Nternal Pirimiphos-methyl Autophagy disulfide bond: C58 – CeIL-23 C58-CC54: connecting to IL-12 IL-IL-12 CIL-IL-23 CIL-6 IL-IL-IL-fIL-23 N-term CHelix 1 C54 -IL-Helix 2 C-termgIL-23C58,70S FLAG IL-23C54S FLAG IL-23C14,22V FLAG + IL-12 L M L M L M + IL-12 L M Hsc70 IL-12 35 FLAG 15 Immunoblot + IL-12 Lysate medium L 70 C-term 55 M L MCCCIL-6 N-term C44 C50 C73 CC74- IL-12 C74 IL-12 N-term C15 C63 C41 C174 C80 C101 C-term MW (kDa)Fig. 1 IL-23 misfolds in cells in the absence of IL-12. a Structure of heterodimeric IL-23. Cysteines in IL-23 (blue) and IL-12 (gray) that type an intermolecular disulfide bond are shown in yellow. b Secretion behavior of FLAG-tagged wild form IL-23 (IL-23wt) in the presence or absence of its interaction companion IL-12. Hsc70 served as loading handle. c IL-23 forms non-native disulfide bonds in isolation (lane three) and IL-12 covalently heterodimerizes with IL-23 (lanes four and 5), concomitantly minimizing misfolding of IL-23. Samples were treated with -Me post-lysisDTT in cells for reduction exactly where indicated and with NEM to conserve redox species. d Structure of IL-23. Cysteines that kind an intramolecular disulfide bond in IL-23 are shown in red, the 1 that engages with IL-12 is highlighted in yellow, and no cost cysteines are depicted in orange. e Structural alignment of IL-23 (blue), IL-6 (cyan) and IL-12 (light gray). The conserved disulfide bond is shown in red and the IL-12 engaging free cysteines of IL-23 and IL-12 in yellow. f Model of IL-23, IL-6 and IL-12 illustrating cysteines and disulfide bonds. Exactly the same color code as in d, e was applied. Numbering is with out signal sequences. g Secretion behavior of FLAG-tagged IL-23 constructs as in b but with all the indicated IL-23 cysteine mutantsisolation andor they’re recognized differently by the ER top quality manage technique. The latter could offer precious insights into how protein folding states are recognized on a molecular level within the ER. All IL-23 mutants that nonetheless contained free cysteines showed a related degree of misfolding and misassembly (Supplementary Fig. 2b, c). We hence proceeded to test the second hypothesis, that the cysteines are recognized differently by chaperones. Unpaired cysteines in secretory pathway proteins might be recognized by protein disulfide isomerase (PDI) family members within the ER30. Due to the fact we didn’t observe any considerable difference in binding of PDI itself to IL-23wt versus IL-23 cysteine mutants (Supplementary Fig. 2d), we assessed interaction with yet another PDI family members member, ERp44. ERp44 serves as an ER recruitment chaperone from the ER olgi intermediate compartment (ERGIC) duringprotein assembly31 and hence was an exciting candidate in terms of IL-23 assembly control. IL-23wt strongly bound to ERp44 (Fig. 2b) and was partially co-localized with ERp44 inside the ERGIC (Supplementary Fig. 2e) indicating a biologically relevant interaction. This was further confirmed by a transient knockdown of ERp44, which led to partial secretion of unassembled IL23 (Supplementary Fig. 2f). Of note, binding of ERp44 was significantly decreased for IL-23C14,22V and IL-23C54S versus IL23wt, whereas binding towards the IL-23C58,70S mutant was not affected (Fig. 2b). Single cysteine mutants in helix 1 of IL-23 (IL23C14S and IL-23C22S) also showed reduced binding to ERp44, which was important for the C14S mutant (Supplementary Fig. 2g). To additionally assess if any chaperones act upstream of ERp44 on IL-23, i.e.: inside the ER, we analyzed binding on the ER HspNATURE COMMUNICATIONS | (2019)10:4.
