GO:0042296 ISG15 transferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0042296 (ISG15 transferase activity) catalyzes the covalent attachment of ISG15 to target proteins, a process known as ISGylation.
• The term is defined as the transfer of ISG15 from one protein to another via a covalent linkage, with both donor and acceptor being ISG15-conjugated.
• Key enzymes include HERC5, which acts as the primary ISG15 E3 ligase, and other HERC family members.
• ISG15 transferase activity regulates innate immune signaling, including STING and cGAS pathways, by modifying key adaptor proteins.
• Dysregulation of ISG15 transferase activity is implicated in autoimmune diseases like dermatomyositis and in cancer progression.
• CRISPR-based models (knockout, knock-in, point mutation) are essential to dissect the specific roles of ISG15 conjugation in health and disease.
Description
ISG15 transferase activity (GO:0042296) is a molecular function that mediates the covalent conjugation of the ubiquitin-like protein ISG15 to target proteins, a process termed ISGylation. This enzymatic activity is central to the interferon-induced antiviral response and broader innate immune regulation. The transfer of ISG15 occurs through a sequential cascade involving E1, E2, and E3 enzymes, ultimately forming an isopeptide bond between the C-terminus of ISG15 and lysine residues on substrate proteins. Understanding this activity is critical because it modulates key signaling pathways, including those involving STING and cGAS, which are essential for detecting cytosolic DNA and mounting immune responses. Moreover, ISG15 transferase activity has been linked to diverse pathological conditions, from autoimmune myopathies to cartilage degradation, underscoring its broad biological significance. Researchers studying this term aim to elucidate how ISGylation alters protein function, stability, and interactions, and to identify therapeutic targets for diseases where ISG15 conjugation is perturbed.
ISG15 transferase activity At A Glance
| GO ID | GO:0042296 |
|---|---|
| GO term | ISG15 transferase activity |
| Ontology | molecular_function |
| Synonym | ISG15 conjugating enzyme activity |
| Major function | Catalyzes the covalent attachment of ISG15 to target proteins |
| Reaction | X-ISG15 + Y = Y-ISG15 + X |
| Covalent linkage | Both X-ISG15 and Y-ISG15 are covalent linkages |
| Related process | ISGylation, innate immune response |
What Is GO:0042296?
ISG15 transferase activity (GO:0042296) is defined as the catalysis of the transfer of ISG15 from one protein to another via the reaction X-ISG15 + Y = Y-ISG15 + X, where both X-ISG15 and Y-ISG15 are covalent linkages. In simpler terms, it is the enzymatic activity that attaches ISG15 to target proteins, forming a covalent bond. This activity is synonymous with ISG15 conjugating enzyme activity and is a key step in the ISGylation pathway.
Why Is ISG15 transferase activity Important in Cell Biology?
ISG15 transferase activity is crucial for innate immunity, as it directly modifies key signaling molecules like STING and cGAS to modulate their function during viral and bacterial infections. Beyond immunity, this activity influences cellular processes such as autophagy, DNA repair, and protein homeostasis, and its dysregulation is associated with autoimmune diseases, cancer, and inflammatory disorders. Therefore, understanding the mechanisms and regulation of ISG15 transferase activity offers insights into fundamental biology and potential therapeutic strategies.
• Regulates antiviral and antibacterial innate immune responses by modifying STING and cGAS.
• Modulates interferon signaling and cytokine production.
• Implicated in autoimmune diseases such as dermatomyositis, where ISG15 pathway genes are dysregulated.
• Plays a role in cartilage degradation and osteoarthritis via ISGylation of MFN1/2.
• Affects cancer progression, including glioma, through interactions with m6A modification machinery.
• Influences protein stability, localization, and interactions through covalent modification.
• Potential target for therapeutic intervention in inflammatory and infectious diseases.
• Essential for understanding cross-talk between ubiquitin-like modifications and other post-translational modifications.
• Provides a model for studying enzymatic cascades involving E1, E2, and E3 enzymes.
• Relevant to host-pathogen interactions, including HIV-1 and HSV-1.
What Happens During ISG15 transferase activity?
Activation of ISG15 by E1 enzyme
In simple terms: First, ISG15 is activated by an E1 enzyme in an ATP-dependent manner.
The ISGylation cascade begins with the activation of ISG15 by the E1 enzyme UBA7 (UBE1L), which forms a thioester bond with ISG15 in an ATP-dependent reaction. This step is analogous to ubiquitin activation and is required for subsequent transfer to E2 enzymes.
Conjugation to E2 enzyme
In simple terms: Activated ISG15 is then passed to an E2 conjugating enzyme.
The E2 enzyme UBE2L6 (UBCH8) receives ISG15 from UBA7 through a trans-thioesterification reaction, forming an E2-ISG15 thioester intermediate. This step is essential for presenting ISG15 to E3 ligases.
Transfer to target protein by E3 ligase
In simple terms: Finally, an E3 ligase attaches ISG15 to a target protein.
HERC5 is the primary E3 ligase for ISG15, catalyzing the transfer of ISG15 from the E2 enzyme to specific lysine residues on substrate proteins, forming an isopeptide bond. Other E3 ligases, such as HERC6, may also participate. This step determines substrate specificity and is critical for the biological effects of ISGylation.
Reversal by deISGylating enzymes
In simple terms: ISG15 can be removed from proteins by specific enzymes.
DeISGylating enzymes, such as USP18, remove ISG15 from conjugated proteins, providing a dynamic regulation of ISGylation. This reversibility allows fine-tuning of signaling pathways.
Key Genes Involved in GO:0042296 ISG15 transferase activity
The following genes encode key components of the ISG15 transferase activity pathway, including enzymes and substrates.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ISG15 | Ubiquitin-like modifier | Substrate for conjugation; central to ISGylation |
| UBA7 | E1 activating enzyme | Activates ISG15 for conjugation |
| UBE2L6 | E2 conjugating enzyme | Transfers ISG15 to E3 ligases |
| HERC5 | E3 ligase | Primary E3 for ISG15; determines substrate specificity |
| HERC6 | E3 ligase | Alternative E3 for ISG15 |
| USP18 | DeISGylase | Removes ISG15 from substrates; regulates pathway |
| STING1 | Substrate | ISGylation modulates STING activity in DNA sensing |
| CGAS | Substrate | ISGylation potentiates cGAS-mediated immunity |
| MFN1 | Substrate | ISGylation affects mitochondrial dynamics |
| MFN2 | Substrate | ISGylation affects mitochondrial dynamics |
| PARP12 | Substrate | ISGylation regulates its function in cartilage degradation |
| MOV10 | Substrate | ISGylation regulates IFN-I production |
| IFIH1 | Substrate | MDA5 ISGylation impacts innate immune detection |
| IRF1 | Transcription factor | Regulates PARP12 expression |
| METTL14 | m6A methyltransferase | Influenced by ISGylation in glioma |
| PINK1 | Kinase | Affected by ISGylation in mitophagy |
| Parkin | E3 ligase | Affected by ISGylation in mitophagy |
How Is ISG15 transferase activity Regulated?
ISG15 transferase activity is regulated at multiple levels. Interferon stimulation strongly induces the expression of ISG15 and its conjugation enzymes, thereby increasing ISGylation. The activity is also controlled by the availability of E1, E2, and E3 enzymes, and by deISGylating enzymes such as USP18 that remove ISG15. Additionally, viral proteins can modulate this pathway; for example, HSV-1 ICP0 suppresses METTL14, affecting m6A reprogramming and potentially influencing ISGylation. Furthermore, cross-talk with ubiquitination and SUMOylation pathways can impact ISG15 conjugation, as seen in cartilage degradation where ISG15 attenuates ubiquitylation and SUMOylation of MFN1/2.
ISG15 transferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ISG15 | Dermatomyositis | Knockout mice or cell lines |
| HERC5 | Antiviral immunity | Overexpression and knockout cells |
| STING1 | DNA sensing disorders | Point mutation knock-in |
| PARP12 | Osteoarthritis | Knockout chondrocytes |
| MFN1/2 | Mitochondrial dynamics | Knock-in of ISGylation sites |
ISG15 transferase activity in autoimmune diseases
Dysregulated ISG15 transferase activity is implicated in autoimmune conditions such as dermatomyositis, where transcriptomic and proteomic analyses reveal shared and distinctive pathways involving ISG15. In juvenile and adult dermatomyositis, increased ISGylation may contribute to muscle inflammation and damage.
ISG15 transferase activity in cancer
In glioma, HSV-1-induced m6A reprogramming via ICP0-mediated suppression of METTL14 potentiates oncolytic activity, and this process may intersect with ISG15 transferase activity. Additionally, ISGylation of key signaling proteins can influence tumor immune surveillance and cancer progression.
ISG15 transferase activity in cartilage degradation
IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin-dependent mitophagy through ISG15, which attenuates ubiquitylation and SUMOylation of MFN1/2. This highlights a role for ISG15 transferase activity in osteoarthritis pathogenesis.
ISG15 transferase activity in viral infections
ISG15 transferase activity is a key antiviral effector mechanism. It restricts replication of viruses such as HIV-1 and HSV-1 by modifying viral and host proteins. For instance, IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA from HIV-1, and ISGylation may modulate this response.
From ISG15 transferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ISG15 conjugation regulate STING signaling? | STING knockout cells with ISG15 transferase overexpression |
| What is the role of HERC5 in antiviral response? | HERC5 knockout cell lines |
| How does ISGylation of cGAS affect DNA sensing? | cGAS point mutants lacking ISGylation sites |
| Does ISG15 modification of MFN1/2 impact mitophagy? | MFN1/2 knock-in with mutated ISGylation sites |
| Can ISG15 transferase activity be targeted in cancer? | Xenograft models with ISG15 pathway knockout |
| What is the interplay between ISGylation and ubiquitination? | Double knockout of ISG15 and ubiquitin enzymes |
How to Study the ISG15 transferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Mass spectrometry | ISG15-conjugated proteins | Identifying substrates in cells |
| RNA-seq | Gene expression changes | Pathway analysis in disease |
| In vitro conjugation assay | Enzymatic activity | Mechanistic studies |
| Immunoprecipitation | Protein interactions | Detecting ISGylation of targets |
| Western blot | ISG15 conjugation levels | Validating ISGylation |
| CRISPR screening | Genes required for ISGylation | Functional genomics |
| Bioinformatics | Pathway enrichment | Data interpretation |
Proteomic identification of ISG15 substrates
Mass spectrometry-based proteomics can identify proteins conjugated to ISG15 under various conditions, revealing substrates and interaction networks. This approach is useful for mapping the ISGylome in disease models.
Transcriptomic analysis of ISG15 pathway genes
RNA-seq can quantify expression changes in ISG15, HERC5, and other pathway components in response to interferon or viral infection. This helps understand transcriptional regulation.
Functional assays for ISG15 conjugation
In vitro conjugation assays using recombinant E1, E2, E3, and substrate proteins can measure ISG15 transferase activity directly. These assays are essential for mechanistic studies.
Imaging of ISG15 localization
Fluorescence microscopy with tagged ISG15 or substrates can visualize subcellular localization and dynamics of ISGylation. This provides spatial context to the activity.
How CRISPR Can Be Used to Study GO:0042296 ISG15 transferase activity
Knockout
CRISPR knockout of ISG15, UBA7, UBE2L6, or HERC5 can abolish ISG15 transferase activity, enabling studies of its loss-of-function phenotypes in immunity and disease.
Point Mutation
Introducing point mutations in ISG15 or substrate proteins (e.g., STING, cGAS) at ISGylation sites can specifically disrupt conjugation without affecting other functions, clarifying the role of ISGylation.
Knock-in
Knock-in of tagged ISG15 (e.g., HA or FLAG) allows for affinity purification and identification of conjugated proteins, as well as live-cell imaging.
Overexpression
Overexpression of HERC5 or ISG15 can enhance ISGylation, useful for studying gain-of-function effects and identifying downstream consequences.
How EDITGENE Supports ISG15 transferase activity Research
Researchers studying ISG15 transferase activity-related genes often need to determine whether a candidate gene is causally involved in ISGylation, innate immunity, or disease pathogenesis. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling functional validation of genes in the ISG15 pathway.
Contact EDITGENE today to design your custom CRISPR model for ISG15 transferase activity research.
Frequently Asked Questions About ISG15 transferase activity
What is ISG15 transferase activity?
ISG15 transferase activity (GO:0042296) is the enzymatic activity that covalently attaches ISG15 to target proteins, a process called ISGylation.
What genes are involved in ISG15 transferase activity?
Key genes include ISG15, UBA7, UBE2L6, HERC5, HERC6, and USP18, as well as substrates like STING1, CGAS, and MFN1/2.
How is ISG15 transferase activity regulated?
It is regulated by interferon signaling, enzyme availability, and deISGylating enzymes like USP18.
What diseases are associated with ISG15 transferase activity?
It is linked to autoimmune diseases like dermatomyositis, cancer, osteoarthritis, and viral infections.
What is the role of HERC5 in ISG15 transferase activity?
HERC5 is the primary E3 ligase that catalyzes the transfer of ISG15 to target proteins.
How can I study ISG15 transferase activity using CRISPR?
CRISPR knockout, point mutation, knock-in, and overexpression models can be used to manipulate the pathway and study its effects.
What are the substrates of ISG15 transferase activity?
Substrates include STING, cGAS, MFN1/2, PARP12, MOV10, and IFIH1, among others.
What is the difference between ISG15 and ubiquitin?
ISG15 is a ubiquitin-like protein, but it is conjugated by a distinct set of enzymes and has unique functions in immunity.
Can ISG15 transferase activity be targeted therapeutically?
Yes, it is a potential target for modulating immune responses in infections, autoimmune diseases, and cancer.
What methods are used to measure ISG15 transferase activity?
Methods include in vitro conjugation assays, mass spectrometry, western blot, and immunoprecipitation.
Conclusion
ISG15 transferase activity (GO:0042296) is a fundamental enzymatic function that covalently attaches ISG15 to target proteins, playing pivotal roles in innate immunity, cellular stress responses, and disease. Its dysregulation contributes to autoimmune conditions, cancer, and degenerative diseases, making it a compelling research focus. By leveraging CRISPR-based models and advanced omics, researchers can dissect the precise mechanisms and therapeutic potential of this pathway.
References
- 1. Lin C et al.. 2023. Regulation of STING activity in DNA sensing by ISG15 modification.. Cell Rep 42(11):113277 PMID: 37864791
- 2. Chu L et al.. 2024. HERC5-catalyzed ISGylation potentiates cGAS-mediated innate immunity.. Cell Rep 43(3):113870 PMID: 38421872
- 3. Sadler AJ et al.. 2008. Interferon-inducible antiviral effectors.. Nat Rev Immunol 8(7):559-68 PMID: 18575461
- 4. Chen Y et al.. 2024. HSV-1-induced N6-methyladenosine reprogramming via ICP0-mediated suppression of METTL14 potentiates oncolytic activity in glioma.. Cell Rep 43(10):114756 PMID: 39325621
- 5. Ward JM et al.. 2023. Shared and Distinctive Transcriptomic and Proteomic Pathways in Adult and Juvenile Dermatomyositis.. Arthritis Rheumatol 75(11):2014-2026 PMID: 37229703
- 6. Deng Z et al.. 2024. IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.. Bone Res 12(1):63 PMID: 39465252
- 7. Mukhopadhyay R et al.. 2026. USP24 is a cross-reactive DUB targeting MOV10 to regulate IFN-I production.. Nat Commun 17(1) PMID: 42321163
- 8. Guney MH et al.. 2024. IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus.. Proc Natl Acad Sci U S A 121(29):e2404349121 PMID: 38985764
