![]() D and E, design of positions for amber suppression ( left panel) in IL-12α and IL-23α subunits, respectively. Analysis of UV-crosslinked adducts can be performed by immunoblots (IBs) and mass spectrometry (MS). DiazK forms a highly reactive carbene intermediate upon photoactivation by UV irradiation, which can insert readily into nearby C–H and heteroatom–H bonds or react with Asp and Glu residues of proximal proteins. B and C, schematic of our in situ photocrosslinking approach to query weak and transient protein–protein interactions. If misfolding occurs or assembly does not take place, ERAD (ER-associated degradation) targets IL-12α/IL-23α for degradation. A, the biogenesis of IL-12 and IL-23 involves assembly induced folding reactions of IL-12α/IL-23α by IL-12β, which are coupled to quality control processes. KeywordsĪbbreviations: ACN ( acetonitrile), ATF6 ( activating transcription factor 6), BiP ( immunoglobulin binding protein), BSA ( bovine serum albumin), CHX ( cycloheximide), co-IP ( coimmunoprecipitation), DSP ( dithiobis(succinimidyl propionate)), eIF2α ( eukaryotic translation initiation factor 2α), ERAD ( ER-associated degradation), ERQC ( ER quality control), FA ( formic acid), GO ( Gene Ontology), HEK293T ( human embryonic kidney 293T cell line), IgG ( immunoglobulin G), IL ( interleukin), IP ( immunoprecipitation), β-Me ( β-mercaptoethanol), MS ( mass spectrometry), NEM ( N-ethylmaleimide), PASEF ( parallel accumulation–serial fragmentation), PDI ( protein disulfide isomerase), pPB ( piggybac), UPR ( unfolded protein response)įigure 1 Establishment of amber suppression for site-specific photocrosslinking of IL-12α and IL-23α. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. In contrast, α:β assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone–client complexes for mass spectrometry. ![]() It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Accordingly, chaperones need to support and control specific assembly processes. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Glycobiology and Extracellular MatricesĬytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits.
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