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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-12 alpha and IL-12 alpha, IL-12 alpha possesses three intramolecular and one intermolecular disulfide bridges. We show that, in isolation, IL-12 alpha fails to form its native structure but, instead, misfolds, forming incorrect disulfide bonds. Co-expression of its beta 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-12 alpha that are critical for assembly-induced secretion and biological activity of IL-12 versus misfolding and degradation of IL-12 alpha. Surprisingly, two of the three disulfide bridges in IL-12 alpha are dispensable for IL-12 secretion, stability, and biological activity. Extending our findings, we show that misfolding also occurs for IL-23 alpha, 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.

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Forster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that – in spite of its low quantum yield – is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

Genetic as well as biochemical data suggest that the yeast fatty-acid synthetase complex has an (AB)6 protein structure where A and B represent multifunctional polypeptide chains with molecular weights of 185000 and 180000 daltons, respectively. Subunit A contains at least 3 and subunit B4 of the 8 known biochemical functions of the multienzyme complex. It is concluded that this complex structure has evolved from the corresponding prokaryotic system of monofunctional enzymes due to a selective advantage regarding the biosynthesis and assembly of the complex components.

Stepwise Edman degradation of the insulin B chain is achieved after reversible protection of the amino groups in A1 and B29 with the acid-stable methylsulfonylethyloxycarbonyl residue. Condensation of the protected N-terminal tetrapeptide of insulin-like growth factor I with the degraded insulin yields an hybrid insulin whose synthesis and biological properties are discussed.