IL36G 152 a.a. Human

What is IL36G and how does it relate to the broader IL-1 cytokine family?

IL36G (Interleukin-36 gamma) is a pro-inflammatory cytokine belonging to the IL-1 family. It was formerly known as IL1F9 (Interleukin 1 Family, Member 9), IL1H1 (Interleukin 1 Homolog 1), or IL1RP2 (Interleukin 1-Related Protein 2). IL36G is one of four members of the IL-36 subfamily, which includes three receptor agonists (IL36A, IL36B, and IL36G) and one receptor antagonist (IL36RA) .

The IL-1 family members share a characteristic 12 β-strand, β-trefoil configuration and are thought to have evolved from a common ancestral gene. Human IL36G shares 23-57% amino acid sequence identity with other IL-1 family members .

What is the molecular structure of recombinant human IL36G (152 a.a.)?

Recombinant human IL36G (152 a.a.) is a single, non-glycosylated polypeptide chain containing 152 amino acids with a molecular mass of approximately 17.0 kDa. The protein has no signal sequence, no prosegment, and no potential N-linked glycosylation sites .

The protein is typically produced in Escherichia coli expression systems and purified using proprietary chromatographic techniques to achieve >95% purity as determined by SDS-PAGE and HPLC analyses .

How does human IL36G compare to IL36G from other species?

Human IL36G shares significant amino acid sequence homology with IL36G from other species:

• 58% identity with mouse IL36G

• 59% identity with rat IL36G

• 68% identity with bovine IL36G

• 69% identity with equine IL36G

What are the optimal reconstitution and storage conditions for lyophilized IL36G?

For optimal reconstitution of lyophilized human IL36G:

• Use sterile 18M-cm H₂O (ultra-pure water)

• Reconstitute at a concentration not less than 100 μg/ml

• The reconstituted solution can be further diluted into other aqueous buffers as needed

For storage:

• Store lyophilized IL36G desiccated below -18°C for long-term stability

• Although stable at room temperature for up to 3 weeks, refrigerated or frozen storage is recommended

• After reconstitution, store at 2-8°C for up to one week or aliquot and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles which may compromise protein activity

How can researchers verify the biological activity of IL36G in experimental systems?

Researchers can verify IL36G biological activity through several complementary approaches:

• Functional Assays: Test the protein's ability to activate NF-κB and MAPK pathways in cells expressing the IL-36 receptor (IL-1Rrp2/IL-1RAcP complex). This can be measured using reporter cell lines or by assessing phosphorylation of pathway components .

• Cytokine Induction: Measure the induction of downstream inflammatory mediators, particularly IL-8 (CXCL8) and IL-6 production in responsive cell types such as keratinocytes and epithelial cells .

• T Cell Activation: Assess the protein's ability to stimulate T cell proliferation and IL-2 release, which are documented biological effects of IL36G .

• Protein Integrity Analysis: Confirm protein integrity and expected molecular weight (approximately 17.0 kDa) using SDS-PAGE, which should show >95% purity .

What technical challenges might arise when working with IL36G and how can they be addressed?

Several technical challenges can arise when working with IL36G:

• Activation Requirement: IL36G must be cleaved at the N-terminus to achieve full biological activity. This processing can be accomplished using neutrophil granule-derived proteases such as elastase and cathepsin G . Researchers should consider using processed forms or including processing steps in their experimental design.

• Receptor Expression: Target cells must express both components of the IL36G receptor (IL-1Rrp2 and IL-1RAcP) for proper response. Verify receptor expression in experimental systems before interpreting negative results .

• Buffer Compatibility: The lyophilization buffer contains components like trehalose, EDTA, and sometimes detergents that may affect certain experimental systems. Consider buffer exchange for sensitive applications .

• Concentration Optimization: Dose-response relationships should be established, as both too low and too high concentrations may yield suboptimal results. Start with concentrations between 10-100 ng/ml and adjust based on system responsiveness .

What is the receptor for IL36G and how does signaling initiate?

IL36G signals through a heterodimeric receptor complex consisting of:

• IL-1Rrp2 (IL1RL2/IL-36 receptor) - the primary binding receptor

• IL-1RAcP (IL-1 receptor accessory protein) - required for signal transduction

The signaling mechanism involves:

• Binding of IL36G to IL-1Rrp2

• Recruitment of IL-1RAcP to form the active receptor complex

• Activation of intracellular signaling cascades

This receptor binding initiates activation of NF-κB and various mitogen-activated protein kinases (MAPKs), including Erk1/2 and JNK. The signaling targets the IL-8 promotor, resulting in IL-6 secretion and induction of various pro-inflammatory mediators .

How does IL36G activation differ from other IL-1 family members?

While IL36G shares signaling pathways with other IL-1 family members, several important distinctions exist:

• Receptor Specificity: IL36G binds specifically to IL-1Rrp2 rather than IL-1R1 (used by IL-1α and IL-1β) or IL-18R (used by IL-18) .

• Activation Requirements: Like other IL-36 family members, IL36G requires N-terminal processing for full activity, but the specific processing enzymes and cleavage sites may differ from those for other IL-1 family members .

• Expression Pattern: IL36G is predominantly expressed in epithelial tissues, particularly keratinocytes and respiratory epithelium, whereas other IL-1 family members may have broader or different expression patterns .

• Potency: IL36G typically has lower biological potency compared to IL-1β, requiring higher concentrations to elicit comparable cellular responses .

• Signaling Dynamics: While activating similar pathways, IL36G may induce distinct temporal patterns of signaling and unique gene expression profiles compared to other family members .

What roles does IL36G play in inflammatory disease processes?

IL36G is implicated in several inflammatory disease processes, particularly those affecting epithelial tissues:

• Psoriasis: IL36G is strongly associated with psoriasis pathogenesis, showing elevated expression in psoriatic skin lesions. It promotes keratinocyte activation and proliferation while stimulating the production of pro-inflammatory mediators .

• Psoriatic Arthritis: Beyond skin involvement, IL36G may contribute to joint inflammation in psoriatic arthritis through effects on synoviocytes and chondrocytes .

• Inflammatory Bowel Disease: IL36G plays a role in intestinal inflammation in ulcerative colitis and Crohn's disease, affecting intestinal epithelial cells and mucosal immune responses .

• Autoimmune Conditions: IL36G has been linked to systemic lupus erythematosus and Sjögren's syndrome, potentially contributing to tissue-specific inflammatory processes .

• Respiratory Inflammation: Given its expression in respiratory epithelium, IL36G may participate in inflammatory lung conditions, though this area requires further investigation .

What cell types and experimental systems are optimal for studying IL36G biology?

Several cell types and experimental systems are particularly suitable for IL36G research:

• Keratinocytes: Primary human keratinocytes or immortalized cell lines (e.g., HaCaT) express IL-36 receptors and respond robustly to IL36G stimulation .

• Epithelial Cell Lines: Respiratory and intestinal epithelial cell lines can be valuable for studying tissue-specific effects of IL36G in different organ systems .

• Immune Cells: Although not the primary targets, T cells, dendritic cells, and macrophages can respond to IL36G and are relevant for studying its immunomodulatory effects .

• 293-T Cells: These cells are useful for transfection studies, as IL36G is efficiently secreted when transfected into 293-T cells .

• 3D Tissue Models: Reconstructed human epidermis and other three-dimensional tissue models provide more physiologically relevant systems than monolayer cultures for studying IL36G effects on tissue architecture and intercellular communication .

How can researchers design experiments to study IL36G processing and activation?

To effectively study IL36G processing and activation, researchers can implement the following experimental design strategies:

• Controlled Processing Experiments:

  • Compare full-length IL36G with enzymatically processed forms

  • Utilize purified neutrophil elastase or cathepsin G to process recombinant IL36G in vitro

  • Confirm processing by SDS-PAGE, mass spectrometry, or N-terminal sequencing

• Cellular Processing Systems:

  • Co-culture IL36G-expressing cells with neutrophils to study physiological processing

  • Use neutrophil-conditioned media to process IL36G

  • Compare activity in the presence of specific protease inhibitors

• Structure-Function Analysis:

  • Generate truncation mutants with different N-termini to identify optimal active forms

  • Create point mutations at potential cleavage sites

  • Measure relative activity using reporter systems or downstream cytokine production

• Comparative Analysis:

  • Conduct parallel experiments with IL36A, IL36B, and IL36G to identify common and distinct processing requirements

  • Compare processing efficiency across species (human vs. mouse IL36G)

What approaches can be used to study the interaction between IL36G and other inflammatory pathways?

To investigate interactions between IL36G and other inflammatory pathways, researchers can employ several sophisticated approaches:

• Combinatorial Cytokine Treatments:

  • Treat cells with IL36G alone or in combination with other inflammatory cytokines (TNF-α, IL-17, IL-22)

  • Perform sequential treatments to assess priming or tolerance effects

  • Measure synergistic or antagonistic effects on downstream readouts

• Pathway Inhibition Studies:

  • Use specific inhibitors of NF-κB, MAPK, or other signaling pathways

  • Apply inhibitors before or after IL36G treatment to distinguish primary and secondary effects

  • Compare pathway dependency profiles between IL36G and other inflammatory mediators

• Multi-omics Approaches:

  • Employ RNA-seq, proteomics, and metabolomics to comprehensively profile cellular responses

  • Apply systems biology approaches to construct network models of IL36G signaling

  • Identify pathway convergence and divergence points with other inflammatory signals

• Genetic Manipulation:

  • Use CRISPR/Cas9 to knockout or modify IL36G, its receptor components, or downstream mediators

  • Perform rescue experiments with wild-type or mutant proteins

  • Create reporter cell lines expressing fluorescent or luminescent indicators of pathway activation

How can researchers effectively troubleshoot when IL36G fails to elicit expected responses?

When IL36G experiments don't yield expected results, researchers should systematically evaluate:

• Protein Quality and Processing:

  • Verify protein integrity by SDS-PAGE

  • Consider that IL36G requires N-terminal processing for full activity; commercial preparations may contain primarily unprocessed protein

  • Process recombinant IL36G with neutrophil elastase or cathepsin G to enhance activity

• Receptor Expression:

  • Confirm expression of both IL-1Rrp2 and IL-1RAcP in target cells

  • Some cell lines may express insufficient receptor levels for robust responses

  • Consider transfecting receptor components if expression is limiting

• Experimental Conditions:

  • Optimize protein concentration (typically effective between 10-100 ng/ml for processed forms)

  • Adjust treatment duration (some responses may require 24-48 hours)

  • Evaluate culture conditions that might affect cell responsiveness

• Presence of Inhibitors:

  • Check for expression of IL36RA (IL-36 receptor antagonist) in the experimental system

  • Consider serum factors that might neutralize IL36G activity

  • Evaluate potential cross-reactivity issues with antibodies or detection reagents

• Readout Selection:

  • Choose appropriate readouts; different cell types may exhibit different response patterns

  • Include positive controls (e.g., IL-1β or TNF-α) to confirm general cell responsiveness

  • Consider kinetic analyses rather than single time points

What considerations are important when designing comparative studies between human and mouse IL36G?

When designing comparative studies between human and mouse IL36G, researchers should consider:

• Sequence and Structure Differences:

  • Human and mouse IL36G share only 58% amino acid identity

  • These differences may affect antibody recognition, processing requirements, and receptor interactions

• Species-Specific Receptor Interactions:

  • Limited cross-species reactivity exists; human IL36G may not optimally activate mouse receptors and vice versa

  • Consider species-matching (using human IL36G with human cells, mouse IL36G with mouse cells)

• Processing Requirements:

  • Both human and mouse IL36G require N-terminal processing for full activity

  • Optimal processing enzymes and cleavage sites may differ between species

• Experimental Systems Selection:

  • For translational studies, consider using humanized mice or human tissue xenografts

  • When comparing, maintain identical experimental conditions apart from the species-specific protein

• Readout Interpretation:

  • Acknowledge species differences when interpreting potency or efficacy data

  • Use multiple readouts to comprehensively compare activities

  • Include within-species positive controls for context

How can researchers investigate the role of IL36G in complex disease models?

To investigate IL36G in complex disease models, researchers can implement these advanced approaches:

• Genetic Models:

  • Utilize IL36G knockout or transgenic overexpression models

  • Consider conditional knockout systems for tissue-specific or inducible deletion

  • Use receptor knockout models to distinguish IL36G-specific effects from other IL-1 family members

• Neutralization Strategies:

  • Apply IL36G-neutralizing antibodies or soluble receptor constructs

  • Use receptor antagonists (IL36RA) to block signaling

  • Compare with broad IL-1 family inhibitors to determine relative contribution

• Ex Vivo Analysis:

  • Isolate tissues from disease models for ex vivo culture and manipulation

  • Perform single-cell analyses to identify IL36G-responsive populations

  • Use patient-derived samples to validate findings in human disease

• Cellular Imaging:

  • Implement intravital imaging to observe IL36G effects in living tissues

  • Use reporter mice expressing fluorescent proteins under IL36G or target gene promoters

  • Apply multiplexed immunofluorescence to map cellular networks in tissue sections

• Multi-System Integration:

  • Study effects across organs (skin-joint interactions in psoriatic disease)

  • Evaluate immune-epithelial cell crosstalk

  • Consider microbiome interactions in barrier tissue inflammation

How do the three IL-36 agonists (IL36A, IL36B, and IL36G) differ in their research applications?

The three IL-36 agonists share the same receptor complex but differ in ways relevant to research applications:

• Expression Patterns:

  • IL36G: Predominantly in epithelial tissues, particularly keratinocytes and respiratory epithelium

  • IL36A: Expressed in monocytes, B cells, and some epithelial tissues

  • IL36B: More restricted expression, mainly in monocytes and specific epithelial cells

• Disease Associations:

  • IL36G: Most strongly linked to psoriasis and psoriatic arthritis

  • IL36A and IL36B: Also implicated in inflammatory diseases but with potentially different tissue specificities

• Biological Potency:

  • Subtle differences in receptor binding affinity and activation kinetics exist

  • Cell type-dependent variations in potency may influence experimental design choices

• Research Applications:

  • IL36G: Most commonly studied in skin inflammation and epithelial biology research

  • Choice among the three should be guided by the specific tissue or disease being studied

  • Comparative studies using all three agonists can reveal unique and redundant functions

What emerging technologies might advance IL36G research in the coming years?

Several emerging technologies show promise for advancing IL36G research:

• CRISPR-Based Technologies:

  • High-throughput CRISPR screens to identify novel regulators of IL36G pathways

  • Base editing for precise modification of IL36G processing sites or receptor binding domains

  • In vivo CRISPR delivery for tissue-specific pathway modulation

• Single-Cell Analysis:

  • Single-cell RNA-seq to map IL36G-responsive cell populations in complex tissues

  • Single-cell proteomics to profile pathway activation at individual cell resolution

  • Spatial transcriptomics to understand tissue organization of IL36G signaling networks

• Advanced Structural Biology:

  • Cryo-EM studies of IL36G-receptor complexes

  • Structure-based design of selective modulators

  • Molecular dynamics simulations to understand processing mechanisms

• Organoid Technologies:

  • Patient-derived organoids for personalized disease modeling

  • Multi-organ-on-chip systems to study systemic effects

  • Bioprinted tissues incorporating immune components for inflammation studies

• Therapeutic Translations:

  • Development of monoclonal antibodies against IL36G for inflammatory disease treatment

  • Small molecule inhibitors of IL36G processing or receptor interaction

  • Cell-type specific targeting strategies for localized pathway modulation

What are the most promising research directions for understanding IL36G in human disease?

The most promising research directions for IL36G in human disease include:

• Precision Medicine Applications:

  • Identification of IL36G-responsive patient subsets in inflammatory diseases

  • Development of IL36G as a biomarker for disease stratification

  • Correlation of genetic variations in IL36G pathway with treatment responses

• Tissue Microenvironment Studies:

  • Investigation of IL36G in epithelial-immune cell crosstalk

  • Role in tissue repair versus pathological inflammation

  • Contribution to barrier function in epithelial tissues

• Pathway Integration:

  • Mapping interaction points between IL36G and other inflammatory cytokine networks

  • Understanding redundancy and unique functions within the IL-36 family

  • Identifying targetable nodes in IL36G signaling networks

• Beyond Known Diseases:

  • Exploration of IL36G in understudied conditions affecting epithelial tissues

  • Investigation of potential roles in cancer immunity

  • Evaluation of contributions to metabolic inflammation

• Translational Models:

  • Development of improved humanized models for IL36G-mediated diseases

  • Validation studies in patient-derived systems

  • Preclinical evaluation of pathway modulation strategies