Recombinant Human Interleukin-36 receptor antagonist protein (IL36RN) (Active)

What is IL36RN and what is its primary biological function?

IL36RN is the Interleukin-36 receptor antagonist protein (IL-36Ra), a member of the IL-1 family that functions as an inhibitor of IL-36 signaling. The protein specifically inhibits the activity of interleukin-36 cytokines (IL36A, IL36B, and IL36G) by competitively binding to the IL-36 receptor (IL1RL2) and preventing its association with the coreceptor IL1RAP, thereby blocking downstream signaling . This antagonism is part of a regulatory mechanism within epithelial barriers that helps control local inflammatory responses. IL36RN is particularly important in skin homeostasis, where it provides negative regulation of inflammatory processes that, when dysregulated, can lead to conditions such as generalized pustular psoriasis . The protein may also participate in innate immune responses to fungal pathogens like Aspergillus fumigatus and potentially activates anti-inflammatory signaling pathways by recruiting SIGIRR .

How does the IL36RN gene structure relate to its function?

The IL36RN gene encodes the IL-36 receptor antagonist, with several common polymorphisms identified in research. The most frequent polymorphism is c.115+6T>C, which has been extensively studied in relation to generalized pustular psoriasis . Other significant polymorphisms include c.140A>G, c.227C>T, c.28C>T, and c.368C>T . These genetic variations can affect protein stability and receptor binding capability, potentially impairing the antagonistic function of IL36RN. The gene structure supports the production of a functional antagonist that regulates IL-36 cytokine activity through direct receptor competition, which is essential for maintaining appropriate inflammatory responses in epithelial tissues. Disruptions in this gene structure through mutations can lead to uncontrolled IL-36 signaling and consequent inflammatory conditions .

What experimental methods are recommended for detecting IL36RN activity?

For detecting IL36RN activity in experimental settings, several methodological approaches are recommended:

• Functional inhibition assays: Measure the ability of IL36RN to inhibit IL-36-induced NF-κB activation in cell-based reporter systems. This approach can quantify the antagonistic activity of the protein against its target receptors .

• Receptor binding assays: Employ surface plasmon resonance (SPR) or enzyme-linked immunosorbent assays (ELISA) to assess direct binding of IL36RN to the IL-36 receptor (IL1RL2) and measure binding affinity parameters .

• Stability assessment methods: Use differential scanning fluorimetry (DSF) or circular dichroism (CD) spectroscopy to evaluate protein stability, which is critical for understanding how mutations might affect IL36RN function .

• IL-36 pathway signaling analysis: Measure downstream effects on the NF-κB and MAPK signaling pathways using phosphorylation-specific antibodies in Western blotting or cell-based assays to confirm functional antagonism .

• Genotyping methods: For clinical samples, Sanger sequencing or restriction fragment length polymorphism PCR (RFLP-PCR) can be used to identify IL36RN mutations associated with inflammatory conditions .

These methods provide complementary data on IL36RN activity and should be selected based on the specific research question being addressed.

How do IL36RN mutations affect protein stability and function in generalized pustular psoriasis?

IL36RN mutations in generalized pustular psoriasis (GPP) affect protein stability and function through several mechanisms based on in silico and in vitro analyses:

These findings demonstrate how IL36RN mutations contribute to GPP pathogenesis by compromising the anti-inflammatory regulation of IL-36 signaling.

What is the relationship between IL36RN polymorphisms and GPP susceptibility?

The relationship between IL36RN polymorphisms and GPP susceptibility has been systematically evaluated through meta-analyses revealing significant associations. The c.115+6T>C polymorphism, in particular, shows strong correlation with GPP susceptibility across multiple genetic models:

These data demonstrate that carriers of the C allele at this position have significantly increased GPP risk compared to individuals with the wild-type T allele . The lack of heterogeneity across studies strengthens the reliability of these associations.

Furthermore, distinct patterns of IL36RN mutations have been observed in different GPP subtypes:

• GPP alone shows significantly higher mutation rates than GPP+PV (OR = 3.82, 95% CI 2.63-5.56)

• Pediatric GPP patients display higher mutation frequencies than adult GPP patients (OR = 0.42, 95% CI 0.23-0.77)

• Similar mutation patterns are observed in both European and Asian populations, suggesting universal pathogenic mechanisms

These relationships provide strong genetic evidence for the critical role of IL36RN in regulating skin inflammation and offer potential for genetic screening to identify at-risk individuals.

What methodological approaches are most effective for studying IL36RN mutational effects?

The most effective methodological approaches for studying IL36RN mutational effects combine computational predictions with experimental validation techniques:

• Computational structure analysis: Using machine-learning tools like Rhapsody to analyze the IL-36Ra three-dimensional structure and predict the impact of amino acid substitutions on protein stability and function . This approach efficiently screens large numbers of potential variants to prioritize experimental testing.

• Protein stability assays: Experimentally characterizing the effects of IL36RN variants on protein stability using thermal shift assays, circular dichroism spectroscopy, or limited proteolysis to identify amino acids critical for structural integrity .

• Receptor binding assays: Employing surface plasmon resonance or cell-based binding assays combined with computational predictions (using tools like mCSM) to assess the impact of mutations on IL-36Ra/IL-36R engagement .

• Functional signaling assays: Investigating the consequences of mutations on IL-36R signaling using reporter systems that monitor NF-κB activation or measuring downstream inflammatory mediator production .

• Genotype-phenotype correlation studies: Conducting case-control studies with clear inclusion criteria (following PRISMA guidelines) and using appropriate genotyping methods like Sanger sequencing or RFLP-PCR to associate specific mutations with clinical presentations .

• Validation of predictions: Experimentally testing representative predictions from computational methods to confirm reliability, as demonstrated by successful validation of three representative predictions of critical amino acids .

This integrated approach has successfully identified both stabilizing and destabilizing mutations and distinguished between mutations affecting protein stability versus those specifically disrupting receptor interactions.

How can researchers differentiate between IL36RN-dependent and IL36RN-independent inflammatory pathways?

Researchers can differentiate between IL36RN-dependent and IL36RN-independent inflammatory pathways through several experimental approaches:

• Genetic profiling: Screening patients with inflammatory conditions for IL36RN mutations to categorize them as either mutation-positive or mutation-negative. This initial stratification helps identify potential IL36RN-dependent cases .

• IL-36 pathway blockade experiments: Using IL-36R antagonists (like spesolimab) or IL-36Ra recombinant proteins in experimental systems to determine if inflammatory responses are suppressed. Conditions that respond to IL-36 pathway inhibition are likely IL36RN-dependent .

• Comparative signaling analysis: Examining activation patterns of NF-κB and MAPK pathways in the presence of wild-type versus mutant IL36RN to identify signaling signatures specific to IL36RN dysfunction .

• Cytokine profiling: Measuring production of downstream inflammatory mediators following IL-36 stimulation in systems with functional versus dysfunctional IL36RN to establish IL36RN-dependent cytokine patterns .

• Clinical phenotyping: Correlating clinical features with IL36RN status, as demonstrated by findings that GPP alone is more frequently associated with IL36RN mutations than GPP+PV (OR = 3.82) , suggesting distinct pathological mechanisms.

• Response to targeted therapies: Monitoring clinical response to IL-36 pathway-targeted biologics (like spesolimab) versus other anti-inflammatory agents can provide insight into pathway dependence in individual patients .

These approaches help researchers understand the heterogeneity of inflammatory conditions and guide the development of personalized therapeutic strategies based on underlying molecular mechanisms.

What are the critical amino acid residues for IL36Ra stability and IL-36R binding?

Research using combined computational and experimental approaches has identified specific amino acid residues critical for IL36Ra stability and IL-36R binding:

For protein stability:
Twenty-one amino acids have been identified as essential for maintaining IL-36Ra structural integrity through integrated in silico and in vitro analyses . Mutations at these positions likely destabilize the protein conformation, leading to loss of function. While the specific residue positions aren't enumerated in the search results, these stability-critical amino acids form the structural foundation necessary for proper protein folding and function.

The identification of these functionally distinct sets of amino acids reveals that IL36RN mutations can affect protein function through at least two mechanisms: destabilization of protein structure or specific disruption of receptor binding capacity. This knowledge has important implications for understanding the molecular pathogenesis of GPP and potentially for designing therapeutic IL-36 inhibitors that mimic the natural antagonist function .

How does IL36RN research inform therapeutic development for generalized pustular psoriasis?

Research on IL36RN has directly informed therapeutic development for generalized pustular psoriasis through multiple translational pathways:

• Biological mechanism elucidation: The discovery of IL36RN disease alleles in GPP patients revealed the mechanistic role of unopposed IL-36 signaling in disease pathogenesis, identifying IL-36 pathway as a therapeutic target .

• Clinical trial design: Understanding of IL36RN mutations has informed the development of IL-36R inhibitors, leading to successful clinical trials of the anti-IL36R antibody spesolimab .

• Regulatory approval: The strong mechanistic rationale from IL36RN research contributed to spesolimab receiving FDA Breakthrough Therapy Designation and subsequent approval for GPP treatment .

• Patient stratification approaches: Research showing differential mutation frequencies between GPP subtypes (OR = 3.82 for GPP alone vs. GPP+PV) and age groups (pediatric vs. adult) provides foundations for precision medicine approaches based on genetic testing .

• Structural insights for drug design: Identification of critical amino acids for IL-36Ra/IL-36R interaction through combined computational and experimental approaches offers potential templates for designing new IL-36 inhibitors with improved specificity or pharmacokinetic properties .

• Expanded therapeutic applications: IL36RN research has catalyzed investigation of IL-36 inhibitors in other inflammatory conditions including hidradenitis suppurativa and atopic dermatitis, broadening the potential clinical impact .

This research exemplifies successful bench-to-bedside translation, where fundamental genetic and molecular insights have directly led to targeted therapies addressing the underlying disease mechanism.

What diagnostic value do IL36RN mutations have in inflammatory skin conditions?

IL36RN mutations provide significant diagnostic value in inflammatory skin conditions through several clinical applications:

• Disease classification: IL36RN mutation analysis helps distinguish between different subtypes of pustular psoriasis, with significantly higher mutation frequencies in GPP alone compared to GPP with psoriasis vulgaris (OR = 3.82, 95% CI 2.63-5.56) . This genetic distinction supports treating these as related but mechanistically distinct conditions.

• Early diagnosis in high-risk groups: The higher prevalence of IL36RN mutations in pediatric GPP cases compared to adult cases (inverse OR = 0.42, 95% CI 0.23-0.77) suggests genetic testing may be particularly valuable for early diagnosis in pediatric patients with inflammatory skin manifestations.

• Population screening approach: Similar mutation patterns observed across European and Asian populations (European OR = 4.03, Asian OR = 3.69) indicate that standardized genetic testing protocols could be applied across diverse populations.

• Targeted therapy selection: With IL-36R inhibitors like spesolimab now available for GPP treatment , identifying patients with IL36RN mutations may help select those most likely to benefit from targeted therapy versus conventional treatments.

• Interpreting variants of uncertain significance: Combined in silico prediction tools (Rhapsody, mCSM) and experimental validation approaches can help interpret novel IL36RN variants in diagnostic settings, distinguishing pathogenic mutations from benign polymorphisms .

The diagnostic value of IL36RN mutation analysis is particularly enhanced in the context of available targeted therapies, where genetic information can directly inform treatment decisions in a precision medicine framework.

How does recombinant IL36RN protein production methodology affect experimental outcomes?

The methodology used for recombinant IL36RN protein production can significantly impact experimental outcomes through several technical factors:

• Expression system selection: The search results indicate successful production of active IL36RN in Escherichia coli systems, yielding >98% pure protein suitable for functional studies . Different expression systems (bacterial, yeast, insect, mammalian) may produce proteins with varying post-translational modifications that affect activity.

• Protein purity requirements: Endotoxin contamination can confound inflammatory response experiments, making the documented endotoxin level (<1 EU/μg) critical for reliable results in cell-based assays and in vivo studies investigating IL36RN's anti-inflammatory properties.

• Protein sequence considerations: The recombinant human IL36RN described spans amino acids 2-155 , representing the mature protein. Inclusion or exclusion of signal peptides or fusion tags can affect protein folding, stability, and interaction with receptors.

• Stability and storage conditions: Given that certain mutations can destabilize IL36RN structure , proper handling and storage of recombinant protein is essential to maintain activity for consistent experimental results.

• Functional validation metrics: Confirming that recombinant IL36RN effectively inhibits IL-36 signaling through appropriate functional assays (NF-κB activation inhibition, binding to IL1RL2) is necessary to ensure experimental validity .

When designing IL36RN studies, researchers should carefully document production methods and validation steps to ensure reproducibility and accurate interpretation of results, particularly when comparing wild-type and mutant protein variants or evaluating potential therapeutic molecules.

What are the optimal experimental controls when studying IL36RN variants?

When studying IL36RN variants, implementing comprehensive experimental controls is essential for reliable interpretation of results:

• Wild-type IL36RN controls: Including properly folded wild-type recombinant IL36RN protein produced under identical conditions as mutant variants provides the baseline for comparative studies of protein stability, receptor binding, and functional inhibition .

• Known pathogenic mutations: Including previously characterized pathogenic mutations (such as those affecting the 21 amino acids identified as critical for stability) provides positive controls for loss-of-function effects .

• Neutral variants: Including variants that don't affect stability or function (such as conservative substitutions in non-critical regions) serves as negative controls to establish the specificity of experimental assays .

• Stability-specific vs. binding-specific mutants: Using mutations that specifically affect either protein stability or receptor binding (without affecting the other) helps distinguish between these two mechanisms of dysfunction .

• Cell-based signaling controls: When measuring IL-36 pathway activation, including positive controls (IL-36 cytokines alone) and negative controls (pathway inhibitors like NF-κB inhibitors) ensures assay functionality .

• Computational prediction validation: For in silico analyses, including variants with known experimental outcomes to validate prediction accuracy, as demonstrated by the successful validation of three representative predictions in the IL36RN structural studies .

These controls allow researchers to distinguish genuine biological effects from technical artifacts and provide a framework for consistent interpretation of variant effects across different experimental platforms.

How can machine learning approaches enhance IL36RN variant interpretation?

Machine learning approaches show significant promise for enhancing IL36RN variant interpretation through several advanced applications:

• Comprehensive variant impact prediction: Building on current work with Rhapsody and mCSM , machine learning models can systematically analyze the potential impact of all possible IL36RN amino acid substitutions on protein stability and receptor binding, creating complete variant effect maps.

• Integration of structural and functional data: Advanced algorithms can integrate three-dimensional structural information with functional assay results to improve prediction accuracy beyond what either approach can achieve alone .

• Variant classification refinement: Machine learning can help classify variants of uncertain significance by recognizing subtle patterns that distinguish pathogenic from benign variants based on structural features and experimental validation datasets.

• Patient outcome prediction: By correlating genetic data with clinical outcomes in patients with different IL36RN variants, machine learning could potentially predict disease severity and treatment response, supporting personalized medicine approaches.

• Novel therapeutic design: Computational approaches could identify potential binding sites for new IL-36 inhibitors by analyzing the interaction surface between IL-36Ra and IL-36R, potentially guiding structure-based drug design .

• Cross-species comparative analysis: Machine learning can leverage evolutionary conservation patterns across species to further refine understanding of which IL36RN regions are functionally critical, providing additional context for human variant interpretation.

The successful application of Rhapsody to identify 21 stability-critical amino acids and mCSM to identify 13 binding-critical residues demonstrates that machine learning approaches are already yielding valuable insights that would be challenging to obtain through experimental approaches alone.

What emerging therapeutic approaches target the IL-36 signaling pathway?

Several emerging therapeutic approaches targeting the IL-36 signaling pathway are being developed based on mechanistic insights from IL36RN research:

• Anti-IL36R monoclonal antibodies: Following the successful development of spesolimab, which has received FDA approval for GPP treatment , other IL-36R-targeting antibodies are likely in development with potential improvements in affinity, half-life, or tissue distribution.

• Small molecule IL-36 pathway inhibitors: Researchers are actively investigating various approaches to IL-36 blockade beyond antibody therapies , potentially including small molecule inhibitors targeting the receptor binding interface or downstream signaling components.

• Recombinant IL-36Ra derivatives: Based on the structural understanding of critical amino acids for IL-36Ra stability and receptor binding , engineered variants with enhanced stability or receptor affinity may be developed as therapeutic proteins.

• Combination therapies: Emerging approaches may combine IL-36 pathway inhibition with targeting of complementary inflammatory pathways identified in IL36RN-independent disease subtypes .

• Localized delivery systems: For skin conditions like GPP, topical or local delivery systems for IL-36 pathway inhibitors may be developed to maximize therapeutic effect while minimizing systemic exposure.

• Expanded disease indications: Current research is exploring IL-36 inhibition in hidradenitis suppurativa and atopic dermatitis , with potential for additional inflammatory conditions as understanding of IL-36 biology continues to expand.

The rapid translation of IL36RN research into approved therapies like spesolimab demonstrates the clinical relevance of this pathway and suggests continued therapeutic innovation targeting different aspects of IL-36 signaling in coming years.