IL 1 alpha Human, His

What is the molecular structure of human IL-1α and how does the His-tag modification impact its biological activity?

Human IL-1α is a 31 kDa pro-protein belonging to the interleukin-1 family. Unlike many cytokines, IL-1α is biologically active in its unprocessed pro-form and contains a functional nuclear localization signal (NLS) in its N-terminal domain. This allows pro-IL-1α to shuttle to the nucleus upon translation in multiple cell types including epithelial cells, myeloid cells, and keratinocytes .

The addition of a histidine tag, typically comprising 6-10 histidine residues, facilitates protein purification via metal affinity chromatography but requires validation to ensure preservation of biological activity. Researchers should implement the following methodological approach:

• Compare the biological activity of His-tagged versus untagged IL-1α in standard bioassays measuring IL-6 or TNF-α production

• Evaluate receptor binding efficiency using surface plasmon resonance

• Assess nuclear translocation capability through immunofluorescence microscopy

• Consider removable His-tags using protease cleavage sites if structural interference is observed

How is IL-1α expression regulated at the transcriptional level and what experimental approaches best capture these regulatory mechanisms?

The IL-1α gene (IL1A) has a distinct promoter structure lacking canonical TATA and CAAT box regulatory regions. Instead, it contains binding sites for the transcription factor Sp1, which mediates basal expression during homeostasis . During inflammatory conditions, IL1A expression is upregulated through binding sites for AP1 and NF-κB transcription factors .

For effective experimental investigation of IL-1α transcriptional regulation:

• Employ chromatin immunoprecipitation (ChIP) assays to identify transcription factor binding to the IL1A promoter under different conditions

• Utilize reporter assays with wild-type and mutated promoter constructs to evaluate the contribution of specific transcription factor binding sites

• Consider the role of the long noncoding antisense IL1A transcript (AS-IL-1α) which is required for IL1A transcription in myeloid cells

• Analyze epigenetic modifications at the IL1A locus using bisulfite sequencing or ATAC-seq

What distinguishes IL-1α from IL-1β despite their shared receptor, and how can researchers experimentally differentiate their biological effects?

While both IL-1α and IL-1β signal through the same IL-1 receptor (IL-1R), they differ substantially in expression patterns, processing requirements, and cellular localization:

To experimentally differentiate their effects:

• Use genetic approaches with IL-1α or IL-1β knockout models, as demonstrated in Ptpn6 spin mice where IL-1α but not IL-1β deletion protects against neutrophilic dermatosis

• Employ neutralizing antibodies specific to each cytokine

• Design cell-type specific deletion strategies to address the different cellular sources

• Analyze the kinetics of expression and release, as IL-1α often precedes IL-1β expression in many inflammatory contexts

How can researchers effectively study the nuclear functions of IL-1α independent of its receptor-mediated signaling?

IL-1α's capacity to localize to the nucleus and potentially regulate gene expression independently of receptor binding represents a unique feature requiring specialized experimental approaches:

• Generate IL-1α constructs with mutations in the nuclear localization signal (residues 79-86) to create cytoplasm-restricted variants

• Develop cell lines expressing IL-1α fused to a regulatable nuclear import system (e.g., hormone-responsive domains)

• Perform nuclear-cytoplasmic fractionation followed by co-immunoprecipitation to identify nuclear binding partners

• Utilize ChIP-seq approaches to map genomic binding sites of nuclear IL-1α, considering its reported interactions with histone acetyltransferases p300, PCAF, and GCN5

• Implement RNA-seq analysis comparing cells expressing wild-type IL-1α versus NLS-mutant forms to identify nuclear-function-dependent gene regulation

The interaction of pro-IL-1α with HAX-1 (HS-1-associated protein X), a protein associated with mitochondrial, endoplasmic reticulum, and nuclear membranes, promotes its nuclear localization and may serve as an additional target for investigation .

What post-translational modifications regulate IL-1α function and what techniques should be employed to investigate their effects?

IL-1α undergoes several post-translational modifications that potentially regulate its function:

• Myristoylation or acetylation on Lys82

• Phosphorylation at Ser90

• Proteolytic processing by calpain and other proteases

To investigate these modifications systematically:

• Employ site-directed mutagenesis to generate modification-resistant variants (e.g., K82R to prevent acetylation, S90A to prevent phosphorylation)

• Use mass spectrometry-based approaches (MS/MS) to identify and quantify modification sites under different conditions

• Develop modification-specific antibodies for immunoblotting and immunoprecipitation

• Apply pharmacological inhibitors of modifying enzymes (e.g., calpain inhibitors, kinase inhibitors) in cellular models

• Assess the impact of modifications on subcellular localization through immunofluorescence microscopy and fractionation approaches

The functional consequences of these modifications remain largely unknown and represent an important frontier in IL-1α research .

How should researchers design experiments to investigate IL-1α's role as an alarmin during sterile inflammation?

IL-1α functions as a critical alarmin released during cell death to initiate inflammatory responses. To effectively study this function:

• Establish necrotic cell administration models, as demonstrated by studies showing that infiltration of neutrophils into the peritoneal cavity upon administration of necrotic cells depends on IL-1α release

• Implement tissue injury models (e.g., microabrasion of footpads) which have been shown to accelerate disease progression in IL-1α-dependent inflammatory conditions

• Compare responses in wild-type versus IL-1α-deficient settings to delineate IL-1α-specific contributions

• Use chimeric models to distinguish the role of IL-1α from hematopoietic versus non-hematopoietic sources

• Analyze downstream inflammatory mediators including G-CSF, CXCL1, CXCL2, and TNF-α to map the inflammatory cascade initiated by IL-1α

Such approaches have revealed that IL-1α from non-hematopoietic cells can trigger robust inflammatory responses acting through myeloid cells in a SYK, MyD88, RIPK1, and TAK1-dependent manner .

What are the optimal models for studying IL-1α's role in inflammatory skin diseases and how should experiments be designed?

IL-1α plays a crucial role in various inflammatory skin conditions. For effective research:

• Utilize the Ptpn6 spin mouse model of neutrophilic dermatosis, where genetic ablation of IL-1α, but not IL-1β, protects mice from disease development

• Consider transgenic models that overexpress IL-1α in epidermal cells, which develop inflammatory skin disease highlighting IL-1α's pathogenic potential

• Implement bone marrow chimeras to determine the cellular source of pathogenic IL-1α (hematopoietic vs. non-hematopoietic)

• Use microabrasion injury models to study how mechanical damage accelerates disease progression through passive release of IL-1α from epidermal cells

• Analyze downstream cytokine production (TNF-α, G-CSF, KC) to map the inflammatory cascade

Key experimental readouts should include:

• Histological assessment of skin pathology

• Flow cytometric analysis of inflammatory infiltrates

• Cytokine profiling in tissue homogenates and serum

• Gene expression analysis of inflammatory mediators

How can researchers effectively investigate the seemingly contradictory roles of IL-1α in cancer?

IL-1α exhibits dual roles in cancer, promoting tumor growth and metastasis in some contexts while enhancing anti-tumor immunity in others. To systematically investigate this dichotomy:

• Compare models of endogenous IL-1α expression versus overexpression approaches:

  • Endogenous IL-1α often promotes tumor growth and angiogenesis

  • Overexpression of cytosolic or membrane-bound forms of IL-1α can lead to tumor regression

• Evaluate different cellular compartments of IL-1α expression:

  • Membrane-bound IL-1α tends to promote anti-tumor immunity

  • Secreted IL-1α often supports tumor progression

• Analyze immune cell populations to determine mechanisms:

  • IL-1α-mediated anti-tumor effects depend on CD8+ T cells, NK cells, and M1-differentiated macrophages

  • Pro-tumorigenic effects may involve myeloid-derived suppressor cells and tumor-associated macrophages

• Study IL-1α in combination with cancer therapies:

  • IL-1α (and IL-1β) maturation by the NLRP3 inflammasome activated by chemotherapeutic drugs can contribute to anti-tumor CD8+ T cell responses

  • Simultaneously, chemotherapy-enhanced production of pro-inflammatory mediators may potentiate tumor invasiveness and metastasis

Clinical trials with IL-1α-blocking monoclonal antibody MABp1 have shown promising outcomes in patients with end-stage cancers, improving survival rate, increasing lean body weight, and decreasing systemic inflammation and cachexia .

What methodological approaches should be used to study IL-1α's role in host defense against microbial infections?

IL-1α plays non-redundant roles in defense against various pathogens. Effective research approaches include:

• Bacterial infection models:

  • Mycobacterium tuberculosis infection, where IL-1α cooperates with TNF to promote protective granuloma formation

  • Pseudomonas aeruginosa colonization with cyclophosphamide-induced immunosuppression to study protection from gut-derived sepsis

  • Staphylococcus aureus or Chlamydia trachomatis infection to examine the inflammatory feedback loop involving IL-1α-induced chemokines

• Viral infection models:

  • Adenovirus interaction with β3 integrin on marginal zone macrophages to study IL-1α upregulation and neutrophil recruitment

  • RSV infection models to examine IL-1α-driven neutrophil infiltration in lungs

• Fungal and protozoan models:

  • Candida albicans infection to study IL-1α production from keratinocytes and subsequent G-CSF production from endothelial cells

  • Cryptococcus neoformans to examine IL-1α expression in the brain following systemic infection

  • Leishmania major infection in susceptible BALB/c mice where IL-1α drives pathology

Key experimental readouts should include:

• Pathogen clearance kinetics

• Inflammatory cell recruitment profiles

• Cytokine and chemokine production measurements

• Tissue damage assessment

• Survival analysis

How should researchers design experiments to elucidate the complex signaling pathways downstream of IL-1α?

IL-1α signaling involves complex interactions with multiple pathways. To effectively study these networks:

• Employ genetic approaches with knockout models for key signaling components:

  • Studies with Ptpn6 spin mice revealed that SHP-1 controls activation of spleen tyrosine kinase (SYK), which regulates MyD88 phosphorylation in myeloid cells

  • Genetic deletion of RIPK1, TAK1, or SYK in the appropriate cellular compartments can help dissect signaling requirements

• Use phosphoproteomic approaches to map signaling cascades:

  • Temporal profiling of phosphorylation events following IL-1α stimulation

  • Comparison of signaling in different cell types (e.g., epithelial vs. immune cells)

• Implement pharmacological inhibition approaches:

  • TAK1 inhibitors to assess transforming growth factor-β activated kinase 1 involvement

  • RIPK1 inhibitors to distinguish between its scaffolding versus kinase functions

• Apply single-cell approaches to address cellular heterogeneity:

  • Single-cell RNA-seq to identify cell-specific responses

  • Single-cell phospho-flow cytometry to quantify signaling at the individual cell level

Studies have shown that IL-1α from non-hematopoietic cells acts on myeloid cells through a pathway involving SYK, MyD88, RIPK1, and TAK1, which is negatively regulated by SHP-1 to restrain excessive IL-1R signaling and associated auto-inflammation .

What are the key considerations for studying IL-1α protein-protein interactions and how can they be effectively investigated?

IL-1α engages in multiple protein-protein interactions that regulate its function and localization:

• Receptor interactions: IL-1α binds to IL-1R1, which then recruits IL-1RAcP to form a signaling complex

• Intracellular binding partners: Pro-IL-1α interacts with HAX-1, p300, PCAF, and GCN5

• Regulatory interactions: Intracellular IL-1R2 can mask the NLS of pro-IL-1α, leading to cytosolic retention

To investigate these interactions systematically:

• Apply proximity-based approaches:

  • BioID or TurboID to identify proteins in close proximity to IL-1α in different cellular compartments

  • FRET or split-luciferase assays to monitor interactions in living cells

• Use co-immunoprecipitation approaches:

  • Standard co-IP followed by mass spectrometry to identify novel binding partners

  • Sequential co-IP to identify multiprotein complexes

• Implement structural biology techniques:

  • X-ray crystallography of IL-1α with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cryo-EM for larger complexes

• Develop domain mapping strategies:

  • Generate truncated variants to identify interaction domains

  • Site-directed mutagenesis of candidate interaction residues

Understanding these interactions is crucial for developing targeted therapeutics and elucidating mechanisms of IL-1α regulation.

What are the most effective strategies for distinguishing between membrane-associated and secreted forms of IL-1α in experimental systems?

IL-1α can exist in multiple forms including nuclear, cytosolic, membrane-associated, and secreted variants. To effectively distinguish between these forms:

• Implement cell fractionation approaches:

  • Separate nuclear, cytosolic, membrane, and extracellular fractions

  • Use appropriate markers to validate fraction purity (histone H3 for nuclear, GAPDH for cytosolic, Na+/K+ ATPase for membrane)

• Generate form-specific IL-1α variants:

  • Membrane-anchored IL-1α by fusion with transmembrane domains

  • Nuclear-restricted forms through strong NLS addition

  • Secretion-enhanced variants through signal peptide addition

• Apply imaging approaches:

  • Live-cell imaging with fluorescently tagged IL-1α variants

  • Super-resolution microscopy to visualize subcellular localization

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

• Develop detection strategies that distinguish the forms:

  • Form-specific ELISA approaches

  • Flow cytometry for cell-surface IL-1α detection

  • Proximity ligation assays to detect specific interactions

Understanding the distinct biological activities of these different forms is critical, as studies suggest membrane-bound IL-1α may promote anti-tumor immunity while secreted forms often contribute to pathological inflammation .

How can researchers effectively address the technical challenges in studying IL-1α's role in the tumor microenvironment?

The tumor microenvironment presents unique challenges for studying IL-1α functions:

• Implement advanced in vivo imaging techniques:

  • Intravital microscopy with fluorescently labeled IL-1α or reporter systems

  • PET imaging with radiolabeled anti-IL-1α antibodies to track distribution

• Develop complex 3D culture systems:

  • Tumor spheroids co-cultured with stromal and immune cells

  • Microfluidic tumor-on-a-chip platforms with controlled cytokine gradients

  • Organoid models derived from patient samples

• Apply spatial transcriptomics and proteomics:

  • Geo-seq or Visium spatial transcriptomics to map IL-1α expression patterns

  • Imaging mass cytometry to visualize protein distribution in tissue sections

  • Digital spatial profiling for high-plex protein and RNA analysis

• Consider heterogeneity through single-cell approaches:

  • Single-cell RNA-seq of tumor and stromal populations

  • Single-cell secretome analysis to identify cellular sources of IL-1α

• Design appropriate therapeutic intervention studies:

  • Local versus systemic IL-1α blockade

  • Combination approaches with immune checkpoint inhibitors

  • Timing considerations for intervention (early versus late-stage disease)

Clinical trials with the IL-1α-blocking monoclonal antibody MABp1 demonstrate that targeting IL-1α can improve outcomes in patients with advanced cancers, suggesting translational potential for these research approaches .