viral infections including dengue virus or vesicular stomatitis indiana virus (PubMed:23131556, PubMed:30142214, PubMed:31375575, PubMed:31578292). Ubiquitinates viral proteins such as dengue virus NS3 thereby limiting infection (PubMed:30844644). In addition, acts as a key mediator of type I interferon induced microtubule stabilization by directly associating to microtubules independently of its E3 ligase activity (PubMed:36251989). Also plays a role in cataract formation together with TP53 (PubMed:30844644). Mechanistically, inhibits UVB-induced cell apoptosis and reactive oxygen species (ROS) production by inducing TP53 ubiquitination (PubMed:30844644). Regulates centrosome dynamics and mitotic progression by ubiquitinating STK3/MST2; leading to its redistribution to the perinuclear cytoskeleton and subsequent phosphorylation by PLK1 (PubMed:37739411)","subcellular_location":"Cytoplasm; Nucleus; Nucleus speckle; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q86WT6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRIM69","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/TRIM69","total_profiled":1310},"omim":[{"mim_id":"616017","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 69; TRIM69","url":"https://www.omim.org/entry/616017"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":33.7}],"url":"https://www.proteinatlas.org/search/TRIM69"},"hgnc":{"alias_symbol":["Trif","TRIMLESS"],"prev_symbol":["RNF36"]},"alphafold":{"accession":"Q86WT6","domains":[{"cath_id":"3.30.40.10","chopping":"28-112","consensus_level":"medium","plddt":84.2675,"start":28,"end":112},{"cath_id":"-","chopping":"114-128_135-154","consensus_level":"medium","plddt":84.3403,"start":114,"end":154},{"cath_id":"2.60.120.920","chopping":"337-498","consensus_level":"high","plddt":94.1496,"start":337,"end":498},{"cath_id":"1.20.5","chopping":"201-287","consensus_level":"high","plddt":94.6372,"start":201,"end":287}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86WT6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86WT6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86WT6-F1-predicted_aligned_error_v6.png","plddt_mean":88.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRIM69","jax_strain_url":"https://www.jax.org/strain/search?query=TRIM69"},"sequence":{"accession":"Q86WT6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86WT6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86WT6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86WT6"}},"corpus_meta":[{"pmid":"36423520","id":"PMC_36423520","title":"HDLBP-stabilized lncFAL inhibits ferroptosis vulnerability by diminishing Trim69-dependent FSP1 degradation in hepatocellular carcinoma.","date":"2022","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/36423520","citation_count":81,"is_preprint":false},{"pmid":"30142214","id":"PMC_30142214","title":"Interferon-stimulated TRIM69 interrupts dengue virus replication by ubiquitinating viral nonstructural protein 3.","date":"2018","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/30142214","citation_count":80,"is_preprint":false},{"pmid":"30844644","id":"PMC_30844644","title":"TRIM69 inhibits cataractogenesis by negatively regulating p53.","date":"2019","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/30844644","citation_count":55,"is_preprint":false},{"pmid":"31375575","id":"PMC_31375575","title":"TRIM69 Inhibits Vesicular Stomatitis Indiana Virus.","date":"2019","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/31375575","citation_count":40,"is_preprint":false},{"pmid":"31578292","id":"PMC_31578292","title":"Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State.","date":"2019","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/31578292","citation_count":33,"is_preprint":false},{"pmid":"37864033","id":"PMC_37864033","title":"TRIM69 suppressed the anoikis resistance and metastasis of gastric cancer through ubiquitin‒proteasome-mediated degradation of PRKCD.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/37864033","citation_count":25,"is_preprint":false},{"pmid":"27031046","id":"PMC_27031046","title":"A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.","date":"2016","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/27031046","citation_count":19,"is_preprint":false},{"pmid":"23131556","id":"PMC_23131556","title":"Characterisation of human RING finger protein TRIM69, a novel testis E3 ubiquitin ligase and its subcellular localisation.","date":"2012","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23131556","citation_count":19,"is_preprint":false},{"pmid":"36251989","id":"PMC_36251989","title":"Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36251989","citation_count":17,"is_preprint":false},{"pmid":"31178136","id":"PMC_31178136","title":"Targeting Trim69 alleviates high fat diet (HFD)-induced hippocampal injury in mice by inhibiting apoptosis and inflammation through ASK1 inactivation.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31178136","citation_count":17,"is_preprint":false},{"pmid":"27050765","id":"PMC_27050765","title":"Trim69 regulates zebrafish brain development by ap-1 pathway.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27050765","citation_count":16,"is_preprint":false},{"pmid":"12837286","id":"PMC_12837286","title":"Forced expression of RNF36 induces cell apoptosis.","date":"2003","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/12837286","citation_count":14,"is_preprint":false},{"pmid":"35397701","id":"PMC_35397701","title":"Mapping of molecular interactions between human E3 ligase TRIM69 and Dengue virus NS3 protease using hydrogen-deuterium exchange mass spectrometry.","date":"2022","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/35397701","citation_count":13,"is_preprint":false},{"pmid":"33021497","id":"PMC_33021497","title":"The RING domain of TRIM69 promotes higher-order assembly.","date":"2020","source":"Acta crystallographica. Section D, Structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/33021497","citation_count":13,"is_preprint":false},{"pmid":"37739411","id":"PMC_37739411","title":"The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation.","date":"2023","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/37739411","citation_count":10,"is_preprint":false},{"pmid":"33273151","id":"PMC_33273151","title":"The testis-specifically expressed gene Trim69 is not essential for fertility in mice.","date":"2020","source":"Journal of biomedical research","url":"https://pubmed.ncbi.nlm.nih.gov/33273151","citation_count":6,"is_preprint":false},{"pmid":"37653392","id":"PMC_37653392","title":"TRIM69: a marker of metastasis and potential sensitizer to 5-Fluorouracil and PD-1 blockers in colon adenocarcinoma.","date":"2023","source":"BMC gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/37653392","citation_count":4,"is_preprint":false},{"pmid":"39804091","id":"PMC_39804091","title":"Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection.","date":"2025","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/39804091","citation_count":3,"is_preprint":false},{"pmid":"41865946","id":"PMC_41865946","title":"TRIM69 potentiates the cGAS-STING signalling pathway by promoting STING ubiquitination.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/41865946","citation_count":1,"is_preprint":false},{"pmid":"41066114","id":"PMC_41066114","title":"Microtubule remodeling by the innate immune factor Trim69 compromises dynein-dependent migration of HIV virion cores toward the nucleus.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41066114","citation_count":0,"is_preprint":false},{"pmid":"41758560","id":"PMC_41758560","title":"Increased expression of TRIM69 in peripheral blood from systemic lupus erythematosus and its clinical significance.","date":"2026","source":"Clinical and experimental rheumatology","url":"https://pubmed.ncbi.nlm.nih.gov/41758560","citation_count":0,"is_preprint":false},{"pmid":"41369736","id":"PMC_41369736","title":"TRIM69 alleviates ultraviolet B irradiation-induced ferroptosis by regulating p53 in human lens epithelial cells.","date":"2025","source":"Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie","url":"https://pubmed.ncbi.nlm.nih.gov/41369736","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.06.636796","title":"Microtubule remodeling by the innate immune factor Trim69 compromises dynein-dependent migration of HIV virion cores towards the nucleus","date":"2025-02-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.06.636796","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13061,"output_tokens":4669,"usd":0.054609,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12782,"output_tokens":4634,"usd":0.08988,"stage2_stop_reason":"end_turn"},"total_usd":0.144489,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"Human TRIM69 (hTRIM69) functions as an E3 ubiquitin ligase in an E2 conjugating enzyme-selective fashion; an intact RING finger domain is indispensable for ubiquitination activity in vitro and in vivo. TRIM69 localizes in both cytoplasm and nucleus in a speckled aggregating pattern, also requiring an intact RING finger domain.\",\n      \"method\": \"In vitro ubiquitination assay, RING domain mutagenesis, proteasome inhibitor treatment, subcellular fractionation/imaging\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus in vivo ubiquitination validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23131556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RNF36 (TRIM69) localizes to nucleus in a speckled pattern, co-localizes and physically interacts with PML; nuclear localization is regulated by phosphorylation potentially mediated by p38, as p38 inhibitor SB203580 causes cytoplasmic translocation. Overexpression induces apoptosis associated with elevated Bax, caspase-2, and RIP.\",\n      \"method\": \"Double immunofluorescence staining, co-immunoprecipitation, in vitro phosphorylation assay, p38 inhibitor treatment, DNA fragmentation assay, flow cytometry, TUNEL staining\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus localization experiments and multiple downstream readouts, single lab\",\n      \"pmids\": [\"12837286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM69 directly interacts with dengue virus NS3 protein, mediates its K48-linked polyubiquitination and proteasomal degradation; the RING domain (E3 ligase activity) is critical for antiviral activity; Lys104 of NS3 is the target ubiquitination site. TRIM69 does not modulate IFN signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RING domain mutants, site-directed mutagenesis of NS3 Lys104, mouse in vivo DENV infection model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination, mutagenesis of both ligase and substrate, plus in vivo mouse model; replicated mechanistically by independent structural study (PMID:35397701)\",\n      \"pmids\": [\"30142214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM69 potently inhibits vesicular stomatitis Indiana virus (VSIV) replication; a single amino acid substitution in the VSIV phosphoprotein governs sensitivity to TRIM69, and TRIM69 exhibits signatures of positive selection consistent with an antiviral role.\",\n      \"method\": \"Arrayed ISG expression screening, viral replication assays, VSIV mutant analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional ISG screen with mechanistic follow-up via single amino acid substitution in viral target, single lab\",\n      \"pmids\": [\"31375575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM69 inhibits VSIV replication by physically associating with the viral phosphoprotein (P), blocking pioneer transcription of incoming virion RNA and preventing viral mRNA synthesis; higher-order TRIM69 multimerization (not proteasomal degradation of P) is required for antiviral activity.\",\n      \"method\": \"Loss-of-function screen (IFN-α vs VSIV), co-immunoprecipitation of TRIM69 with VSIV P, viral transcription assays, multimerization-deficient mutant analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function screen plus Co-IP, mechanistic dissection of transcription inhibition, multimerization mutants; independently consistent with PMID:31375575 and PMID:33021497\",\n      \"pmids\": [\"31578292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM69 interacts with p53, induces p53 ubiquitination, and suppresses UVB-induced apoptosis and ROS production in human lens epithelial cells; Foxo3a acts as a transcription factor driving TRIM69 promoter activity, placing Foxo3a upstream of TRIM69 in the regulatory axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TRIM69 overexpression/knockdown, p53 overexpression epistasis, Foxo3a promoter-activity assay\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus epistasis with p53, single lab\",\n      \"pmids\": [\"30844644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM69 directly interacts with ASK1 in microglial cells and deubiquitinates ASK1, suppressing the MKK4/MKK7/JNK phosphorylation cascade and NF-κB signaling; Trim69 knockout mice show exaggerated HFD-induced hippocampal inflammation and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination/deubiquitination assay, Trim69 KO mice (HFD model), microglial cell-specific Trim69 suppression, LPS challenge\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and biochemical deubiquitination assay with KO mouse model, single lab\",\n      \"pmids\": [\"31178136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The RING domain of TRIM69 mediates higher-order oligomeric assembly (not the B-box domain as seen for other TRIM proteins); crystal structure of the RING domain resolved to 2.1 Å identifies the oligomerization interface and regions outside the four-helix bundle that support assembly.\",\n      \"method\": \"SEC-MALLS, X-ray crystallography (2.1 Å resolution)\",\n      \"journal\": \"Acta crystallographica. Section D, Structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus biophysical assembly analysis; mechanistically consistent with functional requirement for multimerization shown in PMID:31578292\",\n      \"pmids\": [\"33021497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM69 associates directly with microtubules and promotes accumulation of stable microtubules; this microtubule remodeling activity underlies TRIM69's broad antiviral activity (HIV-1, HIV-2, SIVMAC, VSV, SARS-CoV-2) in IFN-stimulated myeloid cells.\",\n      \"method\": \"Direct microtubule binding assay, stable microtubule quantification, TRIM69 loss-of-function, multiple viral replication assays in myeloid cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct microtubule association assay, multiple orthogonal viral restriction assays, functional link between MT remodeling and antiviral activity established\",\n      \"pmids\": [\"36251989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Hydrogen-deuterium exchange mass spectrometry mapped the binding interface between TRIM69 and DENV NS2B-NS3Δpro; NS2B-NS3Δpro is sufficient as a TRIM69 ubiquitination substrate (confirmed by ELISA and in vitro assay); target regions on NS3 are conserved across DENV serotypes and other flaviviruses including Zika, West Nile, and Japanese encephalitis virus.\",\n      \"method\": \"HDXMS (hydrogen-deuterium exchange mass spectrometry), ELISA-based ubiquitination assay, in vitro ubiquitination assay\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — Tier 1 structural mapping (HDXMS) plus in vitro ubiquitination assay, provides molecular resolution of the interaction interface\",\n      \"pmids\": [\"35397701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"lncFAL directly binds FSP1 and competitively abolishes TRIM69-dependent FSP1 polyubiquitination and degradation, thereby reducing ferroptosis vulnerability in hepatocellular carcinoma cells.\",\n      \"method\": \"RNA pulldown, co-immunoprecipitation, ubiquitination assay, in vitro and in vivo ferroptosis assays\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay identifying FSP1 as TRIM69 substrate, with functional rescue, single lab\",\n      \"pmids\": [\"36423520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM69 interacts with PRKCD through its B-box domain and catalyzes K48-linked polyubiquitination of PRKCD, promoting its proteasomal degradation; this suppresses BDNF production and inhibits anoikis resistance and metastasis in gastric cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, K48-linked ubiquitination assay, B-box domain mutants, PRKCD/BDNF overexpression epistasis, in vitro and in vivo metastasis assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with MS identification, linkage-specific ubiquitination, domain mutants, epistasis experiments, in vivo validation\",\n      \"pmids\": [\"37864033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM69 directly binds MST2 and redistributes it to the perinuclear cytoskeleton; TRIM69 promotes MST2 association with PLK1 and stimulates MST2 phosphorylation at S15 (a PLK1 site critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation requiring PRC1 and DYNEIN; TRIM69 ablation causes centrosome scattering and chromosome segregation defects.\",\n      \"method\": \"Co-immunoprecipitation identifying MST2 as binding partner, phosphorylation assay (pS15-MST2), live imaging, TRIM69 knockdown/ablation with centrosome/chromosome phenotype readouts\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, phosphorylation assay, live imaging, multiple orthogonal phenotypic readouts, mechanistic pathway dissection\",\n      \"pmids\": [\"37739411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM69 binding to microtubules is mediated by a basic surface in its SPRY domain that interacts with the C-terminal tails of tubulins; this surface is conserved in mammalian TRIM69s and is critical for MT remodeling and antiviral function. TRIM69-driven MT remodeling inhibits docking and nuclear migration of HIV virion cores by promoting stalling of dynein/dynactin motor complexes.\",\n      \"method\": \"In silico modeling, TIRF microscopy, cell-based HIV infection assays, SPRY domain mutagenesis, dynein/dynactin motor complex analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — structural modeling plus TIRF microscopy plus mutagenesis plus mechanistic cell assays; peer-reviewed publication consistent with preprint\",\n      \"pmids\": [\"41066114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Microtubule acetylation (via αTAT1) is not required for TRIM69-mediated restriction of HIV-1; TRIM69 restricts a diverse range of viruses independently of microtubule acetylation.\",\n      \"method\": \"CRISPR/Cas9 knockout of αTAT1, HIV-1 infectivity assay, TRIM69 restriction assay in αTAT1-KO cells\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with functional viral assay; result is negative/dissociative (acetylation not required), single lab\",\n      \"pmids\": [\"39804091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TRIM69 directly binds STING and promotes its K63-linked ubiquitination, leading to increased STING dimerization and downstream TBK1 activation, thereby potentiating cGAS-STING-mediated type I IFN production during antiviral innate immunity.\",\n      \"method\": \"Co-immunoprecipitation, K63-linked ubiquitination assay, STING dimerization assay, TBK1 activation readout, IFN-I production assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus downstream signaling readouts, single lab\",\n      \"pmids\": [\"41865946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Zebrafish trim69 knockdown activates the tp53 apoptosis pathway during development; injection of human TRIM69 mRNA rescues the apoptotic phenotype, demonstrating functional conservation.\",\n      \"method\": \"Morpholino knockdown in zebrafish, tp53 pathway activation readout, human mRNA rescue experiment\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — zebrafish KD with cross-species mRNA rescue, places TRIM69 upstream of p53 apoptosis pathway\",\n      \"pmids\": [\"27031046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRIM69 interacts with c-Jun in human cells; TRIM69 knockdown increases c-Jun expression and overexpression decreases it; double knockdown of c-Jun and trim69 rescues brain development defects in zebrafish, placing TRIM69 upstream of the AP-1 pathway in neurogenesis.\",\n      \"method\": \"Co-immunoprecipitation (TRIM69–c-Jun), overexpression/knockdown in human cells, zebrafish double knockdown epistasis with brain development readout\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus epistasis in zebrafish model, single lab\",\n      \"pmids\": [\"27050765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM69 SPRY domain basic surface binds C-terminal tails of tubulins, promotes stable microtubule accumulation, and inhibits dynein/dynactin-dependent nuclear migration of HIV virion cores (preprint version consistent with published PMID:41066114).\",\n      \"method\": \"In silico modeling, TIRF microscopy, cell-based assays, SPRY domain mutagenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint; findings replicated in peer-reviewed publication PMID:41066114\",\n      \"pmids\": [\"bio_10.1101_2025.02.06.636796\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TRIM69 is an interferon-stimulated E3 ubiquitin ligase whose RING domain mediates higher-order oligomerization and whose activities span multiple mechanisms: it ubiquitinates and degrades specific viral proteins (dengue NS3 at Lys104, VSIV phosphoprotein sequestered via multimerization) and cellular substrates (p53, PRKCD via K48-linkage, FSP1, and STING via K63-linkage); it directly binds microtubules through a basic surface in its SPRY domain to promote stable microtubule accumulation and inhibit dynein/dynactin-dependent nuclear transport of HIV virion cores; it also regulates centrosome dynamics and chromosome segregation by binding MST2, redistributing it to the perinuclear cytoskeleton, and stimulating PLK1-dependent MST2-S15 phosphorylation required for centrosome disjunction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRIM69 is a RING-domain E3 ubiquitin ligase and interferon-stimulated antiviral effector that acts through two distinct modes—substrate ubiquitination and microtubule-based remodeling—to control viral replication, cell death, and mitotic architecture [#0, #8]. Its RING domain is indispensable for E2-selective ubiquitination activity and also mediates higher-order oligomeric assembly, an unusual property resolved by crystallography to 2.1 Å, that is required both for catalysis and for its non-degradative antiviral function [#0, #7]. As a ligase, TRIM69 directly binds and ubiquitinates diverse substrates with defined linkage specificities: it K48-polyubiquitinates dengue virus NS3 at Lys104 to drive proteasomal degradation and restrict flavivirus replication [#2, #9], degrades cellular targets including p53, PRKCD (via its B-box domain, K48-linked), and the ferroptosis suppressor FSP1 [#5, #11, #10], and conversely promotes K63-linked ubiquitination of STING to potentiate STING dimerization, TBK1 activation, and type I interferon production [#15]. In a ubiquitination-independent arm, TRIM69 binds vesicular stomatitis virus phosphoprotein through multimerization to block pioneer viral transcription [#4], and engages microtubules directly via a conserved basic surface in its SPRY domain that contacts tubulin C-terminal tails, promoting stable microtubule accumulation that stalls dynein/dynactin motors and inhibits nuclear transport of HIV virion cores, underlying broad restriction of HIV, SIV, VSV, and SARS-CoV-2 [#8, #13]. TRIM69 additionally regulates mitotic fidelity by binding MST2, redistributing it to the perinuclear cytoskeleton and stimulating PLK1-dependent MST2-S15 phosphorylation required for centrosome disjunction, such that its loss causes centrosome scattering and chromosome segregation defects [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established TRIM69 (RNF36) as a nuclear-speckle protein that interacts with PML and drives apoptosis, the first hint of its cellular role.\",\n      \"evidence\": \"Co-immunoprecipitation, immunofluorescence, in vitro phosphorylation, and apoptosis readouts in human cells\",\n      \"pmids\": [\"12837286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No enzymatic activity assigned at this stage\", \"Functional consequence of PML interaction not mechanistically defined\", \"p38 phosphorylation link inferred from inhibitor treatment only\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined TRIM69 biochemically as a RING-dependent, E2-selective E3 ubiquitin ligase, supplying the catalytic basis for its later substrate-directed functions.\",\n      \"evidence\": \"In vitro ubiquitination assay with RING mutagenesis plus subcellular imaging\",\n      \"pmids\": [\"23131556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified\", \"E2 partners not enumerated\", \"Link between speckled localization and ligase activity unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed TRIM69 upstream of p53-dependent apoptosis and the AP-1/c-Jun pathway in development, with cross-species rescue confirming functional conservation.\",\n      \"evidence\": \"Zebrafish morpholino knockdown with human mRNA rescue and c-Jun epistasis\",\n      \"pmids\": [\"27031046\", \"27050765\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between TRIM69 and tp53/c-Jun in vivo not established\", \"Whether regulation is via ubiquitination unaddressed\", \"Developmental phenotypes not connected to ligase activity\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the first defined viral substrate, showing TRIM69 K48-ubiquitinates dengue NS3 at Lys104 to restrict infection, demonstrating direct antiviral ligase activity.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, RING and NS3-Lys104 mutagenesis, and a mouse DENV infection model\",\n      \"pmids\": [\"30142214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IFN signaling explicitly not modulated, leaving upstream regulation open\", \"Breadth across flaviviruses not yet tested\", \"Structural basis of NS3 recognition unresolved at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a ubiquitination-independent antiviral mechanism: TRIM69 multimerizes and binds VSIV phosphoprotein to block pioneer viral transcription rather than degrade it.\",\n      \"evidence\": \"ISG and loss-of-function screens, Co-IP with VSIV P, viral transcription assays, and multimerization-deficient mutants\",\n      \"pmids\": [\"31375575\", \"31578292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of multimerization not yet defined\", \"How P sequestration blocks transcription mechanistically unclear\", \"Generality of non-degradative mode to other viruses untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected TRIM69 to redox and inflammatory signaling by showing p53 ubiquitination (Foxo3a-driven) and ASK1 deubiquitination, expanding its cellular substrate repertoire.\",\n      \"evidence\": \"Co-IP and ubiquitination/deubiquitination assays plus Trim69 KO mice and epistasis\",\n      \"pmids\": [\"30844644\", \"31178136\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"A single E3 ligase exhibiting deubiquitination of ASK1 is mechanistically unusual and not reconciled\", \"Linkage types and direct vs indirect effects not fully resolved\", \"Tissue-specific relevance limited to lens/microglia models\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the structural basis of TRIM69 assembly, showing the RING domain—not the B-box—mediates higher-order oligomerization, rationalizing the multimerization requirement for antiviral activity.\",\n      \"evidence\": \"SEC-MALLS and 2.1 Å X-ray crystallography of the RING domain\",\n      \"pmids\": [\"33021497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length assembly architecture not visualized\", \"How oligomerization couples to substrate ubiquitination unresolved\", \"Structure does not address microtubule or P-protein binding\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovered a third mechanism—direct microtubule binding and stabilization—as the basis for TRIM69's broad antiviral restriction in myeloid cells.\",\n      \"evidence\": \"Direct microtubule binding assays, stable MT quantification, and multiple viral restriction assays (HIV, SIV, VSV, SARS-CoV-2)\",\n      \"pmids\": [\"36251989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MT-binding determinant not yet localized to a domain\", \"Relationship between MT remodeling and ligase activity unclear\", \"How stable MTs restrict diverse viruses not mechanistically defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped the DENV NS3 interaction interface at molecular resolution and showed flavivirus conservation, generalizing TRIM69's antiviral substrate recognition.\",\n      \"evidence\": \"HDX-MS interface mapping and ELISA/in vitro ubiquitination of NS2B-NS3Δpro\",\n      \"pmids\": [\"35397701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional restriction of Zika/WNV/JEV not directly demonstrated\", \"Co-crystal structure absent\", \"Stoichiometry of ubiquitin transfer unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended TRIM69's degradative ligase role into cancer cell death control by identifying FSP1 as a substrate whose degradation governs ferroptosis vulnerability.\",\n      \"evidence\": \"RNA pulldown, Co-IP, ubiquitination assays, and ferroptosis assays with lncFAL competition\",\n      \"pmids\": [\"36423520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination linkage type and site on FSP1 not defined\", \"Direct vs lncRNA-mediated regulation entangled\", \"Single tumor context (HCC)\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined TRIM69's mitotic function—binding MST2 and stimulating PLK1-dependent S15 phosphorylation for centrosome disjunction—and a B-box-dependent PRKCD-degradation pathway in cancer metastasis.\",\n      \"evidence\": \"Reciprocal Co-IP, MS, phosphorylation and linkage-specific ubiquitination assays, domain mutants, live imaging, and in vivo metastasis/centrosome readouts\",\n      \"pmids\": [\"37739411\", \"37864033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether centrosome role requires ligase activity unclear\", \"How MST2 redistribution couples to microtubule binding unresolved\", \"Connection between mitotic and antiviral functions not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Pinpointed the SPRY-domain basic surface contacting tubulin C-terminal tails as the molecular basis of microtubule remodeling, mechanistically linking MT stabilization to stalled dynein/dynactin and blocked HIV core nuclear transport.\",\n      \"evidence\": \"In silico modeling, TIRF microscopy, SPRY mutagenesis, and HIV infection/motor-complex assays; microtubule acetylation shown dispensable by αTAT1 knockout\",\n      \"pmids\": [\"41066114\", \"39804091\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Co-structure of SPRY–tubulin not solved\", \"How motor stalling is achieved at molecular level incomplete\", \"Generality beyond HIV core transport not fully mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed TRIM69 acts as a positive regulator of innate immune signaling by K63-ubiquitinating STING to enhance dimerization and type I IFN output, distinguishing this from its degradative K48 activities.\",\n      \"evidence\": \"Co-IP, K63-linkage ubiquitination assay, STING dimerization, TBK1 activation, and IFN-I production readouts\",\n      \"pmids\": [\"41865946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"STING ubiquitination site not mapped\", \"Single lab without reciprocal in vivo validation\", \"Reconciliation with TRIM69's own ISG-stimulated expression unaddressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRIM69's three modes—linkage-specific ubiquitination, oligomeric viral-protein sequestration, and SPRY-mediated microtubule remodeling—are coordinated and regulated within a single protein remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model linking ligase, multimerization, and MT-binding activities\", \"Determinants of K48- vs K63-linkage substrate choice unknown\", \"In vivo importance of mitotic vs antiviral roles not disentangled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2, 11, 15]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 5, 11, 15]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [8, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 12, 13]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 4, 8, 15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 5, 10, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NS3\", \"PML\", \"p53\", \"ASK1\", \"PRKCD\", \"FSP1\", \"MST2\", \"STING\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}