Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

Area of Science:

• Immunology
• Molecular Biology
• Protein Engineering

Background:

• Interleukin-12 (IL-12) family cytokines are crucial for immune system regulation.
• These cytokines function as α:β heterodimers, with alpha subunits often requiring beta subunits for proper folding and secretion.
• Shared beta subunits among family members, like those for IL-12 and IL-23, can lead to competition and affect cytokine balance.

Purpose of the Study:

• To engineer a folding-competent human IL-23 alpha subunit (IL-23α) that is independent of its beta subunit for structure formation.
• To investigate if this engineered subunit can form a functional heterodimeric cytokine with altered assembly properties.
• To explore the implications of engineered alpha subunits on the balance of IL-12 and IL-23 production.

Main Methods:

• Rational design of the human IL-23α subunit using protein engineering.
• Assessment of the engineered subunit's folding competence and chaperone dependency.
• Analysis of heterodimer formation and cytokine assembly with the beta subunit.
• Quantification of IL-12 and IL-23 formation to determine the skewing effect.

Main Results:

• An engineered, folding-competent human IL-23α subunit was successfully designed.
• This variant demonstrated reduced chaperone dependency and enhanced affinity for its beta subunit.
• The engineered subunit assembled more efficiently, leading to increased IL-23 formation over IL-12.
• Few mutations were sufficient to create folding-competent alpha subunits compatible with cytokine assembly and function.

Conclusions:

• Folding-competent alpha subunits for the IL-12 family can be generated through targeted mutations.
• Engineered IL-23α enhances IL-23 production, suggesting a potential mechanism for immune response modulation.
• The findings support the hypothesis that natural alpha subunits may have evolved for assembly-dependent folding to regulate cytokine ratios under subunit competition.