GO:0061653 ISG15 conjugating enzyme activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0061653 (ISG15 conjugating enzyme activity) is a molecular function that transfers ISG15 from one protein to another through thioester-linked intermediates, using a cysteine residue of the enzyme.
• The canonical E2 enzyme for ISG15 conjugation is UBE2L6 (UbcH8), which also functions as a ubiquitin-conjugating enzyme.
• ISG15 conjugation modifies substrates such as Ubc13, thereby suppressing its ubiquitin-conjugating activity and altering downstream signaling.
• The activity is reversible: deconjugating enzymes such as USP18 remove ISG15 from targets, and this balance is critical for interferon signaling and immune homeostasis.
• Viral proteins, including African swine fever virus pI215L and SARS-CoV-2 PLpro, can interfere with ISG15 conjugation or deconjugation to evade host immunity.
• Research tools such as semi-synthetic ISG15-Dha probes enable simultaneous capture of conjugating and deconjugating enzymes for mechanistic and drug-discovery studies.
Description
ISG15 conjugating enzyme activity (GO:0061653) is a molecular function that catalyzes the isoenergetic transfer of ISG15 from one protein to another via thioester-linked intermediates. This activity is central to the ISG15 conjugation pathway, a ubiquitin-like modification system induced by type I interferons and involved in antiviral defense, immune regulation, and cellular stress responses. Unlike ubiquitination, which primarily targets proteins for degradation, ISG15 conjugation often modulates protein function, localization, or interaction without directly promoting proteasomal destruction. Understanding this activity is therefore essential for dissecting interferon-driven signaling and host-pathogen interactions. Researchers study GO:0061653 to identify the enzymes that write, read, and erase ISG15 modifications, and to determine how these enzymes shape immune responses and disease outcomes. The canonical E2 enzyme UBE2L6 (UbcH8) was shown to function as the E2 for ISG15 as well as for ubiquitin, linking the two conjugation systems. Substrate-specific effects, such as ISG15 modification of Ubc13 suppressing its ubiquitin-conjugating activity, illustrate how this activity can rewire signaling networks. Moreover, viral pathogens encode proteins that inhibit or exploit ISG15 conjugation, underscoring its importance in infection. This article provides a research-grade overview of GO:0061653, covering its definition, mechanism, key genes, disease relevance, and experimental models. It is intended for scientists who need a concise, citable resource for grant writing, experimental design, and teaching.
ISG15 conjugating enzyme activity At A Glance
| GO ID | GO:0061653 |
|---|---|
| GO term | ISG15 conjugating enzyme activity |
| Ontology | molecular_function |
| Synonym | E2 |
| Major function | Catalyzes the transfer of ISG15 from one protein to another via thioester-linked intermediates |
| Definition source | QuickGO |
| Related activity | Ubiquitin-conjugating enzyme activity (some enzymes, e.g., UBE2L6, perform both) |
| Key enzyme | UBE2L6 (UbcH8) |
| Reversibility | Opposed by ISG15 deconjugating enzymes such as USP18 |
What Is GO:0061653?
GO:0061653 describes the enzymatic activity that transfers ISG15 from a donor protein to an acceptor protein through a thioester bond between the C-terminal amino acid of ISG15 and a cysteine residue of the enzyme. In this reaction, both the donor and acceptor linkages are thioester bonds, and the transfer is isoenergetic. This activity is distinct from ubiquitin-conjugating activity, although some enzymes, such as UBE2L6, can perform both functions.
Why Is ISG15 conjugating enzyme activity Important in Cell Biology?
ISG15 conjugating enzyme activity is a critical node in the interferon-induced ISG15 conjugation pathway, which regulates antiviral immunity, protein stability, and cellular stress responses. Dysregulation of this activity has been linked to viral immune evasion, autoinflammatory conditions, and cancer biology. Because ISG15 modification can alter substrate function without necessarily causing degradation, this activity provides a versatile mechanism for fine-tuning signaling networks.
• Antiviral defense: ISG15 conjugation is induced by type I interferons and restricts replication of diverse viruses.
• Immune signaling: ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity, impacting NF-kB and other pathways.
• Viral evasion: African swine fever virus pI215L inhibits IFN-I signaling through STAT2 degradation, interfering with ISG15-related responses.
• SARS-CoV-2: PLpro acts as a deconjugase that regulates N-degron recognin-mediated autophagy, highlighting cross-talk with ISG15 pathways.
• Cancer: ISG15 modification of transcriptional corepressor CtBP1 regulates its activity, linking ISG15 conjugation to gene expression.
• Autoinflammation: USP18 deficiency causes severe interferonopathy, demonstrating the importance of balancing ISG15 conjugation and deconjugation.
• Drug discovery: Semi-synthetic ISG15-Dha probes enable simultaneous capture of conjugating and deconjugating enzymes for inhibitor screening.
• Basic cell biology: ISG15 conjugation provides a model for understanding ubiquitin-like modification specificity and cross-talk.
Molecular Mechanism of ISG15 conjugating enzyme activity
Activation and Thioester Formation
In simple terms: The enzyme first grabs ISG15 and holds it tightly using a chemical bond.
ISG15 conjugating enzyme activity begins with the formation of a thioester bond between the C-terminal glycine of ISG15 and the active-site cysteine of the E2 enzyme. This step is isoenergetic and does not require ATP directly at the E2 stage, as the energy is provided by the upstream E1 activating enzyme. The E2 enzyme UBE2L6 (UbcH8) is a canonical example that forms a thioester with ISG15.
Substrate Recognition and Transfer
In simple terms: The enzyme then hands ISG15 over to a target protein.
The E2-ISG15 thioester interacts with a substrate protein, often in concert with an E3 ligase, leading to the transfer of ISG15 to a lysine residue on the substrate. For example, ISG15 modification of Ubc13 occurs on specific lysine residues and suppresses its ubiquitin-conjugating activity. The reaction results in a stable isopeptide bond between ISG15 and the target.
Deconjugation and Reversibility
In simple terms: Another enzyme can remove ISG15, making the process reversible.
ISG15 conjugation is reversed by deconjugating enzymes such as USP18, which cleaves ISG15 from substrates. This reversibility is essential for dynamic regulation of immune signaling. Viral deconjugases, including SARS-CoV-2 PLpro, can also remove ISG15 from host proteins, thereby modulating autophagy and immune responses.
Cross-talk with Ubiquitin System
In simple terms: Some enzymes can work with both ISG15 and ubiquitin, linking two tagging systems.
UBE2L6 (UbcH8) functions as an E2 for both ubiquitin and ISG15, demonstrating cross-talk between the two conjugation pathways. This dual specificity allows cells to integrate signals and may explain why ISG15 modification can compete with ubiquitination on shared substrates.
Viral Interference
In simple terms: Viruses make proteins that block or mimic these enzymes to escape immunity.
African swine fever virus pI215L is a ubiquitin-conjugating enzyme that inhibits IFN-I signaling through STAT2 degradation, indirectly affecting ISG15 responses. SARS-CoV-2 PLpro acts as a deconjugase on ISG15 and other ubiquitin-like modifiers, regulating N-degron recognin-mediated autophagy. These examples highlight how pathogens target ISG15 conjugation machinery.
Key Genes Involved in GO:0061653 ISG15 conjugating enzyme activity
The following genes and proteins are central to ISG15 conjugating enzyme activity, including E2 enzymes, E1, E3 ligases, deconjugases, and key substrates.
| Gene | Major Role | Research Relevance |
|---|---|---|
| UBE2L6 | E2 enzyme for ISG15 and ubiquitin | Canonical ISG15-conjugating enzyme; dual specificity |
| UBA7 | E1 activating enzyme for ISG15 | Required for ISG15 conjugation cascade |
| HERC5 | E3 ligase for ISG15 | Promotes ISG15 conjugation to substrates |
| USP18 | ISG15 deconjugase | Removes ISG15; regulates interferon signaling |
| ISG15 | Ubiquitin-like modifier | Substrate tag transferred by the enzyme |
| UBC13 | Substrate of ISG15 modification | ISG15 modification suppresses its ubiquitin-conjugating activity |
| CTBP1 | Transcriptional corepressor | ISG15 modification regulates its activity |
| STAT2 | Transcription factor | Degraded by ASFV pI215L, affecting IFN-I signaling |
| PLpro | Viral deconjugase | SARS-CoV-2 protease that removes ISG15 |
| pI215L | Viral ubiquitin-conjugating enzyme | Inhibits IFN-I signaling via STAT2 degradation |
| N-degron recognins | Autophagy receptors | Regulated by PLpro-mediated deconjugation |
| UBE2D1 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2D2 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2D3 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2E1 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2E2 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2E3 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
| UBE2G1 | Ubiquitin E2 enzyme | Potential cross-talk with ISG15 system |
How Is ISG15 conjugating enzyme activity Regulated?
ISG15 conjugating enzyme activity is regulated at multiple levels. Transcription of ISG15 and conjugation machinery is induced by type I interferons. The activity is balanced by deconjugating enzymes such as USP18, which is itself an interferon-stimulated gene, creating a negative feedback loop. Viral proteins can inhibit or mimic components of the pathway, as seen with ASFV pI215L and SARS-CoV-2 PLpro. Additionally, cross-talk with the ubiquitin system, exemplified by UBE2L6 dual specificity, provides another layer of regulation.
ISG15 conjugating enzyme activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| USP18 | Interferonopathy, autoinflammation | USP18 knockout cells and mouse models |
| STAT2 | Viral immune evasion | ASFV pI215L overexpression in IFN-treated cells |
| PLpro | COVID-19, autophagy regulation | SARS-CoV-2 PLpro expression in lung epithelial cells |
| CTBP1 | Cancer, transcriptional regulation | ISG15 modification site mutants of CtBP1 |
| UBC13 | NF-kB signaling, cancer | Ubc13 ISG15-modification-deficient knock-in cells |
Viral Infections and Immune Evasion
ISG15 conjugating enzyme activity is a key component of antiviral immunity. African swine fever virus pI215L inhibits IFN-I signaling through STAT2 degradation, counteracting ISG15 responses. SARS-CoV-2 PLpro deconjugates ISG15 from host proteins, regulating autophagy and immune evasion. These examples illustrate how viruses target this activity to establish infection.
Autoinflammatory and Interferonopathies
USP18 deficiency leads to severe interferonopathy, highlighting the importance of balancing ISG15 conjugation and deconjugation. Dysregulated ISG15 conjugating enzyme activity can contribute to excessive interferon signaling and tissue damage.
Cancer and Transcriptional Regulation
ISG15 modification of transcriptional corepressor CtBP1 regulates its activity, linking ISG15 conjugation to gene expression programs relevant to cancer. ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity, which may affect NF-kB signaling and tumor progression.
From ISG15 conjugating enzyme activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does UBE2L6 mediate ISG15 conjugation to a specific substrate? | UBE2L6 knockout cells with ISG15 overexpression |
| What is the role of a specific ISG15 acceptor lysine? | Point mutation of the substrate lysine to arginine |
| How does ISG15 modification affect protein interactions? | Knock-in of tagged ISG15 (e.g., HA-ISG15) |
| Can a viral protein inhibit ISG15 conjugation? | Overexpression of viral protein (e.g., pI215L) in IFN-treated cells |
| What is the impact of USP18 deficiency? | USP18 knockout cells or mouse models |
| How does PLpro regulate autophagy via ISG15? | PLpro overexpression or knockout in SARS-CoV-2 infection models |
How to Study the ISG15 conjugating enzyme activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| ISG15-Dha probe capture | Active conjugating and deconjugating enzymes | Enzyme profiling and inhibitor screening |
| Western blot | ISG15-conjugated proteins | Monitoring conjugation in cells |
| Immunoprecipitation | Specific substrate ISG15 modification | Validating target modification |
| CRISPR knockout screen | Genes required for ISG15 conjugation | Discovery of novel regulators |
| Viral infection assay | Impact of viral proteins on ISG15 pathway | Studying immune evasion |
| qRT-PCR | Expression of ISG15 and related genes | Measuring interferon responses |
| Mass spectrometry | Identification of ISG15-modified sites | Mapping conjugation sites |
| Flow cytometry | ISG15 levels in single cells | Analyzing heterogeneous responses |
Proteomic Capture of ISG15 Conjugating Enzymes
Semi-synthetic ISG15-Dha probes enable simultaneous capture of conjugating and deconjugating enzymes, allowing identification and quantification of active enzymes in cell lysates. This method is useful for profiling enzyme activity under different conditions.
Western Blotting and Immunoprecipitation
ISG15 conjugation can be monitored by Western blotting with anti-ISG15 antibodies, which detect free ISG15 and ISG15-conjugated proteins. Immunoprecipitation of specific substrates followed by anti-ISG15 blotting confirms modification.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes required for ISG15 conjugation and its downstream effects. Such screens are valuable for discovering novel regulators of the pathway.
Viral Infection Models
Infection of cells with viruses expressing ISG15-interfering proteins, such as ASFV pI215L or SARS-CoV-2 PLpro, can reveal how these proteins modulate ISG15 conjugation and immune signaling.
How CRISPR Can Be Used to Study GO:0061653 ISG15 conjugating enzyme activity
Knockout
CRISPR knockout of UBE2L6 or other E2 enzymes can abolish ISG15 conjugating enzyme activity, allowing researchers to test its role in antiviral defense and signaling. Knockout of USP18 leads to hyper-ISG15 conjugation and interferonopathy-like phenotypes.
Point Mutation
Point mutations in the active-site cysteine of UBE2L6 or in ISG15 acceptor lysines on substrates can dissect catalytic mechanisms and substrate specificity. For example, mutating the ISG15-modified lysine on Ubc13 prevents suppression of its ubiquitin-conjugating activity.
Knock-in
Knock-in of tagged ISG15 (e.g., HA-ISG15) enables affinity purification and proteomic identification of ISG15 conjugates. Knock-in of mutant ISG15 that cannot be conjugated can serve as a negative control.
Overexpression
Overexpression of viral proteins such as ASFV pI215L or SARS-CoV-2 PLpro can mimic viral interference with ISG15 conjugation and reveal downstream effects on immune signaling and autophagy.
How EDITGENE Supports ISG15 conjugating enzyme activity Research
Researchers studying ISG15 conjugating enzyme activity-related genes often need to determine whether a candidate gene is causally involved in conjugation, immune signaling, or disease. EDITGENE provides CRISPR-based cell model services to enable such functional studies with high specificity and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for ISG15 conjugating enzyme activity research.
Frequently Asked Questions About ISG15 conjugating enzyme activity
What is ISG15 conjugating enzyme activity?
It is a molecular function (GO:0061653) that transfers ISG15 from one protein to another via thioester-linked intermediates, using a cysteine residue of the enzyme.
What genes are involved in ISG15 conjugating enzyme activity?
Key genes include UBE2L6 (E2), UBA7 (E1), HERC5 (E3), USP18 (deconjugase), and ISG15 itself.
Which enzyme is the canonical E2 for ISG15?
UBE2L6 (UbcH8) is the canonical E2 enzyme for ISG15 and also functions in ubiquitination.
How is ISG15 conjugation reversed?
USP18 and viral deconjugases such as SARS-CoV-2 PLpro remove ISG15 from substrates.
What diseases are linked to ISG15 conjugating enzyme activity?
Viral infections, autoinflammatory interferonopathies, and cancer have been linked to this activity.
How can I study ISG15 conjugating enzyme activity in the lab?
Methods include ISG15-Dha probe capture, Western blotting, immunoprecipitation, and CRISPR screens.
What is the role of ISG15 modification of Ubc13?
ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity, affecting downstream signaling.
Can viruses inhibit ISG15 conjugation?
Yes, African swine fever virus pI215L inhibits IFN-I signaling, and SARS-CoV-2 PLpro deconjugates ISG15.
What is the difference between ISG15 and ubiquitin conjugation?
Both use similar E1/E2/E3 cascades, but ISG15 modification often modulates function rather than targeting for degradation, and some enzymes like UBE2L6 act in both pathways.
How does USP18 regulate ISG15 conjugation?
USP18 removes ISG15 from substrates and is itself interferon-induced, providing negative feedback.
Conclusion
ISG15 conjugating enzyme activity (GO:0061653) is a fundamental molecular function in the interferon-induced ISG15 conjugation pathway, with critical roles in antiviral immunity, immune signaling, and disease. The canonical E2 enzyme UBE2L6 and the opposing deconjugase USP18 exemplify the dynamic regulation of this activity. Viral pathogens such as ASFV and SARS-CoV-2 target this pathway, underscoring its importance in host-pathogen interactions. Continued research using CRISPR models and advanced proteomic tools will further illuminate its mechanistic and therapeutic potential.
References
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- 3. Lim YH et al.. 2024. Transcriptional corepressor activity of CtBP1 is regulated by ISG15 modification.. Anim Cells Syst (Seoul) 28(1):66-74 PMID: 38405356
- 4. Riera E et al.. 2022. African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation.. Front Microbiol 13:1081035 PMID: 36713190
- 5. Ayala-Torres C et al.. 2025. Regulation of N-degron recognin-mediated autophagy by the SARS-CoV-2 PLpro ubiquitin deconjugase.. Autophagy 21(5):1019-1038 PMID: 39723606
- 6. Zhao C et al.. 2004. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein.. Proc Natl Acad Sci U S A 101(20):7578-82 PMID: 15131269
- 7. Bonacci T et al.. 2025. DIG-DUBs: mechanisms and functions of ISG15 deconjugation by human and viral cross-reactive ubiquitin proteases.. Biochem Soc Trans PMID: 40631552
- 8. Kang JA et al.. 2020. Emerging Roles of USP18: From Biology to Pathophysiology.. Int J Mol Sci 21(18) PMID: 32957626
