Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
Members of the IL-12 family perform essential functions in immunoregulation by connecting innate and adaptive immunity and are emerging therapeutic targets. They are unique among other interleukins in forming heterodimers that arise from extensive subunit sharing within the family, leading to the production of at least four functionally distinct heterodimers from only five subunits. This raises important questions about how the assembly of IL-12 family members is regulated and controlled in the cell. Here, using cell-biological approaches, we have dissected basic principles that underlie the biogenesis of the founding member of the family, IL-12. Within the native IL-12 heterodimer, composed of IL-12α and IL-12β, IL-12α possesses three intramolecular and one intermolecular disulfide bridges. We show that, in isolation, IL-12α fails to form its native structure but, instead, misfolds, forming incorrect disulfide bonds. Co-expression of its β subunit inhibits misfolding and thus allows secretion of biologically active heterodimeric IL-12. On the basis of these findings, we identified the disulfide bonds in IL-12α that are critical for assembly-induced secretion and biological activity of IL-12versusmisfolding and degradation of IL-12α. Surprisingly, two of the three disulfide bridges in IL-12α are dispensable for IL-12 secretion, stability, and biological activity. Extending our findings, we show that misfolding also occurs for IL-23α, another IL-12 family protein. Our results indicate that assembly-induced folding is key in IL-12 family biogenesis and secretion. The identification of essential disulfide bonds that underlie this process lays the basis for a simplified yet functional IL-12 cytokine.
From the Center for Integrated Protein Science at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, 85748 Garching, Germany and.
Susanne
S
Reitberger
the Center of Allergy and Environment, Technical University of Munich and Helmholtz Zentrum München, 80802 Munich, Germany.
Pascal
P
Haimerl
From the Center for Integrated Protein Science at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, 85748 Garching, Germany and.
Isabel
I
Aschenbrenner
the Center of Allergy and Environment, Technical University of Munich and Helmholtz Zentrum München, 80802 Munich, Germany.
Julia
J
Esser-von Bieren
From the Center for Integrated Protein Science at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, 85748 Garching, Germany and matthias.feige@tum.de.
Matthias J
0000-0001-5435-4312
MJ
Feige
D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D053765:Interleukin-12 Subunit p35 / Q000378:metabolism*; D053762:Interleukin-12 Subunit p40 / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D010084:Oxidation-Reduction; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D015398:Signal Transduction
D018076:DNA, Complementary; D004220:Disulfides; C508858:IL12A protein, human; C500879:IL12B protein, human; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40
interleukin; protein assembly; protein folding; protein misfolding; protein secretion; protein turnover