A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared alpha and beta subunits. A common theme is that human IL-12 family alpha subunits remain incom-pletely structured in isolation until they pair with a designate beta subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely under-stood, which chaperones are involved in IL-12 family biogen-esis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 alpha subunits (IL-12 alpha and IL-23 alpha) for stabilization of transient chaperone-client com-plexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12 alpha and IL-23 alpha. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely charac-terized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12 alpha and IL-23 alpha. Despite this, PDI binding generally stabilizes unassembled IL-12 alpha and IL-23 alpha against degradation. In contrast, alpha:beta assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our under-standing of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

Automated summary

This study investigates the molecular chaperones involved in the biogenesis of cytokines in the interleukin 12 (IL-12) family. The authors found that multiple endoplasmic reticulum chaperones interact with IL-12 alpha and IL-23 alpha subunits, and members of the protein disulfide isomerase family play a role in regulating the assembly of IL-12 family subunits. Furthermore, they discovered that specific endoplasmic reticulum chaperones can modulate cytokine secretion.