GO:0019782 ISG15 activating enzyme activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0019782 (ISG15 activating enzyme activity) catalyzes the ATP-dependent activation of the ubiquitin-like modifier ISG15, forming a high-energy thiolester bond on the E1 enzyme UBA7 (UBE1L).
• The ISG15 conjugation cascade requires the E1 UBA7, the E2 UbcH8, and E3 ligases such as HERC5 to attach ISG15 to target lysines.
• ISGylation modulates protein stability, activity, and interactions, impacting antiviral defense, autophagy, and cancer progression.
• Dysregulation of ISG15 activation is linked to hepatocellular carcinoma resistance, breast cancer initiation, and viral immune evasion.
• Key experimental approaches include knockout of UBA7, point mutations in the catalytic cysteine, and knock-in of tagged ISG15 to track conjugation.
• EDITGENE provides CRISPR services to dissect ISG15 activating enzyme activity in disease models, from KO to overexpression and library screening.
Description
ISG15 activating enzyme activity (GO:0019782) is the molecular function that initiates the covalent attachment of the ubiquitin-like protein ISG15 to target proteins, a process known as ISGylation. This activity is essential for the first step of the ISG15 conjugation cascade, where the E1 enzyme UBA7 (also called UBE1L) forms an ATP-dependent thiolester bond with ISG15. Researchers study this term to understand how cells regulate interferon-stimulated gene responses, antiviral immunity, and cancer biology. The importance of GO:0019782 extends to viral pathogenesis, as viruses like SARS-CoV-2 can counteract ISGylation through deconjugating enzymes. Moreover, ISG15 activation has been implicated in modulating autophagy and ferroptosis, offering therapeutic targets in oncology. This article synthesizes authoritative QuickGO data and verified PubMed literature to provide a research-grade overview of the genes, mechanisms, and methods surrounding ISG15 activating enzyme activity.
ISG15 activating enzyme activity At A Glance
| GO ID | GO:0019782 |
|---|---|
| GO term | ISG15 activating enzyme activity |
| Ontology | molecular_function |
| Synonym | UBE1L |
| Major function | ATP-dependent activation of ISG15 via thiolester bond formation |
| Cellular location | Cytoplasm (inferred from E1 enzyme localization) |
| Pathway | ISG15 conjugation cascade (ISGylation) |
| Key enzyme | UBA7 (UBE1L) |
| Conjugate | ISG15 |
What Is GO:0019782?
ISG15 activating enzyme activity (GO:0019782) is defined as the catalysis of ISG15 activation through the formation of an ATP-dependent high-energy thiolester bond, a prerequisite for subsequent ISG15 conjugation to substrate proteins. This activity is carried out by the E1 enzyme UBA7, which adenylates ISG15 and transfers it to a catalytic cysteine residue, enabling downstream transfer to E2 and E3 enzymes.
Why Is ISG15 activating enzyme activity Important in Cell Biology?
ISG15 activating enzyme activity is critical for the initiation of ISGylation, a post-translational modification that regulates protein stability, activity, and localization in response to interferon and viral infection. Dysregulation of this activity contributes to cancer progression, viral immune evasion, and altered autophagy, making it a focal point for therapeutic intervention.
• Initiates ISGylation, a key antiviral defense mechanism against SARS-CoV-2 and other viruses.
• Modulates protein function in cancer, including hepatocellular carcinoma and breast cancer.
• Regulates autophagy through ISGylation of N-degron recognins.
• Impacts ferroptosis by regulating NCOA4 deISGylation in drug resistance.
• Controls transcriptional corepressor activity of CtBP1 via ISG15 modification.
• Suppresses ubiquitin-conjugating activity of Ubc13 through ISG15 modification.
• Involved in interferon signaling and immune response.
• Potential target for overcoming acquired resistance in cancer therapy.
• Essential for understanding cross-talk between ubiquitin and ubiquitin-like modifiers.
• Provides a model for studying E1 enzyme mechanisms and specificity.
Molecular Mechanism of ISG15 activating enzyme activity
ATP-dependent activation of ISG15
In simple terms: The enzyme uses ATP to activate ISG15, forming a high-energy bond.
The E1 enzyme UBA7 binds ISG15 and ATP, catalyzing adenylation of ISG15's C-terminal glycine, releasing pyrophosphate. This step is essential for subsequent thiolester bond formation.
Thiolester bond formation
In simple terms: Activated ISG15 is transferred to a cysteine on the E1 enzyme.
The adenylated ISG15 is transferred to the catalytic cysteine of UBA7, forming a high-energy thiolester bond. This E1-ISG15 thiolester is the activated intermediate for downstream conjugation.
Transfer to E2 enzyme UbcH8
In simple terms: ISG15 is handed off to a second enzyme, UbcH8.
The activated ISG15 is transferred from UBA7 to the E2 enzyme UbcH8 (UBE2L6), which is also the E2 for ubiquitin conjugation. This dual specificity highlights the interplay between ubiquitin and ISG15 pathways.
E3 ligase-mediated conjugation to substrates
In simple terms: A third enzyme, an E3 ligase, attaches ISG15 to target proteins.
E3 ligases such as HERC5 facilitate the transfer of ISG15 from UbcH8 to specific lysine residues on substrate proteins, completing ISGylation. This step confers substrate specificity and is regulated by interferon signaling.
Deconjugation by USP18
In simple terms: USP18 removes ISG15 from proteins, reversing the modification.
USP18 is a protease that specifically removes ISG15 from conjugated proteins, balancing ISGylation levels. This regulation is critical for preventing excessive immune activation and is exploited by viruses like SARS-CoV-2.
Key Genes Involved in GO:0019782 ISG15 activating enzyme activity
The following genes and proteins are central to ISG15 activating enzyme activity and its downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| UBA7 (UBE1L) | E1 activating enzyme for ISG15 | Catalyzes ATP-dependent activation; knockout abolishes ISGylation |
| UbcH8 (UBE2L6) | E2 conjugating enzyme | Accepts ISG15 from UBA7; also functions in ubiquitination |
| HERC5 | E3 ligase for ISGylation | Mediates ISG15 conjugation to viral proteins like SARS-CoV-2 nsp8 |
| ISG15 | Ubiquitin-like modifier | Substrate for activation; conjugated to target proteins |
| USP18 | DeISGylase | Removes ISG15; regulates ISGylation and immune response |
| CtBP1 | Transcriptional corepressor | Regulated by ISG15 modification |
| Ubc13 | E2 enzyme for ubiquitination | ISG15 modification suppresses its activity |
| NCOA4 | Autophagy receptor | DeISGylation regulates ferroptosis in HCC |
| CYP4Z1 | Cytochrome P450 | ISGylation stabilizes it in breast cancer |
| SARS-CoV-2 PLpro | Viral protease | Deconjugates ISG15 to evade immunity |
| nsp8 | SARS-CoV-2 nonstructural protein | ISGylated by HERC5 for degradation |
| UBE2L6 | E2 enzyme | Alternative name for UbcH8 |
| HERC6 | E3 ligase | Potential ISG15 E3 ligase (inferred from family) |
| RNF213 | E3 ligase | May regulate ISGylation (inferred) |
| TRIM25 | E3 ligase | Involved in interferon signaling (inferred) |
| STAT1 | Transcription factor | Regulates ISG15 expression (inferred) |
| IRF3 | Transcription factor | Induces interferon-stimulated genes (inferred) |
| NF-kB | Transcription factor | Modulates immune responses (inferred) |
How Is ISG15 activating enzyme activity Regulated?
ISG15 activating enzyme activity is regulated at multiple levels. Interferon signaling induces the expression of UBA7 and ISG15, enhancing ISGylation capacity. USP18 negatively regulates ISGylation by removing ISG15 from substrates, and its expression is itself interferon-inducible, creating a feedback loop. Viral proteins such as SARS-CoV-2 PLpro can deconjugate ISG15, counteracting the activating enzyme's function. Additionally, ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity, linking ISGylation to regulation of ubiquitin signaling.
ISG15 activating enzyme activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| UBA7 | Cancer, viral infection | Knockout cell lines, xenograft models |
| USP18 | Hepatocellular carcinoma resistance | Knockout or overexpression in HCC cells |
| HERC5 | SARS-CoV-2 replication | Overexpression in lung epithelial cells |
| CYP4Z1 | Breast cancer initiation | Knock-in of ISGylation sites |
| Ubc13 | Ubiquitin signaling | Point mutation of ISGylation site |
Cancer
ISG15 activating enzyme activity is implicated in cancer progression and therapy resistance. In hepatocellular carcinoma, targeting USP18 overcomes acquired resistance by regulating NCOA4 deISGylation and ferroptosis. In breast cancer, ISGylation-mediated stabilization of CYP4Z1 fuels tumor initiation and progression. These findings suggest that modulating ISG15 activation could be a therapeutic strategy.
Viral Infection
ISGylation is a key antiviral mechanism. HERC5-mediated ISGylation of SARS-CoV-2 nsp8 facilitates its degradation and inhibits viral replication. Conversely, SARS-CoV-2 PLpro deconjugates ISG15 to evade host immunity, highlighting a tug-of-war between the virus and the ISG15 activating enzyme pathway.
Autophagy and Neurodegeneration
ISG15 activating enzyme activity influences autophagy through ISGylation of N-degron recognins, as shown for SARS-CoV-2 PLpro. Dysregulation of autophagy is linked to neurodegeneration, suggesting a potential role for ISGylation in neuronal health, though direct evidence is limited.
From ISG15 activating enzyme activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does UBA7 knockout abolish ISGylation? | UBA7 KO cell lines (e.g., HEK293, HeLa) |
| What is the effect of catalytic cysteine mutation? | Point mutation (C173S) knock-in |
| How does ISG15 conjugation affect substrate stability? | Knock-in of tagged ISG15 (e.g., HA-ISG15) |
| Can overexpression of HERC5 enhance ISGylation? | Overexpression of HERC5 in interferon-treated cells |
| What is the role of USP18 in ISGylation? | USP18 KO or overexpression |
| How does SARS-CoV-2 PLpro affect ISGylation? | Infection with SARS-CoV-2 or PLpro overexpression |
How to Study the ISG15 activating enzyme activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Western blot | ISG15 conjugates and free ISG15 | Validation of ISGylation in cells |
| Immunoprecipitation | Specific ISGylated proteins | Identification of substrates |
| Mass spectrometry | ISGylation sites and target proteins | Global profiling of ISGylation |
| CRISPR knockout screen | Genes regulating ISGylation | Discovery of pathway components |
| Fluorescence microscopy | Localization of ISG15/UBA7 | Live-cell imaging |
| qPCR | mRNA levels of ISG15, UBA7 | Interferon response assessment |
| Luciferase reporter | Interferon promoter activity | Signaling pathway analysis |
| Co-immunoprecipitation | Protein-protein interactions | E1-E2-E3 complex formation |
Proteomics for ISGylation
Mass spectrometry-based proteomics can identify ISG15-conjugated proteins and map conjugation sites, providing a global view of ISGylation targets. This approach is useful for understanding the downstream effects of ISG15 activating enzyme activity.
Western Blotting and Immunoprecipitation
Western blotting with anti-ISG15 antibodies detects free ISG15 and ISG15 conjugates, while immunoprecipitation can isolate specific ISGylated proteins. These methods are standard for validating ISGylation in cell models.
CRISPR Screening
Genome-wide CRISPR knockout screens can identify genes that regulate ISG15 activating enzyme activity or ISGylation, such as E3 ligases and deISGylases. This unbiased approach reveals novel components of the pathway.
Fluorescence Microscopy
Imaging with fluorescently tagged ISG15 or UBA7 allows visualization of subcellular localization and dynamics of ISGylation in live cells. This method complements biochemical assays.
How CRISPR Can Be Used to Study GO:0019782 ISG15 activating enzyme activity
Knockout
CRISPR knockout of UBA7 or ISG15 abolishes ISG15 activating enzyme activity, providing a clean background to study ISGylation-dependent processes. Such models are essential for validating the role of ISGylation in antiviral defense and cancer.
Point Mutation
Introducing point mutations in the catalytic cysteine of UBA7 (e.g., C173S) via CRISPR knock-in can dissect the enzymatic activity from scaffolding functions. This approach helps distinguish activation from other roles.
Knock-in
Knock-in of tagged ISG15 (e.g., HA or GFP) allows tracking of ISG15 conjugation and localization in real time. This model is valuable for studying dynamic ISGylation in response to stimuli.
Overexpression
CRISPR activation or cDNA overexpression of UBA7, HERC5, or ISG15 can enhance ISGylation, enabling gain-of-function studies. Overexpression models are useful for identifying downstream effects and therapeutic potential.
How EDITGENE Supports ISG15 activating enzyme activity Research
Researchers studying ISG15 activating enzyme activity-related genes often need to determine whether a candidate gene is causally involved in ISGylation, antiviral responses, or cancer progression. EDITGENE provides comprehensive CRISPR services to create precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for ISG15 activating enzyme activity research.
Frequently Asked Questions About ISG15 activating enzyme activity
What is ISG15 activating enzyme activity?
ISG15 activating enzyme activity (GO:0019782) is the ATP-dependent activation of ISG15 by the E1 enzyme UBA7, forming a thiolester bond essential for ISGylation.
What genes are involved in ISG15 activating enzyme activity?
Key genes include UBA7 (E1), UbcH8 (E2), HERC5 (E3), ISG15, and USP18 (deISGylase).
How is ISG15 activating enzyme activity regulated?
It is regulated by interferon signaling, USP18-mediated deconjugation, and viral proteins like SARS-CoV-2 PLpro.
What diseases are associated with ISG15 activating enzyme activity?
It is linked to cancer (hepatocellular carcinoma, breast cancer), viral infections, and autophagy-related disorders.
What is the role of UBA7 in ISG15 activation?
UBA7 is the E1 enzyme that catalyzes ATP-dependent activation of ISG15, forming a thiolester bond.
How can I study ISG15 activating enzyme activity in the lab?
Use CRISPR knockout of UBA7, point mutations, knock-in of tagged ISG15, and proteomics to track ISGylation.
What is the difference between ISG15 and ubiquitin?
ISG15 is a ubiquitin-like modifier with distinct conjugation machinery, including UBA7 and UbcH8, and is induced by interferon.
Can ISG15 activating enzyme activity be targeted for cancer therapy?
Yes, targeting USP18 to modulate ISGylation has shown promise in overcoming hepatocellular carcinoma resistance.
What methods detect ISGylation?
Western blot, immunoprecipitation, mass spectrometry, and fluorescence microscopy are commonly used.
What CRISPR models are available for ISG15 research?
EDITGENE offers knockout, point mutation, knock-in, overexpression, and library screening for ISGylation genes.
Conclusion
ISG15 activating enzyme activity (GO:0019782) is a fundamental molecular function that initiates ISGylation, a critical post-translational modification in antiviral immunity, cancer, and autophagy. Understanding its mechanism and regulation provides insights into disease pathogenesis and potential therapeutic targets. EDITGENE's CRISPR services empower researchers to dissect this pathway with precision, from knockout to knock-in models, accelerating discoveries in ISG15 biology.
References
- 1. Ye S et al.. 2025. Targeting USP18 overcomes acquired resistance in hepatocellular carcinoma by regulating NCOA4 deISGylation and ferroptosis.. Cell Death Dis 16(1):448 PMID: 40514377
- 2. 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
- 3. 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
- 4. Hou Y et al.. 2025. HERC5-mediated ISGylation of SARS-CoV-2 nsp8 facilitates its degradation and inhibits viral replication.. Int J Biol Macromol 315(Pt 2):144546 PMID: 40409630
- 5. Yuan Y et al.. 2026. ISGylation-mediated stabilization of CYP4Z1 fuels breast cancer initiation and progression.. Exp Mol Med 58(7):2202-2222 PMID: 42393313
- 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. Takeuchi T et al.. 2005. ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity.. Biochem Biophys Res Commun 336(1):9-13 PMID: 16112642
- 8. Kang JA et al.. 2020. Emerging Roles of USP18: From Biology to Pathophysiology.. Int J Mol Sci 21(18) PMID: 32957626
