{"gene":"HERC5","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2006,"finding":"HERC5 (also known as Ceb1) is an IFN-induced HECT-type E3 protein ligase that mediates ISGylation. Its active-site cysteine (Cys-994) in the HECT domain is required for E3 ligase activity, as a C994A substitution completely abrogates ISGylation. HERC5 coexpression with Ube1L (E1) and UbcH8 (E2) reconstitutes ISG15 conjugation in vivo independent of IFN stimulation.","method":"Active-site mutagenesis (C994A), siRNA knockdown, co-transfection reconstitution, in vivo ISGylation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis combined with reconstitution assay, independently replicated in a concurrent paper (PMID:16407192)","pmids":["16815975"],"is_preprint":false},{"year":2005,"finding":"Herc5 is required for ISG15 conjugation to a broad spectrum of target proteins in human cells. siRNA knockdown of Herc5 abolishes the vast majority of ISG15 conjugation. Co-transfection of ISG15, Ube1L, UbcH8, and Herc5 reconstitutes robust ISG15 conjugation in non-IFN-treated cells. The active-site cysteine mutant of Herc5 and a mutant lacking the RCC1 repeat region do not support ISG15 conjugation.","method":"siRNA knockdown, co-transfection reconstitution, active-site mutagenesis, domain deletion mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with multiple mutants, siRNA loss-of-function, replicated across two labs (PMID:16815975)","pmids":["16407192"],"is_preprint":false},{"year":2004,"finding":"HERC5 possesses ubiquitin ligase activity in vitro and requires the ubiquitin-conjugating enzyme UbcH5a for its activity. HERC5 protein is subject to enhanced degradation upon LPS stimulation of endothelial cells despite upregulation of its mRNA.","method":"In vitro ubiquitin ligase assay, subcellular fractionation/western blot, differential mRNA/protein quantification","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro assay from single lab, no mutagenesis or replication","pmids":["15331633"],"is_preprint":false},{"year":2006,"finding":"Herc5 functions as a general E3 ISG15 ligase that physically binds target proteins and stimulates their ISGylation in a UBE1L- and UbcH8-dependent manner. Six novel ISGylation substrates were identified by proteomics and confirmed by immunoblot.","method":"Proteomic identification of substrates, co-immunoprecipitation, immunoblot, E1/E2 dependency assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional stimulation assay, single lab with multiple substrate validations","pmids":["16884686"],"is_preprint":false},{"year":2010,"finding":"HERC5 binds IRF3 (identified by immunoprecipitation) and catalyzes ISGylation of IRF3 at Lys193, Lys360, and Lys366. This ISGylation attenuates the interaction between Pin1 and IRF3, preventing IRF3 polyubiquitination and degradation, thereby sustaining IRF3 activation and antiviral gene expression.","method":"Co-immunoprecipitation, site-directed mutagenesis of IRF3 lysines, siRNA knockdown, overexpression, Sendai virus infection assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, lysine-site mutagenesis, loss- and gain-of-function with defined functional readout, single lab with multiple orthogonal methods","pmids":["20308324"],"is_preprint":false},{"year":2010,"finding":"Herc5 catalyzes ISGylation of influenza A virus NS1 protein at Lys20, Lys41, Lys108, Lys110, Lys126, Lys217, and Lys219. ISGylated NS1 fails to form homodimers and its antiviral inhibitory functions are blocked. Knockdown of Herc5 partially alleviates IFN-β-induced antiviral activity, while ectopic expression of the Herc5-mediated ISGylation system potentiates antiviral effects.","method":"Mass spectrometry mapping of ISGylation sites, mutagenesis, homodimerization assay, siRNA knockdown, viral replication assay, mouse infection model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mass spectrometry site mapping, site-directed mutagenesis, functional assays in cells and mice, single lab with multiple orthogonal approaches","pmids":["20385878"],"is_preprint":false},{"year":2011,"finding":"HERC5 inhibits HIV-1 Gag particle production. E3 ligase activity of HERC5 is required for this restriction, which correlates with ISGylation of HIV-1 Gag. HERC5 interacts with HIV-1 Gag but does not alter Gag trafficking to the plasma membrane. Electron microscopy shows HIV-1 Gag particle assembly is arrested at the plasma membrane at an early stage. HERC5 also restricts murine leukemia virus Gag particle production.","method":"Overexpression, siRNA knockdown, co-immunoprecipitation, electron microscopy, Gag particle production assay, active-site mutant analysis","journal":"Retrovirology","confidence":"High","confidence_rationale":"Tier 2 / Strong — electron microscopy, Co-IP, active-site mutant, multiple viruses tested, single lab with multiple orthogonal methods","pmids":["22093708"],"is_preprint":false},{"year":2014,"finding":"HERC5 inhibits HIV-1 particle production by a second mechanism distinct from ISGylation: it targets nuclear export of Rev/RRE-dependent RNA via its RCC1-like domain (N-terminal), without requiring E3 ligase activity. This inhibition correlates with reduced intracellular RanGTP levels and/or impaired RanGTP–RanBP1 interaction, and with altered subcellular localization of HIV-1 Rev. A region in the RCC1-like domain under strong positive evolutionary selection is required for this activity.","method":"Domain deletion/mutagenesis, Rev/RRE-dependent nuclear export assay, RanGTP level measurement, RanBP1 interaction assay, subcellular localization imaging, positive selection analysis","journal":"Retrovirology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mutagenesis combined with functional nuclear export assay and localization studies, single lab with multiple orthogonal methods","pmids":["24693865"],"is_preprint":false},{"year":2017,"finding":"HERC5 physically interacts with β-catenin (immunoprecipitation) and mediates ISGylation of β-catenin, promoting its proteasomal degradation. CYP1B1 suppresses Herc5 expression, thereby stabilizing β-catenin and activating Wnt/β-catenin signaling in HeLa cells.","method":"Co-immunoprecipitation, RT-PCR, western blot, siRNA knockdown, CYP1B1 overexpression/inhibition","journal":"Toxicological research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP showing interaction, functional pathway inference from expression modulation, single lab","pmids":["28744352"],"is_preprint":false},{"year":2021,"finding":"HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein (HBx) at Lys91, Lys95, and Lys140. This ISGylation promotes pro-viral HBV replication and contributes to IFN-α resistance, as silencing ISG15 markedly reduced HBV replication in IFN-α-treated cells while silencing USP18 (de-ISGylase) increased replication.","method":"Immunoblot, site-directed mutagenesis, expression plasmids for E3 ligases, siRNA knockdown, HBV replication assay","journal":"The Journal of general virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site mutagenesis, E3 identification, functional replication assay, single lab","pmids":["34661519"],"is_preprint":false},{"year":2021,"finding":"HERC5 recruits an adaptor protein (CREB-binding protein) to ubiquitinate CtBP1 in non-cancerous colon cells. Downregulation of HERC5 in colorectal cancer attenuates CtBP1 ubiquitination, allowing CtBP1 to accumulate and assemble a transcriptional complex with HDAC1 and c-MYC that represses pro-apoptotic genes (BAX, BIK, PUMA).","method":"Co-immunoprecipitation, ubiquitination assay, western blot, siRNA knockdown, overexpression, promoter binding assay, in vivo xenograft","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, loss- and gain-of-function with defined downstream readout, single lab","pmids":["34147029"],"is_preprint":false},{"year":2023,"finding":"HERC5 (human) and mHERC6 (mouse) mediate ISGylation of the phosphatase PTEN in macrophages, promoting PTEN degradation and thereby increasing PI3K-AKT signaling and proinflammatory cytokine synthesis, enhancing antimycobacterial responses.","method":"Co-immunoprecipitation, ISGylation assay, siRNA knockdown, KO macrophages, PI3K-AKT pathway analysis, bacterial growth assay in vitro and in vivo","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional KO assay with bacterial load readout, single lab","pmids":["37279284"],"is_preprint":false},{"year":2023,"finding":"African swine fever virus MGF-360-10L recruits HERC5 to mediate K48-linked ubiquitination (not ISGylation) of JAK1 at Lys245 and Lys269, leading to JAK1 proteasomal degradation and suppression of STAT1/2 signaling.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis, viral deletion mutant, in vivo virulence assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site mutagenesis, in vivo viral model, single lab","pmids":["37417777"],"is_preprint":false},{"year":2024,"finding":"HERC5 ISGylates cGAS at Lys21, Lys187, Lys219, and Lys458, promoting cGAS oligomerization and enhancing cGAS enzymatic activity. The interaction depends on the cGAS C-terminal domain and RCC1-4/RCC1-5 domains of HERC5. ISGylation deficiency attenuates cGAS-STING-mediated inflammatory gene expression and antiviral defense in mouse and human cells.","method":"Co-immunoprecipitation, mass spectrometry site identification, mutagenesis, cGAS activity assay, oligomerization assay, ISGylation assay, KO mouse model (HSV-1 infection)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mass spectrometry, mutagenesis, enzymatic activity assay, domain mapping, in vivo mouse model, single lab with multiple orthogonal methods","pmids":["38421872"],"is_preprint":false},{"year":2024,"finding":"HERC5 ISGylates SARS-CoV-2 nucleocapsid (N) protein at Lys266, Lys355, Lys387, and Lys388 (preferentially the phosphorylated form), impeding N protein oligomerization and inhibiting viral RNA synthesis. This ISGylation is reversed by PLpro (NSP3) deISGylation activity. Mutant N-4KR abolishes ISGylation and alleviates restriction in a replicon system.","method":"Mass spectrometry, site-directed mutagenesis, SARS-CoV-2 replicon system, oligomerization assay, ISGylation assay, PLpro activity assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mass spectrometry site identification, mutagenesis in replicon system, oligomerization assay, single lab with multiple orthogonal approaches; replicated in preprint PMID:39149229","pmids":["39194248","39149229"],"is_preprint":false},{"year":2024,"finding":"hHERC5 broadly ISGylates proteins cotranslationally as a ribosome-associated HECT E3 ligase. Over 2,000 modified lysines in over 1,100 proteins were characterized in IFN-β-stimulated cells. hHERC5 and mouse HERC6 have distinct amino acid sequence context preferences surrounding ISGylation sites. mHERC6 also cotranslationally modifies nascent polypeptides.","method":"Mass spectrometry ISGylome profiling, IFN-β stimulation, sequence context analysis, comparison with mHERC6","journal":"iScience","confidence":"High","confidence_rationale":"Tier 1 / Strong — large-scale mass spectrometry with rigorous controls, direct comparison of substrate selectivity, establishes cotranslational mechanism","pmids":["38303729"],"is_preprint":false},{"year":2022,"finding":"HERC5 interacts with IFI16 and mediates ISGylation of IFI16 at Lys274, facilitating IFI16 proteasomal degradation. IFI16 acts as a tumor suppressor downstream of HERC5, and IFI16 is positively correlated with p53 expression, defining a HERC5/IFI16/p53 signaling pathway in breast cancer cells.","method":"Co-immunoprecipitation, proteomic analysis, western blot, siRNA knockdown, site mutagenesis, cell proliferation/migration assays","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-directed mutagenesis, functional KD assay, single lab","pmids":["35671810"],"is_preprint":false},{"year":2021,"finding":"HERC5 inhibits Ebola virus (EBOV) VLP replication by depleting EBOV mRNAs. The RCC1-like domain of HERC5 is necessary and sufficient for this inhibition and does not require zinc finger antiviral protein (ZAP). EBOV glycoprotein (Zaire strain) antagonizes HERC5 early during infection.","method":"Transcription- and replication-competent VLP system, domain deletion analysis, ZAP-deficient cell assay, EBOV GP antagonism assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — VLP replication assay with domain mapping and negative control (Marburg GP), single lab","pmids":["34572049"],"is_preprint":false},{"year":2025,"finding":"HERC5 ISGylates SARS-CoV-2 nsp8 (a viral replication factor) at its N2 domain lysine residues, facilitating proteasome-dependent nsp8 degradation and suppressing viral replication. SARS-CoV-2 PLpro counteracts this by deconjugating ISG15 from nsp8, preventing its degradation. The full ISGylation system (HERC5, UBA7, ISG15) suppresses replication of multiple SARS-CoV-2 variants including Omicron.","method":"Mass spectrometry, mutagenesis, overexpression, PLpro deISGylation assay, proteasome inhibition, viral replication assay","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry, mutagenesis, functional viral replication assay, single lab","pmids":["40409630"],"is_preprint":false},{"year":2025,"finding":"HERC5 catalyzes ISGylation of UGDH (UDP-glucose 6-dehydrogenase) via its HECT domain active-site Cys994, facilitating UGDH phosphorylation and enhancing SNAI1 mRNA stability, thereby promoting OSCC cell invasion and cisplatin resistance.","method":"Co-immunoprecipitation, ISGylation assay, site mutagenesis (C994A), siRNA knockdown, overexpression, in vivo xenograft","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, active-site mutagenesis, functional in vivo assay, single lab","pmids":["40045362"],"is_preprint":false},{"year":2025,"finding":"HERC5 inhibits LINE-1 (L1) retrotransposition through an ISGylation-independent mechanism. HERC5 interacts with L1 RNA and selectively reduces ORF1p protein levels, decreasing L1 translation efficiency and altering L1 RNP composition.","method":"L1 retrotransposition assay, HERC5 domain/catalytic mutant analysis, RNA immunoprecipitation, ORF1p level quantification, RNP composition analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional retrotransposition assay, RNA-IP, catalytic mutant confirming ISGylation-independence, single lab","pmids":["42055552"],"is_preprint":false},{"year":2025,"finding":"UbcH8~ISG15 exhibits striking specificity for HECT-family E3 ligases (particularly HERC5) but is inactive with RING or RBR E3s, in contrast to UbcH8~Ub which preferentially engages RBR E3s. A unique closed conformation of UbcH8~ISG15 enables trans-thiolation selectively mediated by HECT E3s. HERC5's C-lobe specifically recognizes donor ISG15 for lysine conjugation, explaining its exclusive ISGylation activity and lack of ubiquitination function.","method":"Structural analysis (biochemical studies), in vitro thiolation assay, comparative E3 ligase specificity assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural and biochemical studies from single lab, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.09.26.678755"],"is_preprint":true},{"year":2025,"finding":"SAXS analysis reveals that the HERC5 HECT domain predominantly adopts a trimeric assembly in solution, suggesting trimerization may play a regulatory role in HERC5 functional activity.","method":"Small-angle X-ray scattering (SAXS), size-exclusion chromatography, Guinier/Kratky analysis, ab initio shape reconstruction","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 1 / Weak — SAXS structure from single lab, preprint, functional consequence of trimerization not directly demonstrated","pmids":["bio_10.1101_2025.03.22.644726"],"is_preprint":true},{"year":2024,"finding":"HERC5 (human) and HERC6 (mouse functional homolog) are the primary E3 ligases responsible for ISGylation of MDA5 at Lys23 and Lys43 within its CARD domain. ISGylation at these sites promotes MDA5 oligomeric assembly and downstream antiviral signaling; knock-in mice with K23R/K43R MDA5 mutations show abrogated ISGylation, impaired MDA5 oligomerization, blunted cytokine responses, and increased mortality upon EMCV infection.","method":"MDA5 K23R/K43R knock-in mice, ISGylation assay, MDA5 oligomerization assay, cytokine measurement, viral infection survival assay, E3 ligase identification","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in mouse with mutagenesis, functional oligomerization and infection assay, preprint from single lab","pmids":["bio_10.1101_2024.09.20.614144"],"is_preprint":true},{"year":2025,"finding":"HERC5 interacts with and ISGylates IRF3, preventing IRF3 ubiquitination and degradation. This stabilizes IRF3 and sustains IFN-β production and overactivation in podocytes, contributing to lupus nephritis pathology.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, ISGylation assay, IFN-β quantification, podocyte injury assay","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional loss/gain-of-function with defined readout, consistent with prior IRF3 ISGylation findings (PMID:20308324), single lab","pmids":["40505230"],"is_preprint":false}],"current_model":"HERC5 is an interferon-induced HECT-type E3 ligase that functions as the primary cellular ISG15 conjugating enzyme (E3) in humans, working sequentially with E1 (UBE1L/UBA7) and E2 (UbcH8) to broadly ISGylate nascent polypeptides cotranslationally on ribosomes; its catalytic Cys994 in the HECT domain is essential for ISGylation, while its N-terminal RCC1-like domain mediates a second antiviral mechanism by blocking Rev/RRE-dependent nuclear RNA export and reducing RanGTP levels; established substrates include IRF3, cGAS, MDA5, PTEN, IFI16, β-catenin, CtBP1, and multiple viral proteins (influenza NS1, HIV Gag, SARS-CoV-2 N protein and nsp8, HBx), with ISGylation variously promoting target stabilization (IRF3, cGAS), degradation (PTEN, nsp8), or functional disruption (viral homodimers, oligomers), thereby broadly potentiating innate antiviral and antibacterial immunity."},"narrative":{"mechanistic_narrative":"HERC5 is the principal cellular E3 ligase for ISG15 conjugation (ISGylation) in human cells and a central effector of interferon-induced innate antiviral immunity [PMID:16407192, PMID:38303729]. It is an interferon-induced HECT-type ligase whose catalytic activity depends on an active-site cysteine, Cys994, in its C-terminal HECT domain; this residue is essential, since its substitution abolishes ISGylation, and HERC5 reconstitutes robust ISG15 conjugation together with the E1 UBE1L and E2 UbcH8 [PMID:16815975, PMID:16407192]. As a ribosome-associated ligase, HERC5 ISGylates a broad spectrum of nascent polypeptides cotranslationally, modifying thousands of lysines across over a thousand proteins, with sequence-context preferences distinct from the mouse homolog HERC6 [PMID:38303729]. Through this activity HERC5 amplifies innate immune signaling by ISGylating pathway components: it modifies IRF3 to block its Pin1-dependent degradation and sustain antiviral gene expression [PMID:20308324, PMID:40505230], promotes cGAS oligomerization and enzymatic activity [PMID:38421872], and drives MDA5 CARD-domain oligomerization required for downstream signaling [PMID:bio_10.1101_2024.09.20.614144]. HERC5 also directly restricts viruses by ISGylating viral proteins to disrupt their function — blocking influenza NS1 and SARS-CoV-2 nucleocapsid oligomerization and targeting SARS-CoV-2 nsp8 for degradation [PMID:20385878, PMID:39194248, PMID:39149229, PMID:40409630]. A second, ISGylation-independent antiviral activity resides in its N-terminal RCC1-like domain, which inhibits Rev/RRE-dependent nuclear RNA export and reduces RanGTP levels to restrict HIV-1 [PMID:24693865]. Beyond antiviral defense, HERC5-mediated ISGylation regulates cellular substrates including PTEN, β-catenin, IFI16, and UGDH, linking it to inflammatory signaling and cancer-associated pathways [PMID:37279284, PMID:28744352, PMID:35671810, PMID:40045362].","teleology":[{"year":2004,"claim":"Established that HERC5 is an active ubiquitin-pathway E3 ligase, providing the first biochemical evidence of its enzymatic activity before its ISG15-specific role was known.","evidence":"In vitro ubiquitin ligase assay with UbcH5a and protein/mRNA quantification in endothelial cells","pmids":["15331633"],"confidence":"Medium","gaps":["Did not identify ISG15 as the relevant conjugate","No active-site mutagenesis","Physiological substrates unknown"]},{"year":2005,"claim":"Identified HERC5 as the enzyme required for the bulk of cellular ISG15 conjugation, defining its function as a general ISG15 E3 ligase rather than a generic ubiquitin ligase.","evidence":"siRNA knockdown, four-component reconstitution, and active-site plus RCC1-deletion mutants in human cells","pmids":["16407192"],"confidence":"High","gaps":["Substrate selection mechanism unresolved","Did not map specific modified lysines"]},{"year":2006,"claim":"Defined the catalytic and enzymatic logic of HERC5 ISGylation by pinpointing the essential HECT active-site Cys994 and showing it physically binds and stimulates ISGylation of targets with E1/E2 dependence.","evidence":"C994A mutagenesis, co-transfection reconstitution, proteomic substrate identification and reciprocal Co-IP","pmids":["16815975","16884686"],"confidence":"High","gaps":["Mechanism conferring ISG15 over ubiquitin specificity not explained","Functional consequence of ISGylation on substrates not yet defined"]},{"year":2010,"claim":"Showed that HERC5 ISGylation can stabilize a substrate to amplify signaling, establishing a positive-regulatory role in innate immunity through IRF3 modification.","evidence":"Co-IP, IRF3 lysine-site mutagenesis, and Sendai virus infection assays linking ISGylation to blocked Pin1-mediated degradation","pmids":["20308324"],"confidence":"High","gaps":["Did not establish in vivo physiological requirement","Interplay with deISGylases not addressed"]},{"year":2010,"claim":"Demonstrated a direct antiviral mechanism whereby ISGylation of a viral protein disrupts its function, exemplified by influenza NS1.","evidence":"Mass spectrometry site mapping, mutagenesis, homodimerization assay, and viral replication assays in cells and mice","pmids":["20385878"],"confidence":"High","gaps":["Generality of oligomerization-blocking mechanism across viral substrates untested at the time"]},{"year":2014,"claim":"Revealed a second, catalytically independent antiviral activity in the N-terminal RCC1-like domain, separating HERC5's ligase function from its control of nuclear RNA export.","evidence":"Domain mutagenesis, Rev/RRE nuclear export assays, RanGTP and RanBP1 measurements, and localization imaging for HIV-1","pmids":["22093708","24693865"],"confidence":"High","gaps":["Molecular target of the RCC1-like domain on the Ran cycle not defined","Generality beyond HIV-1 untested in these studies"]},{"year":2023,"claim":"Extended HERC5 ISGylation to destabilizing substrates and to antibacterial defense, showing ISGylation can target proteins for degradation to reprogram signaling.","evidence":"Co-IP, KO macrophages, PI3K-AKT analysis, and bacterial growth assays for PTEN; viral-hijacking Co-IP and in vivo assays for JAK1 ubiquitination","pmids":["37279284","37417777"],"confidence":"Medium","gaps":["JAK1 ubiquitination represents virus-directed hijacking distinct from ISGylation","Determinants of degradative vs stabilizing outcome unclear"]},{"year":2024,"claim":"Established HERC5 as a ribosome-associated cotranslational ISGylation machine acting on nascent polypeptides at proteome scale, defining the operating mode of the enzyme.","evidence":"Large-scale mass spectrometry ISGylome profiling in IFN-β-stimulated cells with sequence-context analysis and HERC6 comparison","pmids":["38303729"],"confidence":"High","gaps":["How cotranslational targeting reconciles with selective regulatory substrates is unresolved","Determinants of site selection beyond local sequence unclear"]},{"year":2024,"claim":"Showed ISGylation can activate immune sensors by promoting oligomerization, generalizing the positive-regulatory role across the cGAS-STING and RIG-I-like receptor pathways.","evidence":"Mass spectrometry site mapping, domain mapping, oligomerization and enzymatic activity assays, and KO/knock-in mouse infection models for cGAS and MDA5","pmids":["38421872","bio_10.1101_2024.09.20.614144"],"confidence":"Medium","gaps":["MDA5 evidence is from a preprint","Stoichiometry of ISGylation needed for oligomerization not quantified"]},{"year":2024,"claim":"Defined the antagonism between HERC5 ISGylation and viral deISGylases, showing SARS-CoV-2 proteins are ISGylated to block oligomerization and target replication factors for degradation, countered by PLpro.","evidence":"Mass spectrometry, mutagenesis, replicon systems, oligomerization assays, and PLpro deISGylation assays for N protein and nsp8","pmids":["39194248","40409630"],"confidence":"Medium","gaps":["Relative contribution of N vs nsp8 ISGylation to restriction unresolved","In vivo relevance not established"]},{"year":2025,"claim":"Provided a structural basis for HERC5's exclusive ISGylation specificity, explaining why UbcH8~ISG15 engages HECT but not RING/RBR E3s.","evidence":"Structural and biochemical analysis of UbcH8~ISG15 conformation and trans-thiolation specificity (preprint)","pmids":["bio_10.1101_2025.09.26.678755"],"confidence":"Medium","gaps":["Preprint not peer-reviewed","No high-resolution complex structure of HERC5 with charged E2","Awaits independent confirmation"]},{"year":2025,"claim":"Implicated HERC5 in non-antiviral cellular contexts including cancer-associated substrate regulation and retrotransposon control via ISGylation-independent RNA binding.","evidence":"Co-IP and ISGylation/xenograft assays for UGDH; retrotransposition, RNA-IP, and catalytic-mutant analyses for LINE-1 ORF1p","pmids":["40045362","42055552"],"confidence":"Medium","gaps":["Mechanism of ISGylation-independent RNA binding undefined","Single-lab findings without independent replication"]},{"year":null,"claim":"How HERC5 reconciles broad cotranslational ISGylation of nascent chains with selective, functionally consequential modification of specific regulatory and viral substrates, and how its HECT trimerization and RCC1-like domain are coordinated, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking cotranslational targeting to substrate-specific outcomes","Functional role of HECT trimerization undemonstrated","Regulation of the two distinct antiviral mechanisms not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,13,15]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[1,15]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[20]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[15]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,5,13,23]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,9,14,18]}],"complexes":[],"partners":["ISG15","UBA7","UBCH8","IRF3","CGAS","MDA5","PTEN","CTNNB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UII4","full_name":"E3 ISG15--protein ligase HERC5","aliases":["Cyclin-E-binding protein 1","HECT domain and RCC1-like domain-containing protein 5"],"length_aa":1024,"mass_kda":116.9,"function":"Major E3 ligase for ISG15 conjugation (PubMed:26355087, PubMed:27534820, PubMed:27564865, PubMed:34572049, PubMed:37279284). Acts as a positive regulator of innate antiviral response in cells induced by interferon. Functions as part of the ISGylation machinery that recognizes target proteins in a broad and relatively non-specific manner. Catalyzes ISGylation of IRF3 which results in sustained activation, it attenuates IRF3-PIN1 interaction, which antagonizes IRF3 ubiquitination and degradation, and boosts the antiviral response. Mediates ISGylation of the phosphatase PTEN leading to its degradation, thus alleviating its suppression of the PI3K-AKT signaling pathway and promoting the production of cytokines that facilitate bacterial clearance (PubMed:37279284). Interferes with the function of key viral structural proteins such as ebolavirus structural protein VP40 or HIV-1 protein GAG (PubMed:22093708, PubMed:34572049). Catalyzes ISGylation of influenza A viral NS1 which attenuates virulence; ISGylated NS1 fails to form homodimers and thus to interact with its RNA targets. Catalyzes ISGylation of papillomavirus type 16 L1 protein which results in dominant-negative effect on virus infectivity. Physically associated with polyribosomes, broadly modifies newly synthesized proteins in a cotranslational manner. In an interferon-stimulated cell, newly translated viral proteins are primary targets of ISG15. Promotes parkin/PRKN ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation (PubMed:27534820) (Microbial infection) Functions as an E3 ligase for ISGylation of hepatitis B virus protein X leading to enhanced viral replication due to increased interferon resistance","subcellular_location":"Cytoplasm, perinuclear region; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9UII4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HERC5","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":[{"gene":"NPM1","stoichiometry":0.2},{"gene":"RACK1","stoichiometry":0.2},{"gene":"SRP14","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HERC5","total_profiled":1310},"omim":[{"mim_id":"609249","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 6; HERC6","url":"https://www.omim.org/entry/609249"},{"mim_id":"608242","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 5; HERC5","url":"https://www.omim.org/entry/608242"},{"mim_id":"605200","title":"HECT DOMAIN AND RCC1-LIKE DOMAIN 3; HERC3","url":"https://www.omim.org/entry/605200"},{"mim_id":"147571","title":"UBIQUITIN-LIKE MODIFIER ISG15; ISG15","url":"https://www.omim.org/entry/147571"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in 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Its active-site cysteine (Cys-994) in the HECT domain is required for E3 ligase activity, as a C994A substitution completely abrogates ISGylation. HERC5 coexpression with Ube1L (E1) and UbcH8 (E2) reconstitutes ISG15 conjugation in vivo independent of IFN stimulation.\",\n      \"method\": \"Active-site mutagenesis (C994A), siRNA knockdown, co-transfection reconstitution, in vivo ISGylation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis combined with reconstitution assay, independently replicated in a concurrent paper (PMID:16407192)\",\n      \"pmids\": [\"16815975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Herc5 is required for ISG15 conjugation to a broad spectrum of target proteins in human cells. siRNA knockdown of Herc5 abolishes the vast majority of ISG15 conjugation. Co-transfection of ISG15, Ube1L, UbcH8, and Herc5 reconstitutes robust ISG15 conjugation in non-IFN-treated cells. The active-site cysteine mutant of Herc5 and a mutant lacking the RCC1 repeat region do not support ISG15 conjugation.\",\n      \"method\": \"siRNA knockdown, co-transfection reconstitution, active-site mutagenesis, domain deletion mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with multiple mutants, siRNA loss-of-function, replicated across two labs (PMID:16815975)\",\n      \"pmids\": [\"16407192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"HERC5 possesses ubiquitin ligase activity in vitro and requires the ubiquitin-conjugating enzyme UbcH5a for its activity. HERC5 protein is subject to enhanced degradation upon LPS stimulation of endothelial cells despite upregulation of its mRNA.\",\n      \"method\": \"In vitro ubiquitin ligase assay, subcellular fractionation/western blot, differential mRNA/protein quantification\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro assay from single lab, no mutagenesis or replication\",\n      \"pmids\": [\"15331633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Herc5 functions as a general E3 ISG15 ligase that physically binds target proteins and stimulates their ISGylation in a UBE1L- and UbcH8-dependent manner. Six novel ISGylation substrates were identified by proteomics and confirmed by immunoblot.\",\n      \"method\": \"Proteomic identification of substrates, co-immunoprecipitation, immunoblot, E1/E2 dependency assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional stimulation assay, single lab with multiple substrate validations\",\n      \"pmids\": [\"16884686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HERC5 binds IRF3 (identified by immunoprecipitation) and catalyzes ISGylation of IRF3 at Lys193, Lys360, and Lys366. This ISGylation attenuates the interaction between Pin1 and IRF3, preventing IRF3 polyubiquitination and degradation, thereby sustaining IRF3 activation and antiviral gene expression.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of IRF3 lysines, siRNA knockdown, overexpression, Sendai virus infection assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, lysine-site mutagenesis, loss- and gain-of-function with defined functional readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20308324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Herc5 catalyzes ISGylation of influenza A virus NS1 protein at Lys20, Lys41, Lys108, Lys110, Lys126, Lys217, and Lys219. ISGylated NS1 fails to form homodimers and its antiviral inhibitory functions are blocked. Knockdown of Herc5 partially alleviates IFN-β-induced antiviral activity, while ectopic expression of the Herc5-mediated ISGylation system potentiates antiviral effects.\",\n      \"method\": \"Mass spectrometry mapping of ISGylation sites, mutagenesis, homodimerization assay, siRNA knockdown, viral replication assay, mouse infection model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mass spectrometry site mapping, site-directed mutagenesis, functional assays in cells and mice, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"20385878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HERC5 inhibits HIV-1 Gag particle production. E3 ligase activity of HERC5 is required for this restriction, which correlates with ISGylation of HIV-1 Gag. HERC5 interacts with HIV-1 Gag but does not alter Gag trafficking to the plasma membrane. Electron microscopy shows HIV-1 Gag particle assembly is arrested at the plasma membrane at an early stage. HERC5 also restricts murine leukemia virus Gag particle production.\",\n      \"method\": \"Overexpression, siRNA knockdown, co-immunoprecipitation, electron microscopy, Gag particle production assay, active-site mutant analysis\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — electron microscopy, Co-IP, active-site mutant, multiple viruses tested, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22093708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HERC5 inhibits HIV-1 particle production by a second mechanism distinct from ISGylation: it targets nuclear export of Rev/RRE-dependent RNA via its RCC1-like domain (N-terminal), without requiring E3 ligase activity. This inhibition correlates with reduced intracellular RanGTP levels and/or impaired RanGTP–RanBP1 interaction, and with altered subcellular localization of HIV-1 Rev. A region in the RCC1-like domain under strong positive evolutionary selection is required for this activity.\",\n      \"method\": \"Domain deletion/mutagenesis, Rev/RRE-dependent nuclear export assay, RanGTP level measurement, RanBP1 interaction assay, subcellular localization imaging, positive selection analysis\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mutagenesis combined with functional nuclear export assay and localization studies, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24693865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HERC5 physically interacts with β-catenin (immunoprecipitation) and mediates ISGylation of β-catenin, promoting its proteasomal degradation. CYP1B1 suppresses Herc5 expression, thereby stabilizing β-catenin and activating Wnt/β-catenin signaling in HeLa cells.\",\n      \"method\": \"Co-immunoprecipitation, RT-PCR, western blot, siRNA knockdown, CYP1B1 overexpression/inhibition\",\n      \"journal\": \"Toxicological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP showing interaction, functional pathway inference from expression modulation, single lab\",\n      \"pmids\": [\"28744352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein (HBx) at Lys91, Lys95, and Lys140. This ISGylation promotes pro-viral HBV replication and contributes to IFN-α resistance, as silencing ISG15 markedly reduced HBV replication in IFN-α-treated cells while silencing USP18 (de-ISGylase) increased replication.\",\n      \"method\": \"Immunoblot, site-directed mutagenesis, expression plasmids for E3 ligases, siRNA knockdown, HBV replication assay\",\n      \"journal\": \"The Journal of general virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site mutagenesis, E3 identification, functional replication assay, single lab\",\n      \"pmids\": [\"34661519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HERC5 recruits an adaptor protein (CREB-binding protein) to ubiquitinate CtBP1 in non-cancerous colon cells. Downregulation of HERC5 in colorectal cancer attenuates CtBP1 ubiquitination, allowing CtBP1 to accumulate and assemble a transcriptional complex with HDAC1 and c-MYC that represses pro-apoptotic genes (BAX, BIK, PUMA).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, western blot, siRNA knockdown, overexpression, promoter binding assay, in vivo xenograft\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, loss- and gain-of-function with defined downstream readout, single lab\",\n      \"pmids\": [\"34147029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HERC5 (human) and mHERC6 (mouse) mediate ISGylation of the phosphatase PTEN in macrophages, promoting PTEN degradation and thereby increasing PI3K-AKT signaling and proinflammatory cytokine synthesis, enhancing antimycobacterial responses.\",\n      \"method\": \"Co-immunoprecipitation, ISGylation assay, siRNA knockdown, KO macrophages, PI3K-AKT pathway analysis, bacterial growth assay in vitro and in vivo\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional KO assay with bacterial load readout, single lab\",\n      \"pmids\": [\"37279284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"African swine fever virus MGF-360-10L recruits HERC5 to mediate K48-linked ubiquitination (not ISGylation) of JAK1 at Lys245 and Lys269, leading to JAK1 proteasomal degradation and suppression of STAT1/2 signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis, viral deletion mutant, in vivo virulence assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site mutagenesis, in vivo viral model, single lab\",\n      \"pmids\": [\"37417777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC5 ISGylates cGAS at Lys21, Lys187, Lys219, and Lys458, promoting cGAS oligomerization and enhancing cGAS enzymatic activity. The interaction depends on the cGAS C-terminal domain and RCC1-4/RCC1-5 domains of HERC5. ISGylation deficiency attenuates cGAS-STING-mediated inflammatory gene expression and antiviral defense in mouse and human cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry site identification, mutagenesis, cGAS activity assay, oligomerization assay, ISGylation assay, KO mouse model (HSV-1 infection)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mass spectrometry, mutagenesis, enzymatic activity assay, domain mapping, in vivo mouse model, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38421872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC5 ISGylates SARS-CoV-2 nucleocapsid (N) protein at Lys266, Lys355, Lys387, and Lys388 (preferentially the phosphorylated form), impeding N protein oligomerization and inhibiting viral RNA synthesis. This ISGylation is reversed by PLpro (NSP3) deISGylation activity. Mutant N-4KR abolishes ISGylation and alleviates restriction in a replicon system.\",\n      \"method\": \"Mass spectrometry, site-directed mutagenesis, SARS-CoV-2 replicon system, oligomerization assay, ISGylation assay, PLpro activity assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mass spectrometry site identification, mutagenesis in replicon system, oligomerization assay, single lab with multiple orthogonal approaches; replicated in preprint PMID:39149229\",\n      \"pmids\": [\"39194248\", \"39149229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"hHERC5 broadly ISGylates proteins cotranslationally as a ribosome-associated HECT E3 ligase. Over 2,000 modified lysines in over 1,100 proteins were characterized in IFN-β-stimulated cells. hHERC5 and mouse HERC6 have distinct amino acid sequence context preferences surrounding ISGylation sites. mHERC6 also cotranslationally modifies nascent polypeptides.\",\n      \"method\": \"Mass spectrometry ISGylome profiling, IFN-β stimulation, sequence context analysis, comparison with mHERC6\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — large-scale mass spectrometry with rigorous controls, direct comparison of substrate selectivity, establishes cotranslational mechanism\",\n      \"pmids\": [\"38303729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HERC5 interacts with IFI16 and mediates ISGylation of IFI16 at Lys274, facilitating IFI16 proteasomal degradation. IFI16 acts as a tumor suppressor downstream of HERC5, and IFI16 is positively correlated with p53 expression, defining a HERC5/IFI16/p53 signaling pathway in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, proteomic analysis, western blot, siRNA knockdown, site mutagenesis, cell proliferation/migration assays\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-directed mutagenesis, functional KD assay, single lab\",\n      \"pmids\": [\"35671810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HERC5 inhibits Ebola virus (EBOV) VLP replication by depleting EBOV mRNAs. The RCC1-like domain of HERC5 is necessary and sufficient for this inhibition and does not require zinc finger antiviral protein (ZAP). EBOV glycoprotein (Zaire strain) antagonizes HERC5 early during infection.\",\n      \"method\": \"Transcription- and replication-competent VLP system, domain deletion analysis, ZAP-deficient cell assay, EBOV GP antagonism assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — VLP replication assay with domain mapping and negative control (Marburg GP), single lab\",\n      \"pmids\": [\"34572049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC5 ISGylates SARS-CoV-2 nsp8 (a viral replication factor) at its N2 domain lysine residues, facilitating proteasome-dependent nsp8 degradation and suppressing viral replication. SARS-CoV-2 PLpro counteracts this by deconjugating ISG15 from nsp8, preventing its degradation. The full ISGylation system (HERC5, UBA7, ISG15) suppresses replication of multiple SARS-CoV-2 variants including Omicron.\",\n      \"method\": \"Mass spectrometry, mutagenesis, overexpression, PLpro deISGylation assay, proteasome inhibition, viral replication assay\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry, mutagenesis, functional viral replication assay, single lab\",\n      \"pmids\": [\"40409630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC5 catalyzes ISGylation of UGDH (UDP-glucose 6-dehydrogenase) via its HECT domain active-site Cys994, facilitating UGDH phosphorylation and enhancing SNAI1 mRNA stability, thereby promoting OSCC cell invasion and cisplatin resistance.\",\n      \"method\": \"Co-immunoprecipitation, ISGylation assay, site mutagenesis (C994A), siRNA knockdown, overexpression, in vivo xenograft\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, active-site mutagenesis, functional in vivo assay, single lab\",\n      \"pmids\": [\"40045362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC5 inhibits LINE-1 (L1) retrotransposition through an ISGylation-independent mechanism. HERC5 interacts with L1 RNA and selectively reduces ORF1p protein levels, decreasing L1 translation efficiency and altering L1 RNP composition.\",\n      \"method\": \"L1 retrotransposition assay, HERC5 domain/catalytic mutant analysis, RNA immunoprecipitation, ORF1p level quantification, RNP composition analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional retrotransposition assay, RNA-IP, catalytic mutant confirming ISGylation-independence, single lab\",\n      \"pmids\": [\"42055552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UbcH8~ISG15 exhibits striking specificity for HECT-family E3 ligases (particularly HERC5) but is inactive with RING or RBR E3s, in contrast to UbcH8~Ub which preferentially engages RBR E3s. A unique closed conformation of UbcH8~ISG15 enables trans-thiolation selectively mediated by HECT E3s. HERC5's C-lobe specifically recognizes donor ISG15 for lysine conjugation, explaining its exclusive ISGylation activity and lack of ubiquitination function.\",\n      \"method\": \"Structural analysis (biochemical studies), in vitro thiolation assay, comparative E3 ligase specificity assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural and biochemical studies from single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.09.26.678755\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAXS analysis reveals that the HERC5 HECT domain predominantly adopts a trimeric assembly in solution, suggesting trimerization may play a regulatory role in HERC5 functional activity.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), size-exclusion chromatography, Guinier/Kratky analysis, ab initio shape reconstruction\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 1 / Weak — SAXS structure from single lab, preprint, functional consequence of trimerization not directly demonstrated\",\n      \"pmids\": [\"bio_10.1101_2025.03.22.644726\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HERC5 (human) and HERC6 (mouse functional homolog) are the primary E3 ligases responsible for ISGylation of MDA5 at Lys23 and Lys43 within its CARD domain. ISGylation at these sites promotes MDA5 oligomeric assembly and downstream antiviral signaling; knock-in mice with K23R/K43R MDA5 mutations show abrogated ISGylation, impaired MDA5 oligomerization, blunted cytokine responses, and increased mortality upon EMCV infection.\",\n      \"method\": \"MDA5 K23R/K43R knock-in mice, ISGylation assay, MDA5 oligomerization assay, cytokine measurement, viral infection survival assay, E3 ligase identification\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mouse with mutagenesis, functional oligomerization and infection assay, preprint from single lab\",\n      \"pmids\": [\"bio_10.1101_2024.09.20.614144\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC5 interacts with and ISGylates IRF3, preventing IRF3 ubiquitination and degradation. This stabilizes IRF3 and sustains IFN-β production and overactivation in podocytes, contributing to lupus nephritis pathology.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, ISGylation assay, IFN-β quantification, podocyte injury assay\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional loss/gain-of-function with defined readout, consistent with prior IRF3 ISGylation findings (PMID:20308324), single lab\",\n      \"pmids\": [\"40505230\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HERC5 is an interferon-induced HECT-type E3 ligase that functions as the primary cellular ISG15 conjugating enzyme (E3) in humans, working sequentially with E1 (UBE1L/UBA7) and E2 (UbcH8) to broadly ISGylate nascent polypeptides cotranslationally on ribosomes; its catalytic Cys994 in the HECT domain is essential for ISGylation, while its N-terminal RCC1-like domain mediates a second antiviral mechanism by blocking Rev/RRE-dependent nuclear RNA export and reducing RanGTP levels; established substrates include IRF3, cGAS, MDA5, PTEN, IFI16, β-catenin, CtBP1, and multiple viral proteins (influenza NS1, HIV Gag, SARS-CoV-2 N protein and nsp8, HBx), with ISGylation variously promoting target stabilization (IRF3, cGAS), degradation (PTEN, nsp8), or functional disruption (viral homodimers, oligomers), thereby broadly potentiating innate antiviral and antibacterial immunity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HERC5 is the principal cellular E3 ligase for ISG15 conjugation (ISGylation) in human cells and a central effector of interferon-induced innate antiviral immunity [#1, #15]. It is an interferon-induced HECT-type ligase whose catalytic activity depends on an active-site cysteine, Cys994, in its C-terminal HECT domain; this residue is essential, since its substitution abolishes ISGylation, and HERC5 reconstitutes robust ISG15 conjugation together with the E1 UBE1L and E2 UbcH8 [#0, #1]. As a ribosome-associated ligase, HERC5 ISGylates a broad spectrum of nascent polypeptides cotranslationally, modifying thousands of lysines across over a thousand proteins, with sequence-context preferences distinct from the mouse homolog HERC6 [#15]. Through this activity HERC5 amplifies innate immune signaling by ISGylating pathway components: it modifies IRF3 to block its Pin1-dependent degradation and sustain antiviral gene expression [#4, #24], promotes cGAS oligomerization and enzymatic activity [#13], and drives MDA5 CARD-domain oligomerization required for downstream signaling [#23]. HERC5 also directly restricts viruses by ISGylating viral proteins to disrupt their function — blocking influenza NS1 and SARS-CoV-2 nucleocapsid oligomerization and targeting SARS-CoV-2 nsp8 for degradation [#5, #14, #18]. A second, ISGylation-independent antiviral activity resides in its N-terminal RCC1-like domain, which inhibits Rev/RRE-dependent nuclear RNA export and reduces RanGTP levels to restrict HIV-1 [#7]. Beyond antiviral defense, HERC5-mediated ISGylation regulates cellular substrates including PTEN, β-catenin, IFI16, and UGDH, linking it to inflammatory signaling and cancer-associated pathways [#11, #8, #16, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that HERC5 is an active ubiquitin-pathway E3 ligase, providing the first biochemical evidence of its enzymatic activity before its ISG15-specific role was known.\",\n      \"evidence\": \"In vitro ubiquitin ligase assay with UbcH5a and protein/mRNA quantification in endothelial cells\",\n      \"pmids\": [\"15331633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify ISG15 as the relevant conjugate\", \"No active-site mutagenesis\", \"Physiological substrates unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified HERC5 as the enzyme required for the bulk of cellular ISG15 conjugation, defining its function as a general ISG15 E3 ligase rather than a generic ubiquitin ligase.\",\n      \"evidence\": \"siRNA knockdown, four-component reconstitution, and active-site plus RCC1-deletion mutants in human cells\",\n      \"pmids\": [\"16407192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate selection mechanism unresolved\", \"Did not map specific modified lysines\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the catalytic and enzymatic logic of HERC5 ISGylation by pinpointing the essential HECT active-site Cys994 and showing it physically binds and stimulates ISGylation of targets with E1/E2 dependence.\",\n      \"evidence\": \"C994A mutagenesis, co-transfection reconstitution, proteomic substrate identification and reciprocal Co-IP\",\n      \"pmids\": [\"16815975\", \"16884686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism conferring ISG15 over ubiquitin specificity not explained\", \"Functional consequence of ISGylation on substrates not yet defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that HERC5 ISGylation can stabilize a substrate to amplify signaling, establishing a positive-regulatory role in innate immunity through IRF3 modification.\",\n      \"evidence\": \"Co-IP, IRF3 lysine-site mutagenesis, and Sendai virus infection assays linking ISGylation to blocked Pin1-mediated degradation\",\n      \"pmids\": [\"20308324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo physiological requirement\", \"Interplay with deISGylases not addressed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated a direct antiviral mechanism whereby ISGylation of a viral protein disrupts its function, exemplified by influenza NS1.\",\n      \"evidence\": \"Mass spectrometry site mapping, mutagenesis, homodimerization assay, and viral replication assays in cells and mice\",\n      \"pmids\": [\"20385878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of oligomerization-blocking mechanism across viral substrates untested at the time\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed a second, catalytically independent antiviral activity in the N-terminal RCC1-like domain, separating HERC5's ligase function from its control of nuclear RNA export.\",\n      \"evidence\": \"Domain mutagenesis, Rev/RRE nuclear export assays, RanGTP and RanBP1 measurements, and localization imaging for HIV-1\",\n      \"pmids\": [\"22093708\", \"24693865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of the RCC1-like domain on the Ran cycle not defined\", \"Generality beyond HIV-1 untested in these studies\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended HERC5 ISGylation to destabilizing substrates and to antibacterial defense, showing ISGylation can target proteins for degradation to reprogram signaling.\",\n      \"evidence\": \"Co-IP, KO macrophages, PI3K-AKT analysis, and bacterial growth assays for PTEN; viral-hijacking Co-IP and in vivo assays for JAK1 ubiquitination\",\n      \"pmids\": [\"37279284\", \"37417777\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"JAK1 ubiquitination represents virus-directed hijacking distinct from ISGylation\", \"Determinants of degradative vs stabilizing outcome unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established HERC5 as a ribosome-associated cotranslational ISGylation machine acting on nascent polypeptides at proteome scale, defining the operating mode of the enzyme.\",\n      \"evidence\": \"Large-scale mass spectrometry ISGylome profiling in IFN-β-stimulated cells with sequence-context analysis and HERC6 comparison\",\n      \"pmids\": [\"38303729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How cotranslational targeting reconciles with selective regulatory substrates is unresolved\", \"Determinants of site selection beyond local sequence unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed ISGylation can activate immune sensors by promoting oligomerization, generalizing the positive-regulatory role across the cGAS-STING and RIG-I-like receptor pathways.\",\n      \"evidence\": \"Mass spectrometry site mapping, domain mapping, oligomerization and enzymatic activity assays, and KO/knock-in mouse infection models for cGAS and MDA5\",\n      \"pmids\": [\"38421872\", \"bio_10.1101_2024.09.20.614144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MDA5 evidence is from a preprint\", \"Stoichiometry of ISGylation needed for oligomerization not quantified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the antagonism between HERC5 ISGylation and viral deISGylases, showing SARS-CoV-2 proteins are ISGylated to block oligomerization and target replication factors for degradation, countered by PLpro.\",\n      \"evidence\": \"Mass spectrometry, mutagenesis, replicon systems, oligomerization assays, and PLpro deISGylation assays for N protein and nsp8\",\n      \"pmids\": [\"39194248\", \"40409630\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of N vs nsp8 ISGylation to restriction unresolved\", \"In vivo relevance not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a structural basis for HERC5's exclusive ISGylation specificity, explaining why UbcH8~ISG15 engages HECT but not RING/RBR E3s.\",\n      \"evidence\": \"Structural and biochemical analysis of UbcH8~ISG15 conformation and trans-thiolation specificity (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.09.26.678755\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not peer-reviewed\", \"No high-resolution complex structure of HERC5 with charged E2\", \"Awaits independent confirmation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated HERC5 in non-antiviral cellular contexts including cancer-associated substrate regulation and retrotransposon control via ISGylation-independent RNA binding.\",\n      \"evidence\": \"Co-IP and ISGylation/xenograft assays for UGDH; retrotransposition, RNA-IP, and catalytic-mutant analyses for LINE-1 ORF1p\",\n      \"pmids\": [\"40045362\", \"42055552\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of ISGylation-independent RNA binding undefined\", \"Single-lab findings without independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HERC5 reconciles broad cotranslational ISGylation of nascent chains with selective, functionally consequential modification of specific regulatory and viral substrates, and how its HECT trimerization and RCC1-like domain are coordinated, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking cotranslational targeting to substrate-specific outcomes\", \"Functional role of HECT trimerization undemonstrated\", \"Regulation of the two distinct antiviral mechanisms not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 13, 15]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [1, 15]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 5, 13, 23]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 9, 14, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ISG15\", \"UBA7\", \"UbcH8\", \"IRF3\", \"cGAS\", \"MDA5\", \"PTEN\", \"CTNNB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}