Recombinant Human Interleukin-1 alpha (IL1A) (Active) (GMP)

What is the molecular structure of human IL-1 alpha?

Human IL-1 alpha is a polypeptide consisting of 271 amino acids that shows approximately 61% homology to its murine counterpart but only 27% homology to human IL-1 beta, despite their similar biological activities . The protein contains a functional nuclear localization signal (NLS) sequence LKKRRL that allows for translocation to the nucleus under specific conditions . When expressed recombinantly, the active form typically encompasses amino acids 113-271 of the full-length protein .

How does IL-1 alpha differ functionally from IL-1 beta?

While IL-1 alpha and IL-1 beta are equally potent inflammatory cytokines that activate similar inflammatory processes, they differ significantly in their biogenesis and regulation . Unlike IL-1 beta, IL-1 alpha is constitutively present intracellularly in nearly all resting non-hematopoietic cells . IL-1 alpha can function both as an intracellular transcription regulator and as an extracellular cytokine upon release, while IL-1 beta primarily functions extracellularly after processing . Additionally, IL-1 alpha can act as an alarmin when released during cell death, directly sensing DNA damage and signaling genotoxic stress without requiring proteolytic processing for activation .

What are the key signaling pathways activated by IL-1 alpha?

After binding to its receptor IL1R1 together with accessory protein IL1RAP, IL-1 alpha forms a high-affinity receptor complex that initiates signaling cascades . This signaling involves the recruitment of adapter molecules such as MYD88, IRAK1, or IRAK4, which subsequently mediates the activation of NF-kappa-B and three MAPK pathways: p38, p42/p44, and JNK . These pathways collectively drive the inflammatory response and modulate various cellular functions, including gene expression, proliferation, and differentiation in target cells .

What are the optimal expression systems for producing recombinant human IL-1 alpha?

Recombinant human IL-1 alpha can be successfully produced in several expression systems, each with distinct advantages depending on research requirements:

For research requiring highly purified protein with native-like characteristics, expression in HEK 293 cells is preferred, as it yields protein suitable for SDS-PAGE, functional studies, mass spectrometry, and HPLC applications . For structural studies or applications where post-translational modifications are less critical, E. coli-based expression systems can provide higher yields at lower cost .

How should researchers assess the biological activity of recombinant IL-1 alpha preparations?

Biological activity assessment of recombinant IL-1 alpha should employ multiple complementary assays:

• T-cell proliferation assay: Using responsive cell lines such as D10.G4.1 mouse helper T cells to measure proliferation in response to IL-1 alpha stimulation . Effective concentrations (ED50) should be established for each preparation.

• Fibroblast stimulation: Measuring fibroblast proliferation and the induction of collagenase and prostaglandin production following IL-1 alpha treatment .

• Receptor binding assays: Confirming specific binding to IL1R1 receptor complexes using surface plasmon resonance or competitive binding assays .

• Signaling pathway activation: Monitoring the phosphorylation of downstream signaling molecules (NF-κB, MAPK pathways) through Western blotting or reporter gene assays .

For robust validation, researchers should compare activity across multiple assays and establish dose-response relationships to ensure consistency between preparations .

How do IL-1 alpha knockout models affect experimental design and interpretation?

These discrepancies highlight critical concerns for experimental design:

• Researchers must specify which knockout line they are using and verify its characteristics.

• Phenotypes attributed to IL-1α deficiency should be validated using complementary approaches.

• Studies using the original knockout line should be interpreted cautiously, as observed effects may reflect broader disruptions in IL-1 family cytokine regulation rather than specific IL-1α functions .

For maximum rigor, researchers should consider using both genetic models alongside pharmacological approaches (IL-1α neutralizing antibodies or recombinant protein supplementation) to confirm specific IL-1α-dependent effects .

What factors regulate the intracellular localization and release of IL-1 alpha?

The regulation of IL-1 alpha localization and release involves multiple molecular mechanisms that remain partially understood . In resting cells, pro-IL-1α can translocate to the nucleus via its nuclear localization signal (NLS) where it functions as a transcriptional regulator . Several factors influence this process:

• Calpain-dependent cleavage: While the functional significance remains unclear, calpain-mediated cleavage of pro-IL-1α may facilitate either its release from living cells or promote IL-1α-NTP translocation to the nucleus .

• Cell death pathways: During apoptosis, IL-1α becomes sequestered in the nucleus through mechanisms requiring further investigation, while necrotic cell death leads to its passive release into the extracellular space .

• Membrane translocation: Pro-IL-1α can localize to the outer surface of the plasma membrane, but the factors controlling this translocation remain unidentified .

• Cellular stress responses: Oxidative stress, lipid overload, and genotoxic damage can all trigger changes in IL-1α localization and release, suggesting sophisticated stress-sensing mechanisms .

Understanding these regulatory mechanisms has significant implications for targeting IL-1α in inflammatory diseases, as interventions aimed at specific localization or release pathways could provide more precise therapeutic strategies .

How can contradictory findings in IL-1 alpha literature be reconciled?

The IL-1 alpha research field contains several apparent contradictions that researchers must navigate carefully . To reconcile these conflicting findings:

• Genetic model validation: Authenticate genetic resources by confirming the specific nature of the IL-1α knockout or transgenic model used, as different knockout strategies can yield distinct phenotypes .

• Cell type specificity: Consider that IL-1α regulation varies substantially between cell types. For example, monocytes have a unique mechanism of inducible IL-1α expression involving long noncoding RNA AS-IL-1a, while CD4+ T cells exhibit monoallelic expression regulated by promoter methylation .

• Experimental context: Carefully control inflammatory stimuli, as IL-1α can respond differently to sterile injury versus pathogen-associated molecular patterns .

• Signaling feedback loops: Account for potential self-amplifying positive feedback mechanisms between IL-1α and IL-1β, which may be context-dependent and vary between experimental systems .

• Technical approach diversity: Use complementary techniques (genetic models, neutralizing antibodies, recombinant proteins) to validate key findings and establish causality .

By addressing these methodological considerations, researchers can better interpret seemingly contradictory results and develop more accurate models of IL-1α biology .

What mechanisms control IL-1 alpha gene expression?

The transcriptional regulation of IL-1 alpha involves unique regulatory elements and cell-type specific mechanisms:

• Promoter structure: Unlike many inducible genes, the IL-1a promoter lacks canonical TATA and CAAT box regulatory regions. Instead, it contains a binding site for the Sp1 transcription factor, which typically mediates expression of housekeeping genes at homeostasis .

• Inducible elements: The promoter contains binding sites for AP1 and NF-κB transcription factors, which can upregulate IL-1α expression in a cell-type-specific manner following stimulation .

• Transcriptional repressors: The proximal IL1a promoter region contains a transcriptional-repressor-binding site that reduces its transcriptional activity, suggesting that dissociation of this repressor can increase IL-1α expression upon stimulation .

• Epigenetic regulation: In human CD4+ T cells, IL-1α expression is monoallelic and regulated via hyper- or hypomethylation of CpG nucleotides located in promoter regions proximal to the transcription initiation site .

• Non-coding RNA regulation: Monocytes have a unique mechanism involving upregulation of the long non-coding RNA AS-IL-1a, a natural antisense transcript partially complementary to IL-1α mRNA, which influences expression .

These diverse regulatory mechanisms allow for both constitutive expression in certain cell types and rapid induction in response to various stimuli, positioning IL-1α as a versatile inflammatory mediator .

What stimuli induce IL-1 alpha expression, and through which pathways?

IL-1 alpha expression can be rapidly induced by a remarkably diverse array of physiological stimuli through multiple signaling pathways:

This responsiveness to such a broad spectrum of stimuli underlies IL-1α's central role in both sterile inflammation and pathogen-induced inflammatory responses . The molecular integration of these diverse signals enables IL-1α to function as a cellular decision-making nexus that gauges the magnitude of stress, damage, or infection severity to initiate appropriate inflammatory responses .

What critical questions remain unanswered about IL-1 alpha biology?

Despite over 30 years of research, several fundamental aspects of IL-1 alpha biology remain poorly understood:

• Membrane translocation mechanisms: The factors controlling pro-IL-1α translocation from the cytosol to the outer surface of the plasma membrane, allowing it to signal as a membrane-bound cytokine, are still unknown .

• Calpain-mediated processing: The functional significance of calpain-dependent cleavage of pro-IL-1α within cells remains unclear—whether it facilitates release from living cells or is necessary only for nuclear translocation of IL-1α-NTP .

• Nuclear sequestration during apoptosis: The identity of factors that control IL-1α sequestration in the nucleus during apoptosis requires further investigation .

• Age-related expression: Factors allowing for increased IL-1α expression in aged and senescent cells need further study, particularly given the importance of inflammation in aging-related diseases .

• Cell-type specificity: The molecular basis for differential regulation and function of IL-1α across diverse cell types remains to be fully elucidated .

Resolving these questions will likely reveal new therapeutic opportunities for inflammatory diseases and provide deeper insights into fundamental immune system regulation .

What methodological advances are needed to resolve current controversies in IL-1 alpha research?

Addressing the current knowledge gaps and controversies in IL-1 alpha research will require several methodological advances:

• Improved genetic models: Development of conditional and inducible IL-1α knockout systems to overcome limitations of constitutive knockouts and better isolate cell-type specific functions .

• Live-cell imaging techniques: Advanced microscopy approaches to track IL-1α localization and release in real-time within living cells and tissues .

• Single-cell analysis: Application of single-cell transcriptomics and proteomics to understand cell-to-cell variability in IL-1α expression and response .

• Structural biology approaches: High-resolution structures of IL-1α in various cellular compartments and in complex with different binding partners to understand context-specific functions .

• Systems biology integration: Computational modeling of IL-1α signaling networks to predict context-dependent outcomes and identify key regulatory nodes .

As these advanced methodologies are applied, researchers will be better positioned to understand the complex biology of IL-1α and its roles in various inflammatory conditions, potentially leading to more targeted therapeutic strategies for inflammatory diseases .