TRIM56

TRIM56 is a RING-type E3 ubiquitin ligase that functions as a central amplifier of innate immune sensing and a versatile regulator of protein turnover across diverse cellular contexts. In the cytosolic DNA-sensing pathway, TRIM56 catalyzes K63-linked ubiquitination of STING to promote its dimerization and TBK1 recruitment, and monoubiquitinates cGAS at Lys335 to enhance cGAS dimerization, DNA binding, and cGAMP synthesis—activities regulated by HDAC6-mediated deacetylation of TRIM56 at Lys110 (PMID:21074459, PMID:29426904, PMID:39747662). Independent of its E3 ligase activity, TRIM56 augments TLR3-TRIF signaling through a phosphorylation-dependent (Ser471/Ser475/Ser710) scaffolding interaction with TRIF, and restricts influenza and Zika viruses through direct C-terminal RNA binding, while it targets other viral polymerases (e.g., CVB3 3D) for K48-linked ubiquitination and proteasomal degradation (PMID:22948160, PMID:38556084, PMID:31251739, PMID:39348396). Beyond innate immunity, TRIM56 ubiquitinates numerous cellular substrates—including FASN, TLE3, vimentin, IQGAP1, TAK1, Src, KLF4, and GCN2—using K48- or K63-linked chains to control lipid metabolism, adipose thermogenesis, cell migration, NF-κB signaling, and ferroptosis, with its coiled-coil domain forming a tetrameric scaffold that positions RING domains for ubiquitin transfer (PMID:38206764, PMID:39928840, PMID:36870986, PMID:35952808, PMID:37168870).

1. 2010 High

   Establishing TRIM56 as an innate immune E3 ligase: it was unknown how STING dimerization was triggered; the demonstration that TRIM56 catalyzes K63-linked ubiquitination of STING to induce dimerization and TBK1 recruitment identified TRIM56 as a critical upstream activator of cytosolic DNA sensing.

   Evidence Co-immunoprecipitation, ubiquitination assay, and IFN-β reporter with overexpression/knockdown in human cells

   PMID:21074459

   Open questions at the time

   • Ubiquitination site on STING not mapped
   • No in vivo validation in mouse models at this stage
   • Whether TRIM56 acts on other innate sensors was unknown
2. 2011 High

   Defining TRIM56 as a direct-acting antiviral factor: it was unclear whether TRIM56 could restrict viruses beyond the STING pathway; showing E3 ligase-dependent restriction of BVDV but not VSV or HCV revealed virus-specific direct antiviral mechanisms.

   Evidence TRIM56 overexpression/knockdown with E3-dead and C-terminal deletion mutants, viral replication assays

   PMID:21289118

   Open questions at the time

   • Viral substrate targeted by TRIM56 not identified
   • Mechanism of C-terminal requirement unclear
3. 2012 High

   Separating TRIM56's scaffolding from ligase functions: the discovery that TRIM56 positively regulates TLR3-TRIF signaling independent of E3 ligase activity, via direct C-terminal binding to TRIF, revealed a second, non-catalytic mode of innate immune regulation.

   Evidence Reciprocal Co-IP, C-terminal domain mapping, IRF3 and IFN-β reporter assays

   PMID:22948160

   Open questions at the time

   • Post-translational modifications governing TRIF binding not identified
   • In vivo relevance of TLR3 arm not tested
4. 2014 High

   Broadening antiviral scope and dissecting domain requirements: TRIM56 restricted flaviviruses (YFV, DENV2) requiring both E3 and C-terminal domains but restricted HCoV-OC43 via E3 activity alone, indicating virus-specific mechanistic modules.

   Evidence Conditional cell lines with domain mutants, intracellular viral RNA quantification across multiple viruses

   PMID:25253338

   Open questions at the time

   • Exact viral targets for ubiquitination not identified for flaviviruses
   • Host cofactors for C-terminal-mediated restriction unknown
5. 2016 High

   Revealing an E3-independent RNA-binding antiviral mechanism: TRIM56 restriction of influenza A/B required only a 63-residue C-terminal tail and was independent of the RING, B-box, and coiled-coil domains, establishing a minimal antiviral module.

   Evidence Domain mutant expression with viral RNA synthesis assays; C-terminal segment sufficient for restriction

   PMID:26889027

   Open questions at the time

   • Whether the C-terminal tail directly contacts viral RNA not shown
   • Structural basis of the 63-residue tail function unresolved
6. 2018 High

   Identifying TRIM56 as the activating E3 ligase for cGAS: TRIM56-catalyzed monoubiquitination of cGAS at Lys335 enhanced cGAS dimerization and DNA binding, and TRIM56-knockout mice were highly susceptible to HSV-1, placing TRIM56 as an essential upstream regulator of cGAS-STING signaling in vivo.

   Evidence Site-directed mutagenesis (K335R), in vitro monoubiquitination assay, cGAS biochemical assays, TRIM56-KO mice with HSV-1 challenge

   PMID:29426904

   Open questions at the time

   • Structural basis of TRIM56-cGAS recognition unknown
   • Whether additional E3 ligases redundantly activate cGAS not addressed
7. 2019 High

   Demonstrating direct RNA-binding as the mechanism for Zika virus restriction: TRIM56's C-terminal 392 residues directly bound ZIKV RNA in a cell-free system, and a short C-terminal tail deletion abolished both RNA binding and antiviral activity, confirming RNA binding as the effector mechanism independent of Dicer.

   Evidence Recombinant protein–RNA binding assay, RNA immunoprecipitation, Dicer-KO epistasis

   PMID:31251739

   Open questions at the time

   • RNA sequence/structure specificity not defined
   • No crystal structure of RNA-binding domain
8. 2019 Medium

   Expanding TRIM56 substrates to non-immune targets: TRIM56 was shown to ubiquitinate ERα (K63-linked, stabilizing) and SAP18 (proteasomal degradation via KSHV vFLIP hijacking), linking TRIM56 to cancer cell proliferation and viral immune evasion beyond innate sensing.

   Evidence Co-IP with domain mapping, ubiquitination assays, xenograft and functional assays in breast cancer and KSHV-infected cells

   PMID:30670829 PMID:31000690

   Open questions at the time

   • ERα ubiquitination site not mapped
   • vFLIP-TRIM56 interaction not reconstituted with purified components
   • Physiological relevance in non-cancer settings not established
9. 2022 Medium

   Consolidating TRIM56 as a multi-substrate E3 ligase across signaling pathways: studies identified IQGAP1 (K48→K63 switch activating CDC42), TAK1 (M1-linked ubiquitination activating NF-κB), IκBα (ubiquitination promoting NF-κB against HBV), and vimentin (degradation controlling migration) as TRIM56 substrates, revealing its versatility in ubiquitin chain-type specification.

   Evidence Co-IP with linkage-specific ubiquitin analysis, domain mapping, knockdown/overexpression with functional readouts across glioma, HBV, and ovarian cancer models

   PMID:28771721 PMID:35952808 PMID:36084850 PMID:36870986

   Open questions at the time

   • In vitro reconstitution of chain-type specificity switching not performed
   • Mechanism determining K48 vs K63 vs M1 chain selection by TRIM56 unknown
   • Most findings from single laboratories
10. 2022 High

    Genetic confirmation that TRIM56 is required for both TLR3 and cGAS-STING pathways but dispensable for IFN-I downstream signaling: TRIM56-KO HeLa cells showed severely impaired ISG induction by extracellular dsRNA and cytosolic dsDNA but normal IFN-α response, clarifying TRIM56's position upstream of IFN production.

    Evidence CRISPR/Cas9 KO with dsRNA, dsDNA, and IFN-α stimulation and ISG/antiviral bioactivity readouts

    PMID:35062293

    Open questions at the time

    • Single cell line (HeLa); generalizability across cell types not tested
    • Relative contributions of STING vs cGAS ubiquitination not separated
11. 2023 High

    Structural basis for TRIM56 E3 activity: the crystal structure of the coiled-coil domain revealed an antiparallel dimer-of-dimers tetramer that positions two RING domains on each side for productive E2-ubiquitin transfer, explaining how quaternary organization supports catalysis.

    Evidence X-ray crystallography of the TRIM56 coiled-coil domain

    PMID:37168870

    Open questions at the time

    • Full-length structure unavailable
    • How C-terminal substrate-recruiting domains are oriented relative to RING not resolved
    • No structure of RING-E2~Ub complex
12. 2024 High

    Establishing TRIM56 as a metabolic regulator: TRIM56 directly ubiquitinates FASN (K48-linked) for proteasomal degradation to limit lipogenesis, and ubiquitinates TLE3 (K48-linked) to activate thermogenic gene programs in white adipose tissue, connecting TRIM56 to NAFLD protection and adaptive thermogenesis.

    Evidence Hepatocyte- and adipocyte-specific conditional KO/OE mice, NAFLD/obesity/cold models, ubiquitination assays

    PMID:38206764 PMID:39928840

    Open questions at the time

    • Ubiquitination sites on FASN not mapped
    • Upstream signals regulating TRIM56 in metabolic tissues unknown
    • Whether metabolic and immune functions of TRIM56 are coordinately regulated is unexplored
13. 2024 High

    Revealing regulation of TRIM56 itself: HDAC6 deacetylates TRIM56 at Lys110, impairing its ability to monoubiquitinate cGAS, and HSV-1 US3 protein exploits this axis by phosphorylating HDAC6, establishing TRIM56 acetylation as a regulatory switch for cGAS activation.

    Evidence HDAC6-KO cells and mice, deacetylation assay, cGAS ubiquitination and DNA-binding assays, HSV-1 US3 mechanism

    PMID:39747662

    Open questions at the time

    • Acetyltransferase responsible for TRIM56 K110 acetylation not identified
    • Whether acetylation affects TRIM56 activity on substrates other than cGAS unknown
14. 2024 High

    Defining phosphorylation codes for TLR3 signaling: Ser471, Ser475, and Ser710 phosphorylation of TRIM56 were shown to be required for TLR3-TRIF augmentation; Ser710 specifically governs TRIF association, and Ser471/Ser475 phosphorylation is biphasic after poly(I:C) stimulation, providing the first post-translational regulatory map for TRIM56's scaffolding function.

    Evidence Alanine-substitution mutants, phospho-specific antibodies, IFN-β and NF-κB reporters, Co-IP in Tet-regulated cell lines

    PMID:38556084

    Open questions at the time

    • Kinase(s) responsible for Ser471/Ser475/Ser710 phosphorylation not identified
    • Whether phosphorylation regulates E3 ligase activity in other contexts not tested
15. 2024 High

    Demonstrating TRIM56 as a direct antiviral E3 ligase targeting viral polymerases: TRIM56 ubiquitinates CVB3 RNA-dependent RNA polymerase 3D at Lys220 (K48-linked) for proteasomal degradation, and the virus counters by 3C protease-mediated cleavage of TRIM56, revealing an arms race dynamic.

    Evidence Pull-down, site-directed mutagenesis (K220R), ubiquitination assay, viral yield measurement, 3C cleavage assay

    PMID:39348396

    Open questions at the time

    • Whether TRIM56 directly ubiquitinates polymerases of other viruses (flaviviruses, coronaviruses) remains untested
    • Cleavage site on TRIM56 not mapped

• Key unresolved questions include: the structural basis for TRIM56's dual RNA-binding and E3 ligase activities in a full-length context; the kinases and acetyltransferases that regulate TRIM56 post-translational modifications; how TRIM56 achieves ubiquitin chain-type specificity (K48, K63, M1, monoubiquitin) for different substrates; and the physiological integration of TRIM56's immune, metabolic, and cytoskeletal functions.
• No full-length TRIM56 structure
• Chain-type specification mechanism unknown
• Upstream kinases for Ser471/Ser475/Ser710 unidentified
• Acetyltransferase for K110 unidentified