ISG15 Human

What is ISG15 and how does it function in human immunity?

ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like molecule that exists in two distinct functional states: as a free molecule (both intracellular and extracellular) or conjugated to target proteins (ISGylation) . Unlike ubiquitin's near-perfect cross-species conservation, ISG15 shows relatively low conservation across species, ranging from 98% (chimpanzee to human) to 42% (opossum to human) in mammals .

ISG15 functions in humans differ significantly from those in mice:

• Free extracellular ISG15 acts as a cytokine crucial for IFN-γ-dependent antimycobacterial immunity

• Free intracellular ISG15 is essential for USP18-mediated downregulation of IFN-α/β signaling

• ISG15 conjugation (ISGylation) affects diverse cellular pathways including RNA splicing, transcription, cytoskeleton organization, stress responses, and translation

Interestingly, ISG15-deficient patients do not display enhanced susceptibility to viral infections, contrasting sharply with observations in Isg15-deficient mice .

How do research findings on ISG15 differ between humans and mice?

The species-specific differences in ISG15 function represent a critical consideration for researchers designing translational studies:

Human-Mouse ISG15 Function Comparison

These differences highlight why mouse models may provide misleading information about ISG15 function in humans . When studying ISG15, researchers should carefully consider which aspects of ISG15 biology are conserved between species and which represent divergent evolutionary paths.

What methodologies are most effective for studying ISG15 conjugation in human samples?

Research on ISG15 conjugation requires specialized techniques:

Recommended Approaches:

• Western blotting: Using reducing conditions and Immunoblot Buffer Group 2, researchers can detect ISG15 as a specific band at approximately 15-20 kDa . For analyzing conjugated forms, higher molecular weight bands should be examined.

• Double-affinity selection followed by mass spectroscopy: This methodology, as employed by researchers identifying 158 ISG15 target proteins, provides the most comprehensive approach for identifying ISGylated proteins .

• Immunohistochemistry: ISG15 localization can be detected in fixed paraffin-embedded tissue sections using monoclonal antibodies (e.g., at 0.5 μg/mL), with specific staining typically localized to the cytoplasm .

• Simple Western™ system: For more automated quantitative analysis, the 12-230 kDa separation system can effectively detect ISG15 .

When designing ISG15 conjugation experiments, it's crucial to include appropriate controls including both IFN-β-treated and untreated cells to distinguish constitutive versus IFN-induced conjugation patterns.

What is the repertoire of proteins targeted for ISGylation in humans?

ISG15 conjugation affects an extensive network of proteins, with 158 identified target proteins across diverse cellular pathways :

ISG15 Target Categories:

• IFN-α/β-induced antiviral proteins: Including PKR, MxA, HuP56, and RIG-I

• RNA splicing factors

• Chromatin remodeling and polymerase II transcription components

• Cytoskeleton organization and regulatory proteins

• Stress response proteins

• Translation machinery components

This broad targeting profile indicates that ISG15 conjugation significantly extends the range of cellular functions affected by IFN-α/β signaling . Researchers investigating specific pathways should consider how ISGylation might modify their proteins of interest, particularly in the context of interferon responses.

How does ISG15 deficiency manifest in human patients?

ISG15 deficiency in humans presents with distinctive clinical manifestations:

Clinical Manifestations:

• Necrotizing skin lesions: Patients with novel ISG15 mutations present with skin lesions initially managed as dermatologic disease

• Type I IFN inflammation: Systemic inflammation linked to dysregulated IFN signaling

• Normal antiviral immunity: Unlike mouse models, no enhanced susceptibility to viral infections is observed in vivo

• Mycobacterial susceptibility: Due to impaired IFN-γ-dependent antimycobacterial immunity

When investigating patient samples, researchers should recognize that ISG15 deficiency represents a type I interferonopathy rather than a primary immunodeficiency affecting antiviral responses. This makes ISG15 deficiency mechanistically distinct from many other innate immunity defects.

How does ISG15 regulate USP18 and the type I interferon response?

The relationship between ISG15 and USP18 represents a critical species-specific regulatory mechanism:

Mechanistic Details:

• In humans, free intracellular ISG15 is essential for stabilizing USP18, a negative regulator of type I IFN signaling

• Without ISG15, human USP18 levels decrease, leading to enhanced and prolonged STAT1 phosphorylation after IFN-α/β stimulation

• This mechanism does not operate in mice, explaining why ISG15-deficient humans show enhanced rather than impaired antiviral protection

This species-specific stabilization mechanism explains the "species-specific gain-of-function in antiviral immunity" observed in ISG15-deficient patients . When designing experiments to study this pathway, researchers should include measurements of both ISG15 and USP18 protein levels, as well as downstream STAT phosphorylation kinetics.

What experimental approaches can identify novel ISG15 functions?

Advancing ISG15 research requires specialized methodological approaches:

Recommended Experimental Strategies:

• CRISPR-based ISG15 knockout models: Creating isogenic human cell lines with and without ISG15 expression

• Proteomics approaches: Mass spectrometry-based techniques to identify ISGylated proteins under different conditions

• Time-course experiments: Monitoring ISG15 conjugation dynamics following interferon stimulation

• Proximity labeling approaches: Identifying proteins that interact with ISG15 transiently

• Domain-specific mutants: Creating ISG15 variants that can distinguish conjugation-dependent from conjugation-independent functions

When designing these experiments, researchers should account for the substantial induction of ISG15 and its conjugation machinery by type I interferons, potentially requiring pretreatment to fully characterize the ISGylation landscape.

How does ISG15 contribute to antiviral immunity in human cells?

Despite ISG15-deficient patients showing no enhanced viral susceptibility in vivo, ISG15 conjugation has demonstrable antiviral effects in certain contexts:

Antiviral Mechanisms:

• ISG15 conjugation inhibits influenza A virus replication in cell culture models

• Several key antiviral proteins are targets of ISG15 conjugation, including PKR, MxA, HuP56, and RIG-I

• The influenza B virus NS1B protein specifically targets ISG15 conjugation, suggesting evolutionary pressure to counteract this pathway

The apparent contradiction between in vitro antiviral effects and in vivo redundancy highlights the complex nature of ISG15 biology. Researchers should carefully design experiments that can distinguish direct ISG15 effects from compensatory mechanisms in the integrated immune response.

What is the role of extracellular ISG15 in human immunity?

Free extracellular ISG15 functions as a cytokine with distinct immunomodulatory properties:

Extracellular ISG15 Functions:

• Acts as a critical mediator of IFN-γ-dependent antimycobacterial immunity

• Promotes IFN-γ production, potentially through interaction with the LFA-1 integrin receptor

• May contribute to the cytokine network regulating immune responses to different pathogens

When studying extracellular ISG15, researchers should include secretion assays and receptor binding studies to fully characterize its immunomodulatory effects. The addition of recombinant ISG15 to immune cell cultures can help determine its direct effects on cytokine production and cellular activation.

How has our understanding of ISG15 biology evolved in recent years?

The field's understanding of ISG15 has undergone significant evolution:

Research Evolution Timeline:

• Initial identification as the first ubiquitin-like protein

• Early focus on antiviral properties based on mouse studies

• Discovery of human ISG15 deficiency revealing species-specific functions

• Recognition of ISG15's role in USP18 regulation in humans

• Expanding roles in diverse cellular processes beyond immunity

Contemporary ISG15 research increasingly focuses on its diverse functions in processes such as protein translation, autophagy, exosome secretion, cytoskeleton dynamics, DNA damage response, and telomere maintenance . As research continues, integrating these diverse functions into a coherent model of ISG15 biology remains an important challenge.