Abstract
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-12 versus misfolding 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.
Highlights
Members of the IL-12 family perform essential functions in immunoregulation by connecting innate and adaptive immunity and are emerging therapeutic targets
More recently, has come into focus again when mass spectrometric studies revealed several IL-12-interacting proteins in mouse plasma [44]. This interaction promiscuity is a general theme within the IL-12 family, where at least four heterodimers (IL-12, IL-23, IL-27, and IL-35) are made up by only five subunits [18, 19]
Because IL-12␣ as well as IL-23␣ secretion are dependent on IL-12 [26, 31, 34, 36], and secreted IL-12 can compete with IL-12 signaling [32, 45], important questions arise about how IL-12 can induce the secretion of multiple ␣ subunits and how this process, and downstream immune reactions, are regulated and controlled in the endoplasmic reticulum (ER)
Summary
The secretion of IL-12 depends on the expression of both subunits of the IL-12 heterodimer (Fig. 1A), IL-12␣ and IL-12 [26, 31]. Because our data revealed that disulfide bridges 1 and 2 in IL-12␣ were dispensable for secretion of a covalent IL-12␣-IL12 heterodimer (Fig. 3C), in the step we combined these deletions (⌬SS1&2), giving rise to an IL-12␣ mutant with only three cysteine residues This mutant populated the least amount of HMW species of all mutants tested (Fig. 3B). Because our data revealed that two of three disulfide bridges in IL-12␣ are dispensable for secretion and did not change the rate of IL-12␣ degradation, we investigated their impact on the stability and biological activity of IL-12 Toward this end, we incubated supernatants of 293T cells co-transfected with IL-12 and either IL-12␣ WT, ⌬SS1, ⌬SS2, or ⌬SS1&2 for extended times at 37 °C (IL-12␣ ⌬SS3 was excluded because it was not secreted with IL-12). For all mutants that were secreted in the presence of IL-12 (Fig. 3C), we observed induction of IFN␥ in hPBMCs (Fig. 5B), indicating that most cysteines within IL-12␣ are not essential for IL-12 activity
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