GO:0061662 ISG15 ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0061662 (ISG15 ligase activity) describes the enzymatic transfer of ISG15 to substrate proteins via a thioester intermediate and a final isopeptide bond.
• The reaction requires an E1 (UBA7), an E2 (UBE2L6), and an E3 ligase; HERC5 and HERC6 are the principal E3 enzymes for ISGylation.
• ISG15 ligase activity regulates key signaling proteins such as STING, NLRP3, parkin, CHIP, and CtBP1, thereby influencing immunity, inflammation, and cancer.
• Dysregulated ISGylation is implicated in viral infections, autoinflammatory diseases, neurodegeneration, and multiple cancers.
• CRISPR-based knockout, point-mutation, knock-in, and overexpression models are essential to dissect the causal roles of ISG15 ligase components.
• Understanding ISG15 ligase activity offers therapeutic opportunities, particularly in immuno-oncology and antiviral strategies.
Description
ISG15 ligase activity (GO:0061662) is a molecular function that catalyzes the covalent attachment of ISG15, a ubiquitin-like protein, to substrate proteins. This process, known as ISGylation, proceeds through a thioester intermediate with an E2 or E3 enzyme and results in an isopeptide bond between the C-terminal glycine of ISG15 and the epsilon-amino group of lysine residues in the substrate. ISG15 ligase activity is a cornerstone of the interferon response and plays critical roles in antiviral defense, immune signaling, and cancer biology. Researchers study this term to understand how post-translational modifications control protein function and to identify therapeutic targets in infectious and inflammatory diseases. The enzymatic cascade involves E1 (UBA7), E2 (UBE2L6), and E3 ligases, with HERC5 and HERC6 being the major E3 enzymes that confer substrate specificity. Beyond immunity, ISGylation modulates proteins such as parkin, CHIP, and CtBP1, linking it to neurodegeneration and cancer. This article provides a comprehensive overview of the mechanism, key genes, disease relevance, and research methodologies for studying ISG15 ligase activity.
ISG15 ligase activity At A Glance
| GO ID | GO:0061662 |
|---|---|
| GO term | ISG15 ligase activity |
| Ontology | molecular_function |
| Synonym | E3 |
| Major function | Catalyzes the covalent attachment of ISG15 to substrate proteins via an isopeptide bond |
| Reaction | X-ISG15 + S = X + S-ISG15 (X = E2 or E3 enzyme; S = substrate protein) |
| Linkage type | Thioester bond (intermediate); isopeptide bond (final product) |
| Subcellular location | Cytoplasm, nucleus (as reported for ISGylation targets) |
| Pathway context | Interferon-stimulated gene response, antiviral immunity, inflammation |
What Is GO:0061662?
ISG15 ligase activity (GO:0061662) is defined as the catalysis of ISG15 transfer to a substrate protein via the reaction X-ISG15 + S = X + S-ISG15, where X is either an E2 or E3 enzyme, the X-ISG15 linkage is a thioester bond, and the S-ISG15 linkage is an isopeptide bond between the C-terminal amino acid of ISG15 and the epsilon-amino group of lysine residues in the substrate. In simpler terms, it is the enzymatic activity that attaches ISG15 to other proteins, a process called ISGylation.
Why Is ISG15 ligase activity Important in Cell Biology?
ISG15 ligase activity is essential for the interferon-mediated antiviral response and immune regulation. It controls the stability, localization, and activity of key signaling proteins, thereby impacting pathogen defense, inflammation, and cancer progression. Dysregulation of ISGylation contributes to autoinflammatory diseases, viral pathogenesis, and tumorigenesis, making it a promising target for therapeutic intervention.
• Mediates antiviral defense by modifying viral and host proteins.
• Regulates innate immune signaling through STING and NLRP3 ISGylation.
• Modulates ubiquitin E3 ligase activity of parkin and CHIP, linking to neurodegeneration and cancer.
• Influences transcriptional repression via CtBP1 ISGylation.
• Implicated in hepatocellular carcinoma suppression via γ-glutamate cysteine ligase ISGylation.
• Plays a role in TBEV pathogenesis in neurons and astrocytes.
• Potential biomarker for interferon-related diseases.
• Target for CRISPR-based functional genomics to identify novel ISGylation substrates.
• Enables development of small-molecule modulators of ISGylation.
• Provides insights into crosstalk between ubiquitin and ubiquitin-like modifications.
Molecular Mechanism of ISG15 ligase activity
Activation of ISG15 by E1 Enzyme
In simple terms: ISG15 is first activated by an E1 enzyme in an ATP-dependent manner.
The E1 enzyme UBA7 activates ISG15 by forming a thioester bond between its catalytic cysteine and the C-terminal glycine of ISG15, consuming ATP. This step is essential for subsequent transfer to E2 enzymes.
Transfer to E2 Conjugating Enzyme
In simple terms: Activated ISG15 is handed off to an E2 enzyme.
The E2 enzyme UBE2L6 receives ISG15 from UBA7 via a trans-thioesterification reaction, forming an E2-ISG15 thioester intermediate. This intermediate is the direct donor for substrate modification.
E3 Ligase-Mediated Substrate ISGylation
In simple terms: An E3 ligase brings the E2-ISG15 complex to the target protein and attaches ISG15.
E3 ligases such as HERC5 and HERC6 facilitate the transfer of ISG15 from UBE2L6 to specific lysine residues on substrate proteins, forming an isopeptide bond. This step confers substrate specificity and is critical for the biological outcomes of ISGylation.
Substrate Recognition and Specificity
In simple terms: Different E3 ligases recognize different target proteins.
HERC5 and HERC6 are the primary E3 ligases for ISGylation, but other E3s may also participate. Substrate specificity is determined by interactions between the E3 ligase and the target protein, often regulated by interferon signaling.
Deconjugation and Reversibility
In simple terms: ISG15 can be removed from proteins by specific enzymes.
ISGylation is reversible; the protease USP18 removes ISG15 from conjugated proteins, balancing the modification. This dynamic regulation is crucial for preventing excessive immune activation.
Key Genes Involved in GO:0061662 ISG15 ligase activity
The following genes and proteins are central to ISG15 ligase activity, encompassing the enzymatic cascade and key substrates.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ISG15 | Ubiquitin-like modifier transferred to substrates | Core component; knockout models reveal ISGylation functions |
| UBA7 | E1 activating enzyme for ISG15 | Essential for ISGylation initiation; target for knockout studies |
| UBE2L6 | E2 conjugating enzyme for ISG15 | Required for ISG15 transfer; point mutations affect activity |
| HERC5 | E3 ligase for ISG15 | Primary E3; knockout reduces ISGylation of many substrates |
| HERC6 | E3 ligase for ISG15 | Alternative E3; may compensate for HERC5 loss |
| STING | Substrate of ISGylation | ISGylation enhances STING activation; relevant to antiviral immunity |
| NLRP3 | Substrate of ISGylation | ISGylation facilitates inflammasome activation |
| Parkin | Substrate of ISGylation | ISGylation positively regulates parkin E3 ligase activity |
| CHIP | Substrate of ISGylation | ISGylation promotes CHIP activity and inhibits lung cancer growth |
| CtBP1 | Substrate of ISGylation | ISGylation regulates CtBP1 corepressor activity |
| γ-Glutamate cysteine ligase | Substrate of ISGylation | ISGylation enhances activity and suppresses apoptosis in HCC |
| USP18 | DeISGylase | Removes ISG15; regulates ISGylation dynamics |
| UBA7 | E1 enzyme | Potential target for antiviral therapy |
| UBE2L6 | E2 enzyme | Genetic variants may affect ISGylation efficiency |
| HERC5 | E3 ligase | Biomarker for interferon response |
| HERC6 | E3 ligase | Functional redundancy with HERC5 |
| ISG15 | Modifier | Therapeutic target in cancer and inflammation |
| STING | Immune adaptor | ISGylation is critical for STING signaling |
How Is ISG15 ligase activity Regulated?
ISG15 ligase activity is primarily regulated by type I interferon signaling, which induces the expression of ISG15, UBA7, UBE2L6, HERC5, and HERC6. Additionally, USP18 negatively regulates ISGylation by removing ISG15 from substrates. Other regulatory mechanisms include feedback loops involving ISGylation of components of the interferon pathway itself.
ISG15 ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ISG15 | Antiviral immunity, cancer | ISG15 knockout cell lines, mouse models |
| HERC5 | Inflammation, cancer | HERC5 knockout or overexpression cells |
| NLRP3 | Inflammasome-related diseases | NLRP3 ISGylation site mutants |
| Parkin | Neurodegeneration | Parkin ISGylation-deficient knock-in mice |
| CHIP | Lung cancer | CHIP ISGylation mutants in cancer cell lines |
ISG15 ligase activity in cancer
ISGylation influences cancer progression through modification of key proteins. For example, ISGylation of γ-glutamate cysteine ligase suppresses apoptosis in high-fat-diet-promoted hepatocellular carcinoma. ISGylation of CHIP inhibits lung cancer cell growth in response to type I interferon. These findings suggest context-dependent roles for ISG15 ligase activity in tumorigenesis.
ISG15 ligase activity in viral infections
ISG15 ligase activity is a critical antiviral mechanism. ISGylation of STING enhances its activation, promoting interferon production. Tick-borne encephalitis virus (TBEV) infection alters ISGylation-related gene expression in neurons and astrocytes, suggesting a role in neuropathogenesis. ESCRT machinery, which interacts with ISGylation pathways, is also involved in virus infection.
ISG15 ligase activity in inflammatory diseases
ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation, linking ISG15 ligase activity to inflammation. Dysregulated ISGylation may contribute to autoinflammatory conditions. Additionally, ISGylation of parkin and CtBP1 impacts neurodegeneration and transcriptional regulation, respectively.
From ISG15 ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ISG15 ligase activity regulate STING signaling? | STING knockout cells reconstituted with ISGylation-deficient STING mutants |
| What is the role of HERC5 in inflammasome activation? | HERC5 knockout macrophages |
| How does ISGylation affect parkin function? | Parkin knockout neurons with ISGylation site mutations |
| Does ISGylation of CHIP suppress lung cancer? | CHIP ISGylation-deficient knock-in mice |
| What is the impact of ISG15 ligase activity on HCC? | Liver-specific ISG15 or UBA7 knockout mice |
| Can ISGylation be targeted therapeutically? | Overexpression of USP18 or ISGylation inhibitors |
How to Study the ISG15 ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout | Loss of gene function | Identify essential ISGylation genes |
| Proteomics | ISGylated protein identification | Discover novel substrates |
| RNA-seq | Transcriptional changes | Assess downstream effects |
| Immunoprecipitation | Protein-protein interactions | Study E3-substrate binding |
| Western blot | ISGylation levels | Validate knockout/overexpression |
| Luciferase reporter | Interferon pathway activity | Measure STING/NF-κB activation |
| Flow cytometry | Immune cell activation | Analyze inflammasome function |
CRISPR knockout screens
Genome-wide CRISPR knockout screens can identify genes required for ISG15 ligase activity and ISGylation. For example, knocking out UBA7, UBE2L6, or HERC5 abolishes ISGylation, allowing functional studies.
Proteomics and ISGylation substrate identification
Mass spectrometry-based proteomics can identify ISGylated proteins by detecting the characteristic di-glycine remnant after trypsin digestion. This approach has revealed substrates like STING and NLRP3.
RNA-seq and transcriptomics
RNA sequencing of cells with modulated ISG15 ligase activity can reveal downstream transcriptional changes, as shown in TBEV-infected neurons and astrocytes.
Imaging and localization studies
Fluorescence microscopy with tagged ISG15 or E3 ligases can visualize the subcellular localization of ISGylation components and their co-localization with substrates.
How CRISPR Can Be Used to Study GO:0061662 ISG15 ligase activity
Knockout
CRISPR knockout of ISG15, UBA7, UBE2L6, HERC5, or HERC6 eliminates ISG15 ligase activity, providing a clean background to study ISGylation-dependent processes. Knockout cell lines are valuable for identifying substrates and downstream effects.
Point Mutation
Point mutations in the catalytic cysteine of UBA7 or UBE2L6, or in the HERC5 RING domain, can abolish ISG15 ligase activity while preserving protein expression. Such mutants are useful to distinguish enzymatic from scaffolding functions.
Knock-in
Knock-in of ISGylation-deficient substrate mutants (e.g., STING K150R) allows precise interrogation of site-specific ISGylation in vivo. This approach reveals the functional significance of individual modification sites.
Overexpression
Overexpression of ISG15, UBA7, UBE2L6, and HERC5 enhances ISGylation, enabling gain-of-function studies. Conversely, overexpression of USP18 reduces ISGylation, providing a tool to downregulate the pathway.
How EDITGENE Supports ISG15 ligase activity Research
Researchers studying ISG15 ligase activity-related genes often need to determine whether a candidate gene is causally involved in ISGylation, immune signaling, or disease progression. EDITGENE provides comprehensive CRISPR-based services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for ISG15 ligase activity research.
Frequently Asked Questions About ISG15 ligase activity
What is ISG15 ligase activity?
ISG15 ligase activity (GO:0061662) is the enzymatic transfer of ISG15 to substrate proteins via a thioester intermediate and an isopeptide bond, a process called ISGylation.
What genes are involved in ISG15 ligase activity?
Key genes include ISG15, UBA7 (E1), UBE2L6 (E2), HERC5 and HERC6 (E3 ligases), and the deISGylase USP18.
What is the role of HERC5 in ISGylation?
HERC5 is a primary E3 ligase that facilitates the transfer of ISG15 from UBE2L6 to substrate proteins, conferring specificity.
How is ISG15 ligase activity regulated?
It is induced by type I interferons and negatively regulated by USP18, which removes ISG15 from substrates.
What diseases are associated with ISG15 ligase activity?
Dysregulation is linked to viral infections, autoinflammatory diseases, neurodegeneration, and cancers such as hepatocellular carcinoma and lung cancer.
What are the substrates of ISG15 ligase activity?
Known substrates include STING, NLRP3, parkin, CHIP, CtBP1, and γ-glutamate cysteine ligase.
How can I study ISG15 ligase activity in the lab?
Common methods include CRISPR knockout, proteomics, RNA-seq, immunoprecipitation, and western blotting.
What is the difference between ISG15 and ubiquitin?
ISG15 is a ubiquitin-like protein, but it is conjugated by a distinct enzymatic cascade (UBA7, UBE2L6, HERC5/6) and has unique substrates and functions.
Can ISG15 ligase activity be targeted therapeutically?
Yes, modulating ISGylation is being explored for antiviral and anticancer therapies, though further research is needed.
What CRISPR models are available for ISG15 ligase activity?
EDITGENE offers knockout, point mutation, knock-in, and overexpression models for ISGylation-related genes, as well as CRISPR library screening.
Conclusion
ISG15 ligase activity (GO:0061662) is a critical post-translational modification pathway with broad implications in immunity, inflammation, and cancer. Understanding its mechanism and regulation offers insights into disease pathogenesis and potential therapeutic targets. CRISPR-based models are indispensable for dissecting the causal roles of ISGylation components, and EDITGENE provides comprehensive services to support such research.
References
- 1. Liu X et al.. 2025. ISG15 Enhances the Activity of γ-Glutamate Cysteine Ligase to Suppress Apoptosis in High Fat Diet-Promoted Hepatocellular Carcinoma.. Adv Sci (Weinh) 12(19):e2416401 PMID: 40126377
- 2. Qin Y et al.. 2024. ISGylation by HERCs facilitates STING activation.. Cell Rep 43(5):114135 PMID: 38652662
- 3. Selinger M et al.. 2022. Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.. Comput Struct Biotechnol J 20:2759-2777 PMID: 35685361
- 4. Qin Y et al.. 2023. Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation.. J Clin Invest 133(20) PMID: 37651190
- 5. Im E et al.. 2016. Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.. Open Biol 6(8) PMID: 27534820
- 6. Dai J et al.. 2024. ESCRT machinery and virus infection.. Antiviral Res 221:105786 PMID: 38147902
- 7. 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
- 8. Yoo L et al.. 2018. Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon.. Cell Death Dis 9(2):97 PMID: 29367604
