GO:0019785 ISG15-specific peptidase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0019785 (ISG15-specific peptidase activity) is a thiol-dependent isopeptidase activity that removes the ubiquitin-like modifier ISG15 from conjugated target proteins.
• The best-characterized enzyme carrying this activity is USP18 (UBP43), which specifically deconjugates ISG15 and is essential for restraining type I interferon signalling.
• USP18 is not the only ISG15-crossreactive deubiquitinase; systematic screening identified additional enzymes that cleave ISG15 conjugates.
• Viral proteases can mimic this activity: Crimean-Congo hemorrhagic fever virus encodes a ubiquitin- and ISG15-specific protease that suppresses innate immunity.
• Loss or dysregulation of ISG15-specific peptidase activity is linked to cancer, viral pathogenesis and interferonopathies, making it a therapeutic target.
• CRISPR knockout, point-mutation, knock-in and overexpression cell models enable causal dissection of ISG15-specific peptidase function in disease.
Description
ISG15-specific peptidase activity (GO:0019785) is a molecular function that reverses the conjugation of the ubiquitin-like protein ISG15 to target proteins. ISG15 modification, often called ISGylation, is a reversible post-translational event, and the enzymes that remove ISG15 are thiol-dependent isopeptidases. This activity is central to the dynamic regulation of ISG15 signalling and is therefore of broad interest to immunologists, virologists and cancer biologists. The prototype enzyme for this activity is USP18 (also known as UBP43), which was shown to specifically remove ISG15 from conjugated proteins. Subsequent work demonstrated that USP18 is not the sole ISG15-crossreactive deubiquitinase, and that other enzymes can cleave ISG15 conjugates. The functional importance of this activity is underscored by its role in type I interferon signalling: ISG15-dependent stabilization of USP18 is necessary but not sufficient to regulate interferon responses in humans. Moreover, pathogens have evolved proteases that mimic this activity to evade host immunity, as seen with Crimean-Congo hemorrhagic fever virus. Because ISG15-specific peptidase activity sits at the interface of innate immunity, viral pathogenesis and cancer, it is a high-value target for mechanistic and translational research.
ISG15-specific peptidase activity At A Glance
| GO ID | GO:0019785 |
|---|---|
| GO term | ISG15-specific peptidase activity |
| Ontology | molecular_function |
| Synonym | ISG15-specific protease activity |
| Major function | Thiol-dependent cleavage of ISG15 from conjugated target proteins |
| Prototype enzyme | USP18 (UBP43) |
| Pathogen example | Crimean-Congo hemorrhagic fever virus ISG15-specific protease |
| Disease relevance | Cancer, viral pathogenesis, interferon-related disorders |
What Is GO:0019785?
According to the Gene Ontology, GO:0019785 (ISG15-specific peptidase activity) is a thiol-dependent isopeptidase activity that cleaves ISG15 from a target protein to which it is conjugated. In other words, it is the enzymatic removal of the ISG15 modifier from ISGylated proteins, reversing a ubiquitin-like conjugation event. The synonym ISG15-specific protease activity reflects this deconjugating function.
Why Is ISG15-specific peptidase activity Important in Cell Biology?
ISG15-specific peptidase activity is important because it controls the steady-state levels of ISGylated proteins, which in turn modulate innate immune signalling, antiviral defence and cell proliferation. Dysregulation of this activity can lead to unrestrained type I interferon responses or, conversely, to immune evasion by viruses. In cancer, USP18 has been proposed as an antineoplastic target, highlighting the therapeutic potential of modulating this activity. Therefore, understanding GO:0019785 is essential for dissecting interferon biology, host-pathogen interactions and cancer cell signalling.
• Regulates type I interferon signalling by controlling ISG15 conjugation status.
• Prototype enzyme USP18 is a key negative regulator of interferon responses.
• Viral proteases with this activity suppress innate immunity, as shown for Crimean-Congo hemorrhagic fever virus.
• USP18 has been proposed as an antineoplastic target in cancer research.
• USP18 abrogates p21-mediated inhibition of HIV-1, linking this activity to viral replication.
• ISG15-crossreactive deubiquitinases expand the repertoire of enzymes with this activity.
• Enzymatic assays for UBP43 provide a foundation for measuring ISG15-specific deconjugation.
• The activity is relevant to interferonopathies and autoimmune conditions.
• It is a potential drug target for antiviral and anticancer therapies.
• CRISPR models enable causal testing of this activity in disease contexts.
Molecular Mechanism of ISG15-specific peptidase activity
Substrate recognition and ISG15 conjugate binding
In simple terms: The enzyme must first grab onto the ISG15 tag that is attached to another protein.
ISG15-specific peptidases recognize ISG15 conjugated to target proteins. USP18 (UBP43) was shown to specifically remove ISG15 from conjugated proteins, indicating selective recognition of the ISG15 moiety. Screening for ISG15-crossreactive deubiquitinases identified enzymes that can bind and cleave ISG15 conjugates, confirming that substrate recognition is a defined step.
Thiol-dependent catalytic cleavage
In simple terms: A chemical reaction in the enzyme's active site snips the ISG15 tag off the target protein.
The activity is thiol-dependent, meaning it relies on a catalytic cysteine residue to cleave the isopeptide bond between ISG15 and the target protein. This mechanism is shared with other ubiquitin-like proteases and is essential for reversing ISGylation. Enzymatic assays for UBP43 have been developed to measure this cleavage activity in vitro.
Regulation by interferon and ISG15 availability
In simple terms: The amount of ISG15 tag and interferon signals can change how active the enzyme is.
ISG15-dependent stabilization of USP18 is necessary but not sufficient to regulate type I interferon signalling in humans, indicating that the activity is embedded in a feedback loop with interferon. USP18 expression itself is interferon-inducible, linking the activity to the broader interferon response. Thus, the availability of ISG15 conjugates and interferon stimulation modulates the functional output of this peptidase activity.
Viral mimicry and immune evasion
In simple terms: Some viruses make their own version of this enzyme to disable the host's immune alarm.
Crimean-Congo hemorrhagic fever virus encodes a ubiquitin and ISG15-specific protease that suppresses innate immune responses, demonstrating that pathogens can exploit this activity for immune evasion. This viral enzyme functionally mimics host ISG15-specific peptidases, highlighting the evolutionary importance of the activity.
Cross-reactivity with ubiquitin and other modifiers
In simple terms: Some enzymes that remove ISG15 can also remove ubiquitin, so specificity varies.
A screen for ISG15-crossreactive deubiquitinases revealed that some enzymes can cleave both ISG15 and ubiquitin conjugates, indicating that ISG15-specific peptidase activity is not always absolute. This cross-reactivity has implications for interpreting knockout phenotypes and for drug design.
Key Genes Involved in GO:0019785 ISG15-specific peptidase activity
The following genes and proteins are experimentally linked to ISG15-specific peptidase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| USP18 | Prototype ISG15-specific deconjugating enzyme (UBP43) | Key negative regulator of type I interferon; antineoplastic target |
| ISG15 | Ubiquitin-like modifier removed by this activity | Substrate for deconjugation; interferon-stimulated gene |
| UBP43 | Alternative name for USP18; ISG15-specific deconjugating enzyme | Enzymatic assays and purification protocols available |
| USP18 (viral homolog) | Crimean-Congo hemorrhagic fever virus protease with ISG15-specific activity | Model for viral immune evasion |
| USP5 | ISG15-crossreactive deubiquitinase identified in screen | Broadens understanding of ISG15 deconjugation |
| USP14 | ISG15-crossreactive deubiquitinase identified in screen | Potential redundancy in ISG15 processing |
| USP21 | ISG15-crossreactive deubiquitinase identified in screen | Candidate for functional studies |
| USP2 | ISG15-crossreactive deubiquitinase identified in screen | Cross-reactivity with ubiquitin pathways |
| USP36 | ISG15-crossreactive deubiquitinase identified in screen | Role in ISG15 conjugate turnover |
| USP13 | ISG15-crossreactive deubiquitinase identified in screen | Potential modulator of ISGylation |
| USP39 | ISG15-crossreactive deubiquitinase identified in screen | Candidate for further validation |
| USP15 | ISG15-crossreactive deubiquitinase identified in screen | Links to ubiquitin and ISG15 pathways |
| USP16 | ISG15-crossreactive deubiquitinase identified in screen | Potential role in cell cycle and immunity |
| USP18 (human) | Regulates type I interferon signalling via ISG15 stabilization | Interferonopathy and antiviral research |
| p21 | Target of USP18-mediated abrogation in HIV-1 | Links ISG15-specific activity to HIV-1 replication |
| TBEV proteins | Viral effectors that may interact with ISG15 pathways | Neuropathogenesis research |
How Is ISG15-specific peptidase activity Regulated?
ISG15-specific peptidase activity is regulated at multiple levels. USP18, the prototype enzyme, is interferon-inducible, and its ISG15-dependent stabilization is necessary but not sufficient to regulate type I interferon signalling in humans. This creates a feedback loop where interferon increases USP18 expression, which then removes ISG15 from targets and dampens the response. Additionally, the availability of ISG15 conjugates and the presence of cross-reactive deubiquitinases can influence the net activity. Viral proteases can also introduce this activity into host cells to suppress immunity.
ISG15-specific peptidase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| USP18 | Cancer; antineoplastic target | USP18 knockout cancer cell lines |
| USP18 | Type I interferon signalling dysregulation | ISG15-dependent stabilization mutants |
| USP18 | HIV-1 replication via p21 abrogation | USP18 overexpression in HIV-1 infection models |
| CCHFV protease | Viral immune evasion | Viral protease expression in human cells |
| TBEV effectors | Neuropathogenesis | Infected neuron and astrocyte cultures |
Cancer and antineoplastic targeting
USP18 has been proposed as an antineoplastic target, suggesting that ISG15-specific peptidase activity contributes to tumour cell survival or proliferation. Modulating this activity could therefore have therapeutic potential in oncology.
Viral pathogenesis and immune evasion
Crimean-Congo hemorrhagic fever virus encodes a ubiquitin and ISG15-specific protease that suppresses innate immune responses, demonstrating that this activity can be exploited by pathogens. Similarly, USP18 abrogates p21-mediated inhibition of HIV-1, linking ISG15-specific deconjugation to viral replication. These findings highlight the activity as a host-pathogen interface.
Interferonopathies and autoimmune signalling
ISG15-dependent stabilization of USP18 is necessary but not sufficient to regulate type I interferon signalling in humans, implying that defects in this pathway may contribute to interferon-related disorders. Dysregulated ISG15-specific peptidase activity could therefore underlie aberrant interferon responses.
Neuropathogenesis
Integrative RNA profiling of TBEV-infected neurons and astrocytes revealed potential pathogenic effectors, suggesting that ISG15-related pathways may be relevant in viral neuropathogenesis. However, direct evidence for ISG15-specific peptidase activity in this context remains to be established.
From ISG15-specific peptidase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does USP18 loss alter ISG15 conjugate levels? | USP18 knockout cell line |
| Is ISG15-dependent USP18 stabilization required for interferon regulation? | Point-mutation knock-in of USP18 |
| Can a tagged USP18 be used to track ISG15 deconjugation? | Tagged knock-in of USP18 |
| Does overexpression of USP18 suppress interferon responses? | USP18 overexpression cell model |
| Which deubiquitinases cross-react with ISG15? | CRISPR library screening |
| Does viral protease expression suppress innate immunity? | Viral protease overexpression |
How to Study the ISG15-specific peptidase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro deconjugation assay | Cleavage of ISG15 from conjugates | Enzyme kinetics and inhibitor testing |
| ISG15-crossreactive DUB screen | Identification of enzymes cleaving ISG15 | Discovery of novel ISG15 peptidases |
| RNA-seq | Transcriptional changes in infection models | Pathogen effector discovery |
| Interferon reporter assay | Type I interferon pathway activity | Functional validation of USP18 variants |
| Western blot for ISG15 conjugates | Steady-state ISGylation levels | Knockout/overexpression validation |
| CRISPR library screening | Gene requirements for ISG15 processing | Functional genomics of deubiquitinases |
| Viral protease activity assay | Immune evasion potential | Antiviral target discovery |
Enzymatic assays for ISG15 deconjugation
In vitro assays using purified UBP43 (USP18) have been developed to measure ISG15-specific deconjugating activity, providing a direct readout of enzyme function. These assays typically use ISG15 conjugates as substrates and detect cleavage products.
Screening for ISG15-crossreactive deubiquitinases
A screen for ISG15-crossreactive deubiquitinases identified multiple enzymes capable of cleaving ISG15 conjugates, demonstrating the utility of unbiased screening approaches. Such screens can be adapted to CRISPR-based libraries for functional genomics.
RNA profiling in infection models
Integrative RNA profiling of TBEV-infected neurons and astrocytes revealed potential pathogenic effectors, illustrating how transcriptomic methods can uncover links to ISG15-related pathways. These approaches can be combined with ISG15-specific peptidase perturbations.
Interferon signalling readouts
ISG15-dependent stabilization of USP18 and its impact on type I interferon signalling can be assessed using interferon-stimulated gene reporters and phosphorylation of STAT proteins. Such readouts are essential for linking enzymatic activity to cellular outcomes.
How CRISPR Can Be Used to Study GO:0019785 ISG15-specific peptidase activity
Knockout
CRISPR knockout of USP18 can be used to assess the loss of ISG15-specific peptidase activity and its impact on interferon signalling and ISG15 conjugate accumulation. Knockout models are essential for establishing causality in cancer and viral infection contexts.
Point Mutation
Point mutations in the catalytic cysteine of USP18 can abolish thiol-dependent isopeptidase activity, allowing separation of enzymatic function from scaffolding roles. Such mutants are valuable for testing whether ISG15 deconjugation is required for a given phenotype.
Knock-in
Knock-in of tagged USP18 (e.g., FLAG or HA) enables affinity purification and tracking of ISG15-specific peptidase complexes in cells. This approach facilitates proteomic and imaging studies of the enzyme in its native context.
Overexpression
Overexpression of USP18 or viral ISG15-specific proteases can suppress innate immune responses and ISG15 conjugation, providing gain-of-function models for studying immune evasion and interferon regulation. Overexpression models are also useful for drug screening.
How EDITGENE Supports ISG15-specific peptidase activity Research
Researchers studying ISG15-specific peptidase activity-related genes often need to determine whether a candidate gene is causally involved in ISG15 deconjugation, interferon regulation or disease phenotypes. EDITGENE provides CRISPR-based cell model services to enable such causal experiments.
Contact EDITGENE today to design your custom CRISPR model for ISG15-specific peptidase activity research.
Frequently Asked Questions About ISG15-specific peptidase activity
What is ISG15-specific peptidase activity?
It is a thiol-dependent isopeptidase activity that cleaves ISG15 from a target protein to which it is conjugated, as defined by GO:0019785.
What genes are involved in ISG15-specific peptidase activity?
USP18 (UBP43) is the prototype gene, and additional ISG15-crossreactive deubiquitinases have been identified.
What is the GO ID for ISG15-specific peptidase activity?
The GO ID is GO:0019785.
Which enzyme specifically removes ISG15 from conjugated proteins?
USP18 (UBP43) specifically removes ISG15 from conjugated proteins.
How is ISG15-specific peptidase activity regulated?
It is regulated by interferon-induced expression of USP18 and by ISG15-dependent stabilization of USP18.
What diseases are linked to ISG15-specific peptidase activity?
It has been linked to cancer, viral pathogenesis including HIV-1 and Crimean-Congo hemorrhagic fever virus, and interferon-related disorders.
Can viruses encode ISG15-specific proteases?
Yes, Crimean-Congo hemorrhagic fever virus encodes a ubiquitin and ISG15-specific protease that suppresses innate immunity.
What methods are used to study ISG15-specific peptidase activity?
In vitro deconjugation assays, ISG15-crossreactive DUB screens, RNA-seq, interferon reporter assays and CRISPR screening are commonly used.
Is USP18 a therapeutic target?
USP18 has been proposed as an antineoplastic target, and modulating its activity is of interest in cancer and antiviral research.
How can CRISPR help study ISG15-specific peptidase activity?
CRISPR knockout, point mutation, knock-in and overexpression models allow causal testing of ISG15-specific peptidase function in disease contexts.
Conclusion
ISG15-specific peptidase activity (GO:0019785) is a critical enzymatic function that reverses ISG15 conjugation and regulates innate immunity, viral pathogenesis and cancer. USP18 is the prototype enzyme, but additional cross-reactive deubiquitinases and viral proteases expand the functional landscape. CRISPR-based cell models provide powerful tools to dissect the causal roles of these enzymes in health and disease.
References
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- 2. Mustachio LM et al.. 2018. Evidence for the ISG15-Specific Deubiquitinase USP18 as an Antineoplastic Target.. Cancer Res 78(3):587-592 PMID: 29343520
- 3. Vasou A et al.. 2025. ISG15-Dependent Stabilisation of USP18 Is Necessary but Not Sufficient to Regulate Type I Interferon Signalling in Humans.. Eur J Immunol 55(2):e202451651 PMID: 39931755
- 4. Catic A et al.. 2007. Screen for ISG15-crossreactive deubiquitinases.. PLoS One 2(7):e679 PMID: 17653289
- 5. Scholte FEM et al.. 2017. Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease.. Cell Rep 20(10):2396-2407 PMID: 28877473
- 6. Osei Kuffour E et al.. 2018. USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1.. J Virol 92(20) PMID: 30068654
- 7. Malakhov MP et al.. 2002. UBP43 (USP18) specifically removes ISG15 from conjugated proteins.. J Biol Chem 277(12):9976-81 PMID: 11788588
- 8. Kim KI et al.. 2005. UBP43, an ISG15-specific deconjugating enzyme: expression, purification, and enzymatic assays.. Methods Enzymol 398:491-9 PMID: 16275353
