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

  • Author Footnotes
    ∗ these authors contributed equally to this work
    Yonatan G. Mideksa
    Footnotes
    ∗ these authors contributed equally to this work
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Author Footnotes
    ∗ these authors contributed equally to this work
    Isabel Aschenbrenner
    Footnotes
    ∗ these authors contributed equally to this work
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Anja Fux
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Dinah Kaylani
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Caroline A.M. Weiß
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Tuan-Anh Nguyen
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Nina C. Bach
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Kathrin Lang
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)

    Laboratory of Organic Chemistry, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland
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  • Stephan A. Sieber
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)
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  • Matthias J. Feige
    Correspondence
    To whom correspondence may be addressed: Tel: 49-89-28913667; Fax: 49-89-28910698;
    Affiliations
    Technical University of Munich, Germany; TUM School of Natural Sciences, Department of Bioscience; Center for Functional Protein Assemblies (CPA)

    Laboratory of Organic Chemistry, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland
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  • Author Footnotes
    ∗ these authors contributed equally to this work
Open AccessPublished:November 03, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102677

      Abstract

      Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis.
      Here, we site-specifically introduce photo-crosslinking 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. Our analysis reveals that a large set of ER chaperones interacts with 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. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:β 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 understanding 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 ER chaperones.

      Keywords

      The abbreviations used are:

      ATF6 (activating transcription factor 6), BiP (immunoglobulin heavy-chain binding protein), CALR (calreticulin), CANX (calnexin), CHX (cycloheximide), DiazK (N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)-L-lysine), DSP (dithiobis(succinimidyl propionate)), DTT (dithiothreitol), eIF2α (eukaryotic translation initiation factor 2α), ER (endoplasmic reticulum), ERAD (ER-associated degradation), IL (interleukin), MS (mass spectrometry), NEM (N-ethylmaleimide), PDI (protein disulfide isomerase), PERK (protein kinase R-like ER kinase), β-Me (β-mercaptoethanol)

      Introduction

      Mammalian cells dedicate one third of their genome to secretory pathway proteins, which allow cells to interact with their environment. These proteins generally acquire their native structure in the endoplasmic reticulum (ER), where a comprehensive chaperone machinery supports and controls each molecular step towards the native state (
      • Braakman I.
      • Hebert D.N.
      Protein folding in the endoplasmic reticulum.
      ). Protein maturation in the ER includes post-translational modifications that render structure formation more robust and tune functionality, but also target proteins to certain chaperone systems. The most prominent modifications are glycosylation and disulfide bond formation, which occur in the majority of ER-produced proteins (
      • Braakman I.
      • Bulleid N.J.
      Protein folding and modification in the mammalian endoplasmic reticulum.
      ). N-linked glycans target proteins to the calnexin/calreticulin cycle that monitors and supports folding processes in secretory pathway proteins (
      • Tannous A.
      • Pisoni G.B.
      • Hebert D.N.
      • Molinari M.
      N-linked sugar-regulated protein folding and quality control in the ER.
      ,
      • Kozlov G.
      • Gehring K.
      Calnexin cycle - structural features of the ER chaperone system.
      ). Disulfide bonds stabilize the native structure and, while unpaired, cysteines provide a handle for the ER quality control (ERQC) system (

      Feige, M. J. (2018) Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, RSC publishing

      ,
      • Anelli T.
      • Sitia R.
      Protein quality control in the early secretory pathway.
      ). Disulfide bond formation, isomerization and reduction are catalyzed by the ER-resident protein disulfide isomerase (PDI) family. This family comprises a surprisingly large number of approximately 20 members in humans (
      • Appenzeller-Herzog C.
      • Ellgaard L.
      The human PDI family: versatility packed into a single fold.
      ,
      • Kanemura S.
      • Matsusaki M.
      • Inaba K.
      • Okumura M.
      PDI Family Members as Guides for Client Folding and Assembly.
      ). The expansion of the PDI family during evolution of more complex cells can likely be explained by functional specialization of certain family members. While PDI is a generic oxidoreductase with additional chaperone functions (
      • Freedman R.B.
      • Klappa P.
      • Ruddock L.W.
      Protein disulfide isomerases exploit synergy between catalytic and specific binding domains.
      ,
      • Wilson R.
      • Lees J.F.
      • Bulleid N.J.
      Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen.
      ,
      • Yu J.
      • Li T.
      • Liu Y.
      • Wang X.
      • Zhang J.
      • Wang X.
      • Shi G.
      • Lou J.
      • Wang L.
      • Wang C.C.
      • Wang L.
      Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.
      ), other family members are more restricted in their clientele and functionalities. For some family members, insights into their specializations have been obtained: ERp57 interacts with calnexin and calreticulin and is thus mostly recruited to glycoproteins (
      • Molinari M.
      • Helenius A.
      Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells.
      ,
      • Frickel E.M.
      • Frei P.
      • Bouvier M.
      • Stafford W.F.
      • Helenius A.
      • Glockshuber R.
      • Ellgaard L.
      ERp57 is a multifunctional thiol-disulfide oxidoreductase.
      ), whereas the membrane integral PDI family member TMX1 prefers membrane proteins as clients (
      • Pisoni G.B.
      • Ruddock L.W.
      • Bulleid N.
      • Molinari M.
      Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides.
      ). The PDI ERp5 interacts with the ER Hsp70 BiP, and thus may have a preference for BiP clients (
      • Jessop C.E.
      • Watkins R.H.
      • Simmons J.J.
      • Tasab M.
      • Bulleid N.J.
      Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins.
      ). TMX4 and ERdj5, the latter being another BiP co-chaperone, are involved in reducing disulfide bonds for ER-associated degradation (ERAD) (
      • Ushioda R.
      • Hoseki J.
      • Araki K.
      • Jansen G.
      • Thomas D.Y.
      • Nagata K.
      ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER.
      ,
      • Sugiura Y.
      • Araki K.
      • Iemura S.
      • Natsume T.
      • Hoseki J.
      • Nagata K.
      Novel thioredoxin-related transmembrane protein TMX4 has reductase activity.
      ) but also dissolving incorrectly formed disulfide bonds (
      • Oka O.B.
      • Pringle M.A.
      • Schopp I.M.
      • Braakman I.
      • Bulleid N.J.
      ERdj5 is the ER reductase that catalyzes the removal of non-native disulfides and correct folding of the LDL receptor.
      ). ERp44, on the other hand, serves as a recruitment factor for immature proteins that leave the ER while their native disulfide bonds have not formed yet (
      • Vavassori S.
      • Cortini M.
      • Masui S.
      • Sannino S.
      • Anelli T.
      • Caserta I.R.
      • Fagioli C.
      • Mossuto M.F.
      • Fornili A.
      • van Anken E.
      • Degano M.
      • Inaba K.
      • Sitia R.
      A pH-regulated quality control cycle for surveillance of secretory protein assembly.
      ,
      • Anelli T.
      • Alessio M.
      • Mezghrani A.
      • Simmen T.
      • Talamo F.
      • Bachi A.
      • Sitia R.
      ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family.
      ). In addition to their role in catalyzing redox reactions in their clients, PDI family members are key regulators of ER stress responses and thus have further broadened their functional spectrum during evolution (
      • Yu J.
      • Li T.
      • Liu Y.
      • Wang X.
      • Zhang J.
      • Wang X.
      • Shi G.
      • Lou J.
      • Wang L.
      • Wang C.C.
      • Wang L.
      Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.
      ,
      • Oka O.B.
      • van Lith M.
      • Rudolf J.
      • Tungkum W.
      • Pringle M.A.
      • Bulleid N.J.
      ERp18 regulates activation of ATF6α during unfolded protein response.
      ).
      The large variety of PDI family members combined with their different roles complicates their functional analysis in the native cellular context, but also renders it particularly relevant to decipher the working principles of the ER folding environment. Previous studies often focused on certain PDI family members and analyzed the fate of selected clients in their absence (
      • Rutkevich L.A.
      • Cohen-Doyle M.F.
      • Brockmeier U.
      • Williams D.B.
      Functional relationship between protein disulfide isomerase family members during the oxidative folding of human secretory proteins.
      ). Alternatively, substrate-trapping mutants of PDI family members were used to define their clients (
      • Jessop C.E.
      • Watkins R.H.
      • Simmons J.J.
      • Tasab M.
      • Bulleid N.J.
      Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins.
      ), which may miss chaperone or oxidase functions of the respective PDI family member. Here we complement these studies by a client-centric crosslinking approach. We focus on key signaling molecules in the immune system, the interleukin (IL) 12 family members IL-12 and IL-23, which both coordinate innate and adaptive immune responses (
      • Vignali D.A.
      • Kuchroo V.K.
      IL-12 family cytokines: immunological playmakers.
      ,
      • Tait Wojno E.D.
      • Hunter C.A.
      • Stumhofer J.S.
      The Immunobiology of the Interleukin-12 Family: Room for Discovery.
      ). These two cytokines are ideally suited to further dissect PDI family member functions in the cell: Our recent work has shown that oxidative folding governs the biogenesis of these cysteine-rich cytokines (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Muller S.I.
      • Aschenbrenner I.
      • Zacharias M.
      • Feige M.J.
      An Interspecies Analysis Reveals Molecular Construction Principles of Interleukin 27.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ). Furthermore, these heterodimeric cytokines form intra- as well as inter-molecular disulfide bonds and populate mis-oxidized species during their biogenesis, which strongly demands for support by PDI family members (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Yoon C.
      • Johnston S.C.
      • Tang J.
      • Stahl M.
      • Tobin J.F.
      • Somers W.S.
      Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
      ,
      • Lupardus P.J.
      • Garcia K.C.
      The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12.
      ). Since IL-12 and IL-23 both share the same β subunit (IL-12β) (
      • Oppmann B.
      • Lesley R.
      • Blom B.
      • Timans J.C.
      • Xu Y.
      • Hunte B.
      • Vega F.
      • Yu N.
      • Wang J.
      • Singh K.
      • Zonin F.
      • Vaisberg E.
      • Churakova T.
      • Liu M.
      • Gorman D.
      • Wagner J.
      • Zurawski S.
      • Liu Y.
      • Abrams J.S.
      • Moore K.W.
      • Rennick D.
      • de Waal-Malefyt R.
      • Hannum C.
      • Bazan J.F.
      • Kastelein R.A.
      Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
      ,
      • Gubler U.
      • Chua A.O.
      • Schoenhaut D.S.
      • Dwyer C.M.
      • McComas W.
      • Motyka R.
      • Nabavi N.
      • Wolitzky A.G.
      • Quinn P.M.
      • Familletti P.C.
      • et al.
      Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
      ,
      • Wolf S.F.
      • Temple P.A.
      • Kobayashi M.
      • Young D.
      • Dicig M.
      • Lowe L.
      • Dzialo R.
      • Fitz L.
      • Ferenz C.
      • Hewick R.M.
      • et al.
      Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.
      ), an analysis of the redox machinery that acts on IL-12 versus IL-23 can provide insights into PDI family member client specificity versus promiscuity. Accordingly, these studies may point toward possible specific ways of modulating IL-12 versus IL-23 assembly, which are both highly relevant molecules for human disease (
      • Tait Wojno E.D.
      • Hunter C.A.
      • Stumhofer J.S.
      The Immunobiology of the Interleukin-12 Family: Room for Discovery.
      ).

      Results

      Establishment of a photo-crosslinking approach to identify IL-12/IL-23 chaperones

      Chaperones and folding enzymes generally only transiently interact with their clients. This is a prerequisite for their function but complicates analyses of chaperone:client complexes, in particular in the biologically relevant context of cells. To analyze the ER chaperone machinery that acts on the different steps of IL-12 and IL-23 biogenesis (Fig. 1A) we thus decided to covalently crosslink chaperone:client complexes for downstream analyses. Toward this end, we site-specifically incorporated the diazirine bearing unnatural amino acid DiazK (Fig. 1B) into various positions of the α subunits of IL-12 and IL-23 (IL-12α and IL-23α, respectively). For this we used an efficient Pyrrolysyl-tRNA synthetase variant together with its amber-suppressor tRNA, a setup which has been thoroughly characterized in very recent studies (
      • Bartoschek M.D.
      • Ugur E.
      • Nguyen T.A.
      • Rodschinka G.
      • Wierer M.
      • Lang K.
      • Bultmann S.
      Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.
      ,
      • Nguyen T.A.
      • Gronauer T.
      • Nast-Kolb T.
      • Sieber S.
      • Lang K.
      Substrate profiling of mitochondrial caseinolytic protease P via a site-specific photocrosslinking approach.
      ). Upon irradiation with UV light (365 nm), DiazK forms a carbene (Fig. 1B) that readily reacts with adjacent proteins to stabilize transient protein:protein complexes for analyses by e.g. immunoblotting and mass spectrometry (Fig. 1C). In this study, we specifically focused on IL-12α and IL-23α since these subunits remain incompletely structured in isolation and are retained in cells until they pair with their shared IL-12β subunit (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Oppmann B.
      • Lesley R.
      • Blom B.
      • Timans J.C.
      • Xu Y.
      • Hunte B.
      • Vega F.
      • Yu N.
      • Wang J.
      • Singh K.
      • Zonin F.
      • Vaisberg E.
      • Churakova T.
      • Liu M.
      • Gorman D.
      • Wagner J.
      • Zurawski S.
      • Liu Y.
      • Abrams J.S.
      • Moore K.W.
      • Rennick D.
      • de Waal-Malefyt R.
      • Hannum C.
      • Bazan J.F.
      • Kastelein R.A.
      Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
      ,
      • Gubler U.
      • Chua A.O.
      • Schoenhaut D.S.
      • Dwyer C.M.
      • McComas W.
      • Motyka R.
      • Nabavi N.
      • Wolitzky A.G.
      • Quinn P.M.
      • Familletti P.C.
      • et al.
      Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
      ,
      • Jalah R.
      • Rosati M.
      • Ganneru B.
      • Pilkington G.R.
      • Valentin A.
      • Kulkarni V.
      • Bergamaschi C.
      • Chowdhury B.
      • Zhang G.M.
      • Beach R.K.
      • Alicea C.
      • Broderick K.E.
      • Sardesai N.Y.
      • Pavlakis G.N.
      • Felber B.K.
      The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit: IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION.
      ). They are thus prime targets for molecular chaperoning. To comprehensively analyze the chaperone repertoire that acts on IL-12α and IL-23α we selected 14 or nine positions within each subunit, respectively, where we individually introduced an amber stop codon to be suppressed by incorporation of DiazK (Fig. 1, D and E, left panels). We focused on positions that were surface-exposed, not predicted to destabilize the respective protein upon mutation and not in the interface with IL-12β (
      • Yoon C.
      • Johnston S.C.
      • Tang J.
      • Stahl M.
      • Tobin J.F.
      • Somers W.S.
      Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
      ,
      • Lupardus P.J.
      • Garcia K.C.
      The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12.
      ). For each construct, we observed expression upon transient transfection into HEK293T cells in the presence of DiazK, but also the presence of polypeptide chains truncated at the intrinsic amber stop codon, as expected for amber suppression (supplemental Fig. S1, A and B). We thus fused a C-terminal FLAG tag to the constructs (supplemental Fig. S1, C and D), which allows for the specific immunoprecipitation only of completely translated polypeptide chains containing the DiazK moiety. Since IL-12α and IL-23α can form homo-dimers in cells (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ), some truncated proteins could still be observed if rather C-terminally located amber codons were used, which give rise to almost fully translated, homo-dimerization competent polypeptide chains if a truncation occurs (e.g. in supplemental Fig. S1, C and D). In general, amber suppression was efficient and ∼20% to 80% of expression of the non-suppressed wild type (wt) constructs was obtained (supplemental Fig. S1, C and D).
      Figure thumbnail gr1
      Figure 1Establishment of amber suppression for site-specific photo-crosslinking of IL-12α and IL-23α. 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. If misfolding occurs or assembly does not take place, ERAD (ER-associated degradation) targets IL-12α/IL-23α for degradation. Each process is dependent on chaperones. B, C) Schematic of our in situ photo-crosslinking approach to query weak and transient protein-protein interactions. 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. Analysis of UV-crosslinked adducts can be performed by immunoblots (IB) and mass spectrometry (MS). D, E) Design of positions for amber suppression (left panel) in IL-12α and IL-23α subunits, respectively. Selected constructs were tested for assembly-induced secretion upon co-expression of IL-12β (right panel). HEK293T cells were transiently co-transfected with the indicated constructs in the presence of DiazK and samples were analyzed by immunoblots. An asterisk denotes the site of Stop codon introduction and amber suppression. The upward shift of IL-12α upon secretion into the medium (M: medium; L: cell lysate) is caused by modifications of its sugar moieties in the Golgi (
      • Bohnacker S.
      • Hildenbrand K.
      • Aschenbrenner I.
      • Müller S.I.
      • Bieren J.E.
      • Feige M.J.
      Influence of glycosylation on IL-12 family cytokine biogenesis and function.
      ). IL-12β populates two species differing in the use of N-glycosylation sites (
      • Bohnacker S.
      • Hildenbrand K.
      • Aschenbrenner I.
      • Müller S.I.
      • Bieren J.E.
      • Feige M.J.
      Influence of glycosylation on IL-12 family cytokine biogenesis and function.
      ).
      For a subset of constructs, we additionally tested wt-like behavior in terms of ERQC. Normally, IL-12α and IL-23α are retained in the ER in isolation and can only pass ERQC and become secreted upon co-expression of IL-12β, including further modification of sugar moieties for IL-12α (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Oppmann B.
      • Lesley R.
      • Blom B.
      • Timans J.C.
      • Xu Y.
      • Hunte B.
      • Vega F.
      • Yu N.
      • Wang J.
      • Singh K.
      • Zonin F.
      • Vaisberg E.
      • Churakova T.
      • Liu M.
      • Gorman D.
      • Wagner J.
      • Zurawski S.
      • Liu Y.
      • Abrams J.S.
      • Moore K.W.
      • Rennick D.
      • de Waal-Malefyt R.
      • Hannum C.
      • Bazan J.F.
      • Kastelein R.A.
      Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
      ,
      • Gubler U.
      • Chua A.O.
      • Schoenhaut D.S.
      • Dwyer C.M.
      • McComas W.
      • Motyka R.
      • Nabavi N.
      • Wolitzky A.G.
      • Quinn P.M.
      • Familletti P.C.
      • et al.
      Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
      ,
      • Jalah R.
      • Rosati M.
      • Ganneru B.
      • Pilkington G.R.
      • Valentin A.
      • Kulkarni V.
      • Bergamaschi C.
      • Chowdhury B.
      • Zhang G.M.
      • Beach R.K.
      • Alicea C.
      • Broderick K.E.
      • Sardesai N.Y.
      • Pavlakis G.N.
      • Felber B.K.
      The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit: IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION.
      ,
      • Bohnacker S.
      • Hildenbrand K.
      • Aschenbrenner I.
      • Müller S.I.
      • Bieren J.E.
      • Feige M.J.
      Influence of glycosylation on IL-12 family cytokine biogenesis and function.
      ). The same behavior was observed for IL-12α and IL-23α containing DiazK at different positions. When expressed alone in HEK293T cells, subunits were retained in cells. When IL-12β was co-transfected, IL-12α and IL-23α were secreted together with IL-12β (Fig. 1, D and E, right panels), showing that DiazK incorporation for photo-crosslinking is a suitable tool to query their chaperone repertoire.
      Using this approach, we could detect several crosslinked species for IL-12α containing a DiazK moiety, which were present exclusively if the cells were UV-irradiated and independent of the presence of the C-terminal FLAG epitope tag (Fig. 2, A and B and supplemental Fig. S2A-C, S3A). Importantly, some distinct crosslinked species could be detected for different positions of DiazK incorporation. Amber suppression did not interfere with immunoprecipitation and the crosslinked species could generally be co-immunoprecipitated (Fig. 2, A and B and supplemental Fig. S2, B and C, S3A). A similar behavior was observed for IL-23α (Fig. 2, C and D and supplemental Fig. S2D-F). Together this setup should thus allow downstream mass spectrometric analyses. For IL-12α, we focused on two constructs that showed the presence of a significant number of crosslinks and covered different positions, whereas for IL-23α one crosslinking position within its first α-helix was used (Fig. 2A-D and supplemental Fig. S3A) since this first α-helix has been shown to serve as a chaperone recognition site (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ). Mass spectrometric analyses revealed a large number of proteins in the immunoprecipitated samples of IL-12α and IL-23α (Fig. 2, E and F, supplemental Fig. S3B and Table S1). Some interactions were dependent on photo-crosslinking, showing that this approach extends the interactome that can be detected by mass spectrometry (supplemental Fig. S3C-E). To identify IL-12α and IL-23α ER chaperones and quality control factors among the identified proteins, we used suitable gene ontology (GO) term annotations to filter the interactomes (for details see Experimental Procedures).
      Figure thumbnail gr2
      Figure 2Photo-crosslinking allows to capture IL-12α and IL-23α interaction partners for mass spectrometry. A-D) Immunoblot verification of UV-crosslinked complexes before and after immunoprecipitation (IP) with FLAG beads. The orthogonal DiazKRS/tRNACUA pair was co-expressed in all panels in the presence of DiazK, for site-specific modification of the α-subunits. Where indicated, cells were irradiated with UV light (UV irr.) to induce photo-crosslinking of DiAzK to adjacent residues. Overexposed blots are shown to highlight weak signals for high molecular weight species; the schematic summarizes the workflow. Truncated protein species for FLAG-immunoprecipitated IL-12α/IL-23α likely originate from homo-dimerization as previously reported (
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ). E, F) Volcano plots derived from LC-MS/MS analysis of the indicated IL constructs compared to control FLAG co-IPs using cells transfected with empty vector (ev), carried out in three replicates. The top-right quadrants list significant hits with cut-off values defined as log2 = 1 (2-fold enrichment) and -log10 (p-value) of 1.3 (p<0.05). ER chaperones and quality control proteins are depicted as orange circles. Those with a possible additional PDI molecular function are shown as orange squares and labeled in bold. Hits were labeled with respective UniProt entry names and for possible PDIs, protein names are additionally given in brackets. IL-12α and IL-23α are shown in violet or green, respectively.

      Multiple overlapping but also distinct ER PDI family members are involved in IL-12 and IL-23 biogenesis

      Our mass spectrometry analyses identified several ER chaperones interacting with IL-12α and IL-23α, including e.g., the ER Hsp70 chaperone BiP, the Hsp90 chaperone Grp94 (ENPL) and the lectin chaperones calreticulin (CALR) and calnexin (CALX) (Fig. 2E and F and supplemental Fig. S3B-E). Among the IL-12α or IL-23α interactors we decided to focus on ER oxidoreductases, due to the key role of oxidative folding in IL-12/IL-23 biogenesis (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Muller S.I.
      • Aschenbrenner I.
      • Zacharias M.
      • Feige M.J.
      An Interspecies Analysis Reveals Molecular Construction Principles of Interleukin 27.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ). We found several overlapping but also distinct PDI family members to interact with IL-12α or IL-23α (Fig. 2E and F). To validate and extend our mass spectrometric data, for each of the identified interacting PDI family members we next assessed interaction with wt IL-12α or IL-23α by co-immunoprecipitation experiments. In some cases, N-ethylmaleimide (NEM), which blocks reshuffling of disulfide bonds, was sufficient to preserve interactions. In other cases, the amine-reactive crosslinker DSP had to be used, suggesting that different interactions between the cytokine subunits and the PDI family members were formed, including non-covalent ones. All significant interactions with PDI family members detected by mass spectrometry could be verified by co-immunoprecipitation for IL-12α (Fig. 3A) as well as for IL-23α (Fig. 3B). Confirming the specificity of our approach but also underlining the need for confirmatory experiments, ERp57, which is recruited to glycoproteins via calnexin/calreticulin, was found to be highly enriched in the interactome of the N-glycoprotein IL-12α but only weakly for IL-23α (Fig. 2E and F), which does not contain N-glycosylation sites (
      • Bohnacker S.
      • Hildenbrand K.
      • Aschenbrenner I.
      • Müller S.I.
      • Bieren J.E.
      • Feige M.J.
      Influence of glycosylation on IL-12 family cytokine biogenesis and function.
      ). Interactions with IL-23α were also not observed in co-immunoprecipitation experiments (Fig. 3B). To further extend our studies, we also included ERp46 into our co-immunoprecipitation experiments due to its recently described role in early protein folding reactions (
      • Hirayama C.
      • Machida K.
      • Noi K.
      • Murakawa T.
      • Okumura M.
      • Ogura T.
      • Imataka H.
      • Inaba K.
      Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation.
      ) and the fact that we could detect it in the IL-12α interactome, although not significantly enriched (Table S1). ERp46 interacted with IL-12α independent of the chemical crosslinker DSP (Fig. 3A) whereas interaction with IL-23α was strongly increased in the presence of DSP (Fig. 3B), showing that although not a significant MS-hit, ERp46 appears to interact with IL-12α and IL-23α and that complementary approaches can further extend the MS interactome.
      Figure thumbnail gr3
      Figure 3Analysis of PDI binding partners by co-immunoprecipitations. A, B) HEK293T transiently expressing either IL-12α or IL-23α, each C-terminally FLAG-tagged, were subjected to FLAG IP with or without an amine-reactive DSP-crosslinker and analyzed using western blots under reducing conditions. CALR (calreticulin), a soluble ER lectin chaperone, was included to benchmark the distinct interaction profile that exists between the N-glycosylated IL-12α versus non-N-glycosylated IL-23α. Arrowheads point to the top band marking the correct size of the CALR protein; EV – empty vector and iso. – isotype control beads. In all cases, interactions with wild type (wt) interleukin subunits not containing any UAA were analyzed. C, D) DSP-independent interactions were further evaluated for covalent complex formation via non-reducing co-IPs/SDS-PAGE. Reducing input blots are included to verify the expression of FLAG-tagged IL-12α/23α in whole cell lysates. E) Each PDI’s interaction profile obtained from results in A-D is summarized in E.
      Of note, for interactions that were observable without DSP as a crosslinker, we could generally detect covalent complexes between IL-12α and the different PDIs (Fig. 3C) or IL-23α and the different PDIs (Fig. 3D), respectively. Taken together, as intended, our workflow succeeded in identifying covalent and non-covalent chaperone:client complexes (Fig. 3E). Of note, among the identified PDIs, our approach revealed well-characterized PDI family members (e.g., PDI, ERp57) to interact with IL-12α and/or IL-23α, but also less well-understood ones, including ERp72 and Sep15, the latter being a selenoprotein involved in protein quality control (
      • Yim S.H.
      • Everley R.A.
      • Schildberg F.A.
      • Lee S.G.
      • Orsi A.
      • Barbati Z.R.
      • Karatepe K.
      • Fomenko D.E.
      • Tsuji P.A.
      • Luo H.R.
      • Gygi S.P.
      • Sitia R.
      • Sharpe A.H.
      • Hatfield D.L.
      • Gladyshev V.N.
      Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.
      ). The overlapping but also partially distinct PDI family repertoire, which contained ill-characterized members, led us to investigate their binding preferences and their role in IL-12/IL-23 biogenesis in more detail.

      Cysteines in IL-12α and IL-23α are recognized differently by PDI family members

      Our photo-crosslinking MS approach and its validation by co-immunoprecipitation experiments revealed multiple PDI family members to interact with IL-12α and IL-23α. This raises the question if the identified PDI family members recognized the same or different cysteines within these clients in cells. To address this question, we generated a panel of IL-12α mutants. We replaced either cysteine 96, which forms an interchain disulfide bond with IL-12β within IL-12, or each pair of cysteines that form one of the three internal disulfide bonds in IL-12α by serines (Fig. 4A) (
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Yoon C.
      • Johnston S.C.
      • Tang J.
      • Stahl M.
      • Tobin J.F.
      • Somers W.S.
      Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
      ). Additionally, in one mutant all cysteines were replaced by serines. These mutants were used to analyze interactions with ERp72, ERp5 and ERp46, which all formed covalent complexes with IL-12α (Fig 3, C and E) and are thus suitable to assess their cysteine binding specificities. For the cysteine-free IL-12α mutant hardly any binding to the three queried PDIs was detectable (Fig. 4B). Since no crosslinkers but only NEM to avoid disulfide bond reshuffling was used in this experiment, this finding indicates absence of any stable chaperone-like interactions between IL-12α and ERp72, ERp5 or ERp46. In contrast, each of the cysteine mutants still bound to the PDIs, but with different effects on binding: ERp72 had a preference for the cysteines forming disulfide bond 1 and 3 within IL-12α, ERp5 preferred cysteines forming disulfide bonds 2 and 3 and ERp46 preferred the cysteines forming disulfide bond 2 (Fig. 4B). None of these three PDIs showed reduced binding upon mutation of the interchain disulfide bond-forming cysteine 96 (Fig. 4, A and B). For IL-23α, we analyzed interaction with ERp5 analogously. In this case, binding was only preserved if the cysteines forming the single disulfide bond in IL-23α were mutated to serines, indicating a preference of ERp5 for the three free cysteines in IL-23α (Fig. 4, C and D).
      Figure thumbnail gr4
      Figure 4PDI family members show specificity for distinct cysteines in IL-12α/IL-23α. A) Schematic of IL-12α. Cysteines are highlighted in red and numbered. Six of the seven cysteines in IL-12α form intramolecular disulfide bonds (denoted as SS1-3, disulfide bonds 1-3), and C96 engages with IL-12β to form an intermolecular disulfide bond. N-terminus (N) and C-terminus (C) are labeled. B) HEK293T cells were transiently transfected with the indicated IL-12α variants in which the indicated cysteines were replaced by serines. Co-IPs of ERp72, ERp5 and ERp46 were analyzed and quantified (n=4-10 ± SEM, *p < 0.05, two-way ANOVA with Dunnett test). C) Schematic of IL-23α. Cysteines are highlighted in red and numbered. C58 and C70 form an intramolecular disulfide bond, and C54 engages with IL-12β to form an intermolecular disulfide bond, and C14 and C22 remain unpaired in IL-23α. N-terminus (N) and C-terminus (C) are labeled. D) HEK293T cells were transiently transfected with the indicated IL-23α variants where the indicated cysteines were replaced by serines. Co-IP of ERp5 was analyzed and quantified (n=5-6 ± SEM , *p < 0.05, one-way ANOVA with Dunnett test).

      PDI family members stabilize unassembled cytokine subunits and improve cytokine secretion

      Our comprehensive mass spectrometric and biochemical analyses revealed several overlapping but also distinct ER PDI family members to interact with IL-12α and IL-23α, respectively. To analyze functional effects of these different PDI family members on IL-12 and IL-23 biogenesis, we performed siRNA-mediated knockdowns of the individual PDI family members. None of the knockdowns caused pronounced ER stress as measured by the activation of the unfolded protein response (supplemental Fig. S4A). We thus assessed effects on protein stability in cycloheximide (CHX) translational shut-off experiments, individually knocking down each PDI family member we had found to interact with IL-12α or IL-23α, respectively. Knockdown of any of the PDI family members interacting with IL-12α led to its faster degradation (Fig. 5A, B and D). For ERp46, however, the effect of knockdown on protein stability was only very weak. For others, e.g., ERp5, degradation was accelerated almost twofold (Fig. 5A, B and D). For IL-23α, we also tested a subset of the PDIs in similar experiments, including all those we found to interact with IL-23α (ERp46, ERp5 and ERp72; Fig. 3E) but also one that we did not find to strongly associate with this subunit (ERp57; Fig. 3E). Similar to what we had observed for IL-12α, knockdown of each of the interacting PDI family members accelerated IL-23α degradation. In contrast, knockdown of ERp57 did not accelerate IL-23α degradation (Fig. 5C and D).
      Figure thumbnail gr5
      Figure 5Effect of PDI knockdowns on IL-12α and IL-23α turnover rates. A-C) CHX chases were performed for the indicated periods of time upon specific depletion of the indicated individual PDIs by siRNA. The levels of IL-12α or IL-23α were determined from lysate immunoblots relative to the 0 h time point. Representative immunoblots and quantifications are shown (n=4-7 ± SEM). D) Summary of protein half-lives and corresponding statistical unpaired t-test analysis with Welch’s corrections for individual PDI knockdowns. Statistical analysis was performed after semi-log decay curve fitting to determine protein half-lives, *p < 0.05. A goodness-of-fit for the linear curve can be assessed based on the given R2 values.
      Based on these findings, we proceeded to analyze secretion levels of heterodimeric IL-12 or IL-23, respectively, under the same PDI knockdown conditions. In contrast to a more rapid degradation of isolated α subunits, no effect of single PDI knockdown on the secretion of the heterodimeric IL-12 or IL-23 was observed (Fig. 6A and B and supplemental Fig. S5A and B). To assess possible compensatory effects of individual PDI members, we thus simultaneously knocked down combinations of three individual PDI family members we had found to interact with IL-12α/IL-23α and assessed secretion of the heterodimeric ILs. Again, no significant ER stress induction was detectable (supplemental Fig. S4B). In this case, when three PDIs were knocked down simultaneously, although IL-23 remained unaffected, a significant decrease in IL-12 secretion by around 20% could be observed (Fig. 6C-E).
      Figure thumbnail gr6
      Figure 6Effect of PDI knockdowns on IL-12 and IL-23 cytokine secretion. A) Graph depicting IL-12α secretion levels upon co-expression of IL-12β, i.e.: secretion of the heterodimeric cytokine, in HEK293T cells treated with the respective PDI siRNAs relative to those in si ctrl (set to 1, dashed line); ns – not significant. B) The same as in A) for IL-23α. C-E) Similar analyses as in A and B, but with siRNA-mediated knockdown of the indicated combination of PDIs. Representative immunoblots on IL-12 secretion (C) and IL-23 secretion (D) are shown. In the quantifications, the dashed line corresponds to si ctrl sample treatment. *p-value < 0.05 for multiple knockdowns versus si ctrl (unpaired two-tailed Student’s t test). F) Model for the role of PDIs in the biogenesis of the pro-inflammatory cytokines IL-12 and IL-23. Each α subunit physically associates with overlapping but also distinct PDI family members (only ER-lumenal, soluble PDIs are shown). Knockdowns of individual interacting PDIs generally led to a faster degradation of the unfolded, unpaired α subunits via ERAD. However, no change in secretion levels was observed after assembly with the β-subunit. Combined siRNA knockdown, in contrast, leads to reduced secretion of heterodimeric IL-12 but not IL-23.

      Discussion

      IL-12 and IL-23 are key cytokines in the human immune systems and highly relevant molecules in the clinics (
      • Tait Wojno E.D.
      • Hunter C.A.
      • Stumhofer J.S.
      The Immunobiology of the Interleukin-12 Family: Room for Discovery.
      ). At the same time, they are demanding clients of the ER folding machinery. The human α subunits, IL-12α and IL-23α, are unfolded in isolation and depend on the shared β subunit IL-12β for structure formation and secretion of the bioactive heterodimeric cytokines (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Oppmann B.
      • Lesley R.
      • Blom B.
      • Timans J.C.
      • Xu Y.
      • Hunte B.
      • Vega F.
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      • Bazan J.F.
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      Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
      ,
      • Gubler U.
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      • McComas W.
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      • et al.
      Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
      ,
      • Wolf S.F.
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      • et al.
      Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.
      ,
      • Jalah R.
      • Rosati M.
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      • Pilkington G.R.
      • Valentin A.
      • Kulkarni V.
      • Bergamaschi C.
      • Chowdhury B.
      • Zhang G.M.
      • Beach R.K.
      • Alicea C.
      • Broderick K.E.
      • Sardesai N.Y.
      • Pavlakis G.N.
      • Felber B.K.
      The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit: IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION.
      ,
      • Hildenbrand K.
      • Aschenbrenner I.
      • Franke F.C.
      • Devergne O.
      • Feige M.J.
      Biogenesis and engineering of interleukin 12 family cytokines.
      ). Both α subunits contain several cysteine residues, five in the case of IL-23α and seven for IL-12α. In IL-12α, these form three intrachain and one interchain disulfide bond to IL-12β (
      • Yoon C.
      • Johnston S.C.
      • Tang J.
      • Stahl M.
      • Tobin J.F.
      • Somers W.S.
      Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
      ). In IL-23α, the five cysteines form one intrachain and one interchain disulfide bond, whereas two cysteines remain unpaired (
      • Lupardus P.J.
      • Garcia K.C.
      The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12.
      ). Correct disulfide bond formation is important for IL-12/IL-23 to be secreted (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ). Their unfolded nature prior to assembly and their complex oxidative folding render IL-12α and IL-23α highly dependent on the ER folding machinery. Using site-specific photo-crosslinking coupled to mass spectrometry, this study is the first comprehensive analysis of the chaperone repertoire that acts on the IL-12 and IL-23 cytokine α subunits, significantly extending previous studies (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Alloza I.
      • Martens E.
      • Hawthorne S.
      • Vandenbroeck K.
      Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates.
      ,
      • McLaughlin M.
      • Alloza I.
      • Quoc H.P.
      • Scott C.J.
      • Hirabayashi Y.
      • Vandenbroeck K.
      Inhibition of secretion of interleukin (IL)-12/IL-23 family cytokines by 4-trifluoromethyl-celecoxib is coupled to degradation via the endoplasmic reticulum stress protein HERP.
      ). Among the interacting chaperones, we find overlapping but also distinct PDI family members to engage IL-12α or IL-23α (Fig. 6F). Of note, our photo-crosslinking approach allows to identify interactions with potential PDI family members that do not covalently engage their clients, e.g., ERp29, and thus extends the repertoire of interactors that can be identified.
      We generally observe a stabilizing effect of PDI family members on the unassembled α subunits, similar to what e.g., had been observed for ERp57 and the prion protein (
      • Torres M.
      • Medinas D.B.
      • Matamala J.M.
      • Woehlbier U.
      • Cornejo V.H.
      • Solda T.
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      • Cartier L.
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      • Hetz C.
      The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein.
      ). A possible explanation is that PDI-binding protects the unfolded α subunits from premature ERAD, a process for which our MS analyses also provide relevant hits, including XTP3B (ERLEC) and OS9 for IL-12α (Fig. 2E and Supplemental Table S1). In contrast to the pronounced effects on isolated α subunits, no effects of individual PDI knockdowns on secretion of heterodimeric IL-12/IL-23 was observed when IL-12β was co-expressed (Fig. 6F). Although this may depend on relative expression levels of individual subunits, it argues that α:β assembly is a fast and efficient process; hence the role of the β subunit as a folding matrix may overcome the need for stabilizing unassembled α subunits for IL-12 and IL-23. For the more labile IL-35, that is not disulfide-linked (
      • Devergne O.
      • Birkenbach M.
      • Kieff E.
      Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.
      ), this may be different. Our findings are in agreement with the observation that although IL-12α and IL-23α misfold in isolation and form incorrect disulfide bonds, misfolding is not observed upon co-expression of IL-12β (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ). Even though our work was performed by transient transfections in non-immune cells and thus awaits further studies in endogenous producers, this raises the question of why such a complex network of chaperones caters for IL-12α and IL-23α if heterodimerization appears to be highly efficient. One explanation may be the ubiquitous expression of IL-12α (
      • Liu J.
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      • Jimenez V.
      • Ma X.
      Interleukin-12: an update on its immunological activities, signaling and regulation of gene expression.
      ) and its pairings with other subunits, e.g., EBI3 to form IL-35 (
      • Devergne O.
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      • Kieff E.
      Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin.
      ,
      • Collison L.W.
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      • Vignali K.M.
      • Cross R.
      • Sehy D.
      • Blumberg R.S.
      • Vignali D.A.
      The inhibitory cytokine IL-35 contributes to regulatory T-cell function.
      ) but possibly also autonomous functions of IL-12α as an anti-inflammatory molecule (
      • Dambuza I.M.
      • He C.
      • Choi J.K.
      • Yu C.R.
      • Wang R.
      • Mattapallil M.J.
      • Wingfield P.T.
      • Caspi R.R.
      • Egwuagu C.E.
      IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease.
      ). Another likely explanation is that immune cells must regulate IL-12 versus IL-23 assembly. Since IL-12 and IL-23 share the same β subunit, and some cells express all three proteins (see e.g., (
      • Dixon K.O.
      • van der Kooij S.W.
      • Vignali D.A.
      • van Kooten C.
      Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members.
      )), their biogenesis has to be chaperoned in the ER to allow for specific downstream immune responses. The large number of ER chaperones, and in particular ER PDI family members our study identifies, testifies to this notion. Our work also shows that combined depletion of several PDI family members can selectively reduce IL-12 secretion without affecting IL-23 secretion (Fig. 6F). It may be explained by the larger number of disulfide bonds IL-12α has to form in comparison to IL-23α (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ), and its dependency on different branches of the ER folding machinery, IL-12α being a N-glycoprotein which IL-23α is not (
      • Bohnacker S.
      • Hildenbrand K.
      • Aschenbrenner I.
      • Müller S.I.
      • Bieren J.E.
      • Feige M.J.
      Influence of glycosylation on IL-12 family cytokine biogenesis and function.
      ).
      In addition to insights into the chaperoning of immune signaling proteins, our study contributes to our understanding of the ER PDI family. A surprising finding we make is that most PDI family members seem to have a stabilizing effect on our two investigated client proteins, arguing against a possible mutual compensation in this function. This is in agreement with recent insights into different binding characteristics of PDI family members (
      • Hirayama C.
      • Machida K.
      • Noi K.
      • Murakawa T.
      • Okumura M.
      • Ogura T.
      • Imataka H.
      • Inaba K.
      Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation.
      ) and synergistic functions in protein folding (
      • Sato Y.
      • Kojima R.
      • Okumura M.
      • Hagiwara M.
      • Masui S.
      • Maegawa K.
      • Saiki M.
      • Horibe T.
      • Suzuki M.
      • Inaba K.
      Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.
      ). A possible explanation is that different folding states, each prone to ERAD, are recognized by the different PDIs or that binding to multiple PDIs shifts the competition between ERAD and stabilizing unfolded proteins toward the latter. This notion, that different PDI recognizes different features of their clients, is in agreement with our findings that mutating cysteines individually or pairwise within IL-12α or IL-23α differentially affects binding to ERp72, ERp5 and ERp46. Protein folding itself also modulates PDI-dependency (
      • Robinson P.J.
      • Kanemura S.
      • Cao X.
      • Bulleid N.J.
      Protein secondary structure determines the temporal relationship between folding and disulfide formation.
      ). The fact that IL-12α and IL-23α cannot fold to a native state autonomously may thus contribute to their strong PDI-dependency in isolation, where misfolding and mispairing of cysteines are to be chaperoned and multiple folding intermediates exist in cells (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ,
      • Reitberger S.
      • Haimerl P.
      • Aschenbrenner I.
      • Esser-von Bieren J.
      • Feige M.J.
      Assembly-induced folding regulates interleukin 12 biogenesis and secretion.
      ,
      • Robinson P.J.
      • Kanemura S.
      • Cao X.
      • Bulleid N.J.
      Protein secondary structure determines the temporal relationship between folding and disulfide formation.
      ). For IL-23α, it is noteworthy that ERp44, an ER-Golgi intermediate compartment (ERGIC) PDI, can recognize the same free cysteines in IL-23α which our study reveals to be bound by ERp5 – and which are close to a BiP binding site (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ). These free cysteines in IL-23α become buried upon folding (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ), together highlighting these as important molecular motifs of folding and assembly control for IL-23. Despite these insights, it should be noted that for several of the PDI family members we find to interact with IL-12α or IL-23α, functions yet remain to be determined. IL-12α or IL-23α may prove to be very valuable and medically relevant clients for this. One example is Sep15, an interactor of UGGT (
      • Korotkov K.V.
      • Kumaraswamy E.
      • Zhou Y.
      • Hatfield D.L.
      • Gladyshev V.N.
      Association between the 15-kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells.
      ), that has been described as a gatekeeper to maintain misfolded immune proteins in the ER (
      • Yim S.H.
      • Everley R.A.
      • Schildberg F.A.
      • Lee S.G.
      • Orsi A.
      • Barbati Z.R.
      • Karatepe K.
      • Fomenko D.E.
      • Tsuji P.A.
      • Luo H.R.
      • Gygi S.P.
      • Sitia R.
      • Sharpe A.H.
      • Hatfield D.L.
      • Gladyshev V.N.
      Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.
      ) but also has redox activity (
      • Ferguson A.D.
      • Labunskyy V.M.
      • Fomenko D.E.
      • Araç D.
      • Chelliah Y.
      • Amezcua C.A.
      • Rizo J.
      • Gladyshev V.N.
      • Deisenhofer J.
      NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family.
      ). Of note, our data show that IL-12α interacts with both, Sep15 and UGGT1/2, which qualifies it as an interesting client to further define the functions of Sep15.
      Taken together, our study reveals a complex network of PDI family members that act on the highly disulfide-bonded glycoprotein IL-12α. The less disulfide-bonded, non-glycosylated IL-23α interacts with significantly less PDI family members. The PDI family members recognize different cysteines in their clients and thus seem to act synergistically, not redundantly, when it comes to stabilizing the unassembled, incompletely folded cytokine subunits. Despite this, only when multiple PDIs are depleted is the secretion of heterodimeric IL-12, but not IL-23, selectively affected which may be relevant in the light of PDI inhibitors entering the clinic (
      • Hoffstrom B.G.
      • Kaplan A.
      • Letso R.
      • Schmid R.S.
      • Turmel G.J.
      • Lo D.C.
      • Stockwell B.R.
      Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins.
      ,
      • Vatolin S.
      • Phillips J.G.
      • Jha B.K.
      • Govindgari S.
      • Hu J.
      • Grabowski D.
      • Parker Y.
      • Lindner D.J.
      • Zhong F.
      • Distelhorst C.W.
      • Smith M.R.
      • Cotta C.
      • Xu Y.
      • Chilakala S.
      • Kuang R.R.
      • Tall S.
      • Reu F.J.
      Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.
      ,
      • Karatas E.
      • Raymond A.A.
      • Leon C.
      • Dupuy J.W.
      • Di-Tommaso S.
      • Senant N.
      • Collardeau-Frachon S.
      • Ruiz M.
      • Lachaux A.
      • Saltel F.
      • Bouchecareilh M.
      Hepatocyte proteomes reveal the role of protein disulfide isomerase 4 in alpha 1-antitrypsin deficiency.
      ).

      Experimental Procedures

      Cloning, DNA constructs and siRNA

      The piggybac (pPB) vector containing DiazKRS with mutations (M. mazei: Y306M, L309A, C348A) (
      • Nödling A.R.
      • Spear L.A.
      • Williams T.L.
      • Luk L.Y.P.
      • Tsai Y.H.
      Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells.
      ) has been described previously (
      • Bartoschek M.D.
      • Ugur E.
      • Nguyen T.A.
      • Rodschinka G.
      • Wierer M.
      • Lang K.
      • Bultmann S.
      Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.
      ). Amber suppression sites were inserted in IL-12α/23α constructs by site-directed mutagenesis PCR using Pfu (Promega) DNA polymerase in a pSVL vector backbone. ‘TAG’ replaced coding sequences were subcloned into the pPB vector as reported previously (
      • Mideksa Y.G.
      • Fottner M.
      • Braus S.
      • Weiß C.A.M.
      • Nguyen T.A.
      • Meier S.
      • Lang K.
      • Feige M.J.
      Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.
      ) in-frame downstream of the EF-1 promoter. Constructs equipped with a C-terminal FLAG tag were subcloned in a similar approach separated by four (IL-12α) or five (IL-23α) GS-linker repeats. Other plasmids used in this study were an IL-12β construct in the pcDNA3.1(+) vector (
      • Mideksa Y.G.
      • Fottner M.
      • Braus S.
      • Weiß C.A.M.
      • Nguyen T.A.
      • Meier S.
      • Lang K.
      • Feige M.J.
      Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.
      ) and immunoglobulin γ1 heavy chain in pSVL, a kind gift from Linda M. Hendershot, St. Jude Children’s Research Hospital. All constructs were verified by sequencing. Custom oligos (Sigma-Aldrich) were designed using the SnapGene tool fulfilling optimal parameters for PCR mutagenesis.
      Tabled 1
      List of siRNAs used in this study
      siRNAID/Cat. no.
      si negative control #14390843
      si Sep15/SEP15s17999
      si ERp18/TXNDC12s27323
      si ERp29/PDIA9AM16708
      si ERp46/TXNDC5s37649
      si ERp5/PDIA6s531609
      si ERp57/PDIA3s6227
      si ERp72/PDIA4s225165

      Mammalian cell culture

      Human embryonic kidney-293T (HEK293T) cells were sub-cultured every two days in Dulbecco’s modified Eagle medium (DMEM) containing L-Ala-L-Gln (AQ™, Sigma-Aldrich) supplemented with 10% v/v fetal bovine serum (FBS, Gibco) and 1% v/v antibiotic/antimycotic solution (25 μg/ml amphotenicin B, 10 mg/ml streptomycin and 10,000 units of penicillin; Sigma-Aldrich) under standard conditions (37°C and 5% CO2 in a humidified incubator). Cells were routinely tested by PCR for absence of mycoplasma contamination.

      Transient transfections

      Transient transfections were carried out using GeneCellin (BioCellChallenge) or Lipofectamine 3000 (ThermoFisher) according to the manufacturers’ instructions. Cells were grown in poly-D-Lysine coated 35mm dishes (Corning) to a confluency of 60-70%. 1 μg of IL-12α or IL-23α construct in combination with 1 μg of DiazKRS or 1 μg of the alpha-subunits alone were delivered to cells for expression tests and CHX chase experiments, respectively. For PDI co-immunoprecipitation (co-IP) (4 μg of IL-12α or IL-23α) or mass spectrometry analysis (1 μg of pPB IL-12α or IL-23α ‘TAG’-replaced constructs or pPB empty vector and 1 μg of DiazKRS), cells were seeded on poly-D-Lysine coated 60 mm (Corning). 2 μg of total DNA in a 1:2 ratio (α-subunit:IL-12β) in the presence of 0.5 μg DiazKRS, where indicated, was transfected for in cellulo secretion and assembly tests. Cells were lysed 24 - 48 h post-transfection.
      For siRNA-mediated knockdown experiments, 25 nM of each individual siRNA was added to cells using Lipofectamine’s RNAiMAX (ThermoFisher) protocol and incubated for another 24 h prior to DNA transfection. Combined knockdowns were achieved by adding three different siRNAs to a final concentration of 50 nM. siRNA stocks at 10 μM were prepared using nuclease-free water.

      Cell harvesting and immunoblotting

      Cells were washed in ice-cold phosphate-buffered saline (PBS, Sigma-Aldrich) and lysed in an appropriate amount of RIPA (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% NaDOC, 1× Roche complete protease inhibitor w/o EDTA) for 20 - 30 min on ice. For UPR activation tests, cell lysis was performed using either Triton lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× Roche complete protease inhibitor w/o EDTA, 1× SERVA phosphatase inhibitor mix) or NP-40 lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% NaDOC, 0.5% NP-40 substitute, 1× Roche complete protease inhibitor w/o EDTA, 1× SERVA phosphatase inhibitor mix). 20 mM N-ethylmaleimide (NEM, Sigma-Aldrich) was added to lysis steps where indicated. Cell debris was pelleted by centrifugation at 20,000 × g at 4°C for 15 min. Whole cell lysate/input or immunoprecipitated samples were supplemented with Laemmli containing either 10% β-mercaptoethanol (β-Me, reducing) or 100 mM NEM (non-reducing) and heated to 95°C for 5 min or, in the case of ATF6 sample processing, to 37°C for 30 min. Proteins were then separated using SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad) by wet-electroblotting (overnight at 30 V and 4°C). Thereafter, membranes were blocked for at least 3 h (at room temperature) with MTBST (25 mM Tris/HCl pH 7.5, 150 mM NaCl, 5% skim milk powder, 0.1% Tween) or 5% w/v bovine serum albumin (BSA) in TBST under constant agitation. Proteins of interest were detected using anti-IL-12α (Abcam ab133751, 1:500/1:1000 in MTBST), anti-IL-23α (Biolegend #511202, 1:500 in MTBST), anti-IL-12β (Abcam ab133752, 1:500 in MTBST), anti-Hsc70 (Santa Cruz sc-7298, 1:1000 in MTBST), anti-Sep15 (Abcam ab124840, 1:200 in MTBST), anti-ERp18 (Abcam ab134938, 1:500 in MTBST), anti-ERp29 (Abcam ab11420, 1:1000 in MTBST), anti-ERp46 (Proteintech 19834-1-AP, 1:1000 in MTBST), anti-PDIA6 (Proteintech 18233-1-AP, 1:1000 in MTBST), anti-ERp57 (Abcam ab13506, 1:1000 in MTBST), anti-CALR (Abcam, 1:1000 in MTBST), anti-ERp72 (Proteintech 14712-1-AP, 1:1000 in MTBST), anti-ATF6 (Abcam ab122897, 1:500 in MTBST), anti-eIF2α (Cell Signaling #9722, 1:1000 in BSA), anti-phospho-eIF2α (Cell Signaling #9721, 1:500 in BSA), anti-BiP (Cell Signaling #3177, 1:500 in MTBST). Membranes were next probed with species-specific secondary antibodies coupled to HRP: goat anti-mouse IgG (Santa Cruz sc-2031), mouse-IgGκ BP (Santa Cruz sc-516102), or goat anti-rabbit IgG (Santa Cruz sc-2054/sc-2357). Bands were detected by enhanced chemiluminescence (ECL prime, Amersham) on a Fusion Pulse 6 imager (Vilber Lourmat).

      Incorporation of DiazK

      Where specified, N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)-L-lysine (DiazK, (
      • Bartoschek M.D.
      • Ugur E.
      • Nguyen T.A.
      • Rodschinka G.
      • Wierer M.
      • Lang K.
      • Bultmann S.
      Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.
      )) was added to the complete DMEM at a concentration of 0.25 mM (mass spectrometry experiments) or 1 mM during DNA transfections. A 100 mM DiazK stock solution was prepared by dissolving powder form of DiazK in 100 mM TFA/H2O, sterile filtered and stored at -20°C. Before incubations, an equivalent amount of NaOH was added to the cell culture medium to neutralize pH.

      Determination of protein removal rates

      Translational arrest (chase) was carried out 24 - 48 h post-gene transfection with 50 μg/ml cycloheximide (Sigma-Aldrich) added to cells for the indicated time points. Linear regression fittings on semi-log curves were used to calculate protein half-lives using plots of protein abundance over time (0 h set to 100%).

      In situ photo-/chemical crosslinking, pull-down and co-immunoprecipitation (co-IP) workflows

      Cells expressing the desired constructs for DiazK incorporation were washed twice with PBS and subjected to a broad-emitting UV lamp (Vilber VL-215.L; 2× 15W, 365 nm) for 30 min in PBS. During this procedure, culture plates were placed on ice under cardboard covers with occasional swirling. Reactions without irradiation served as negative controls. Thiol-cleavable DSP (Dithiobis(succinimidyl propionate), ThermoFisher) crosslinks were also performed in intact cells. A 25 mM stock solution was prepared by reconstituting 1 mg of desiccated DSP in 100 μl dry DMSO. In brief, cells were first washed in PBS and then in crosslinking buffer (25 mM HEPES-KOH, pH 8.3, 125 mM KCl) on ice before incubation with 0.8 mM DSP in the same buffer for 1 h 20 min and quenched using 100 mM Glycine for 20 min on ice checking for even dispersion. Cell lysis was performed as described above.
      To study interactors of IL-12α/23α via enrichment of cell lysates, purification from photo-/chemically crosslinked samples was performed using anti-FLAG affinity gel (Sigma). The same amount of isotype control slurry (mouse IgG-Agarose, Sigma) was used to discriminate positive hits from unspecific binding to antibody and beads. Alternatively, ATF6 samples were initially precleared for 30 min and pulled-down overnight using 2 μg of antibody followed by immobilization on protein A/G agarose beads (ThermoFisher) for 1 h at 4°C, while rotating and eluted with 2× Laemmli with 10% v/v β-Me after washing three times with NP-40 wash buffer (50 mM Tris/HCl, pH 7.5, 400 mM NaCl, 0.5% NaDOC, 0.5% NP-40 substitute) and centrifugation in each round (7000 × g, 1 min at 4°C). Of note, in covalent complex (non-reducing IP) SDS-PAGE, 10% v/v NEM was added instead of β-Me. Beads were washed twice with RIPA buffer and three times with PBS in the case of MS measurements.

      Sample preparation for MS

      After enrichment, proteins were reduced and digested on-beads in 25 μl 50 mM Tris-HCl, pH 8.0 containing 5 ng/μl sequencing grade trypsin (Promega), 2 M urea and 1 mM DTT for 30 min at 25°C and with shaking at 600 rpm. Next, 100 μl 50 mM Tris-HCl, pH 8.0 containing 2 M urea and alkylating 5 mM iodoacetamide were added (30 min incubation at 25°C under shaking at 600 rpm). Digestion took place overnight at 37°C with shaking 600 rpm. The following day, digestion was stopped by addition of formic acid (FA, 0.5% v/v final amount). The beads were pelleted and the supernatant was desalted using double layer C18-stage tips (Agilent Technologies, Empore disk-C18, 47 mm) equilibrated with 70 μl methanol and aqueous 0.5% FA v/v (3×). Samples were loaded and washed three times with 70 μl aqueous 0.5% v/v FA and eluted three times with 30 μl 80% v/v acetonitrile (ACN), 20% v/v H2O and 0.5% v/v FA. The eluate was lyophilized in vacuo, resuspended in 25 μl aqueous 1% v/v FA, pipetted up and down, vortexed and sonicated for 15 min. Finally, the peptide solution was passed through a PVDF filter (Millipore, 0.22 μm pore size).

      MS Analysis

      Three replicates of photo-crosslink/co-IP samples on mutants and wt IL-12α/23α as well as controls transfected with empty vectors were analyzed via LC-MS/MS using an UltiMate 3000 nano HPLC system (ThermoFisher) equipped with an Acclaim C18 PepMap100 75 μm ID x 2 cm trap (ThermoFisher) and an Aurora C18 separation column (75 μm ID x 25 cm, 1.6 μm; Ionoptics) coupled to a CaptiveSpray source equipped TimsTOF Pro mass spectrometer (Bruker). Samples were loaded onto the trap column at a flow rate of 5 μl/min with aqueous 0.1% TFA and then transferred onto the separation column at 0.4 μl/min. Buffers for the nano-chromatography pump were aqueous 0.1% FA (buffer A) and 0.1% FA in ACN (buffer B). The gradient length on the TimsTOF Pro was 73 mins, while acetonitrile in 0.1 % FA was step wise increased from 5 to 28% in 60 mins and from 28 to 40% in 13 mins, followed by a washing and equilibration step of the column. The TimsTOF Pro was operated in PASEF mode. Mass spectra for MS and MS/MS scans were recorded between 100 and 1700 m/z. Ion mobility resolution was set to 0.85–1.40 V·s/cm over a ramp time of 100 ms. Data-dependent acquisition was performed using 10 PASEF MS/MS scans per cycle with a near 100% duty cycle. A polygon filter was applied in the m/z and ion mobility space to exclude low m/z, singly charged ions from PASEF precursor selection. An active exclusion time of 0.4 min was applied to precursors that reached 20,000 intensity units. Collisional energy was ramped stepwise as a function of ion mobility (
      • Meier F.
      • Brunner A.D.
      • Koch S.
      • Koch H.
      • Lubeck M.
      • Krause M.
      • Goedecke N.
      • Decker J.
      • Kosinski T.
      • Park M.A.
      • Bache N.
      • Hoerning O.
      • Cox J.
      • Räther O.
      • Mann M.
      Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer.
      ). The acquisition of all MS spectra on the TimsTOF instrument was performed with the Compass HyStar software version 6.0 (Bruker).

      Unfolded protein response (UPR) activation tests

      To observe possible effect of PDI siRNAs on ER stress, cells transfected with each siRNA were checked for upregulation of intracellular BiP levels, phosphorylation of eIF2α (PERK branch) and ATF6 N-terminal cleavage (ATF6 branch) using immunoblots. HEK293T cells incubated with 10 mM DTT (Sigma, 1 h) or 5 μg/ml Tunicamycin (Sigma, 4-6 h) before cell lysis served as positive controls.

      Software and statistical analyses

      IL-12 and IL-23 structures were modeled in silico with YASARA Structure (
      • Land H.
      • Humble M.S.
      YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations.
      ) for missing loops and energy minimized (steepest descent). Sites for replacement to amber codons were selected on the basis of residue solvent accessibility (PDBePISA server (
      • Krissinel E.
      • Henrick K.
      Inference of macromolecular assemblies from crystalline state.
      )), mutation stability prediction (SDM (
      • Pandurangan A.P.
      • Ochoa-Montano B.
      • Ascher D.B.
      • Blundell T.L.
      SDM: a server for predicting effects of mutations on protein stability.
      ) and DynaMut servers (
      • Rodrigues C.H.
      • Pires D.E.
      • Ascher D.B.
      DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability.
      )) and interfaces with the IL-12β subunit and/or IL-23 receptor (
      • Yoon C.
      • Johnston S.C.
      • Tang J.
      • Stahl M.
      • Tobin J.F.
      • Somers W.S.
      Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12.
      ,
      • Lupardus P.J.
      • Garcia K.C.
      The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12.
      ,
      • Bloch Y.
      • Bouchareychas L.
      • Merceron R.
      • Skladanowska K.
      • Van den Bossche L.
      • Detry S.
      • Govindarajan S.
      • Elewaut D.
      • Haerynck F.
      • Dullaers M.
      • Adamopoulos I.E.
      • Savvides S.N.
      Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rbeta1.
      ). Other known experimental constraints like secondary structure flexibility/lesions/chaperone binding sites (
      • Meier S.
      • Bohnacker S.
      • Klose C.J.
      • Lopez A.
      • Choe C.A.
      • Schmid P.W.N.
      • Bloemeke N.
      • Ruhrnossl F.
      • Haslbeck M.
      • Bieren J.E.
      • Sattler M.
      • Huang P.S.
      • Feige M.J.
      The molecular basis of chaperone-mediated interleukin 23 assembly control.
      ) were also taken into account. Available crystal structural data (PDB codes: 3HMX, 1F45, 3DUH, 5MXA and 5MZV) were inputs for the aforementioned analyses and visualized with PyMOL (www.pymol.org). Western blot raw images were processed for brightness and contrast in ImageJ (
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • Kaynig V.
      • Longair M.
      • Pietzsch T.
      • Preibisch S.
      • Rueden C.
      • Saalfeld S.
      • Schmid B.
      • Tinevez J.Y.
      • White D.J.
      • Hartenstein V.
      • Eliceiri K.
      • Tomancak P.
      • Cardona A.
      Fiji: an open-source platform for biological-image analysis.
      ) or Adobe Photoshop. Chemiluminescence band intensity quantifications were performed using the Bio-1D (Vilber Lourmat) software. For normalization of PDI co-IP, IP signals were background subtracted if unspecific signals were detected for empty vector controls. IP signals of wildtype were set to 1 and chaperone IP was divided by respective FLAG signals, thus amount of interleukin subunit, IP to obtain normalized PDI co-IP ratios. Statistical analyses and graph fittings were performed with Prism 7 (GraphPad) software as stated in the figure legends.

      Statistical Analyses of MS Data

      MS raw files were analyzed with MaxQuant software (version 2.1.0.0) and the default settings for TimsTOF files were applied except that the TOF MS/MS match tolerance was set to 0.05 Da. Searches were performed with the Andromeda search engine embedded in the MaxQuant environment against the UniProt human protein database (taxon identifier: 9606; downloaded September 2021, number of entries: 20371). The following parameter settings were used: PSM and protein FDR 1%; enzyme specificity trypsin/P; minimal peptide length: 7; variable modifications: methionine oxidation, N-terminal acetylation; fixed modification: carbamidomethylation. The minimal number of unique peptides for protein identification was set to 2. For label-free protein quantification, the MaxLFQ algorithm was used as part of the MaxQuant environment: (LFQ) minimum ratio count: 2; peptides for quantification: unique. Resulting data were further analyzed using Perseus software version 1.6.15.0 (
      • Tyanova S.
      • Temu T.
      • Sinitcyn P.
      • Carlson A.
      • Hein M.Y.
      • Geiger T.
      • Mann M.
      • Cox J.
      The Perseus computational platform for comprehensive analysis of (prote)omics data.
      ). The rows were filtered (only identified by site, potential contaminant, reverse) and LFQ intensities log2 transformed. Replicates (n=3) were grouped, filtered for at least two valid values in at least one group and missing values were imputed for total matrix using default settings. A both sided, two-sample Student’s t-test was performed and derived p-values were corrected for multiple testing by the method of Benjamini and Hochberg with a significance level of p = 0.05. Volcano plots were generated by plotting log2 (fold change) against -log10 (p-value). ER-chaperones were detected searching for GO terms cellular compartment = endoplasmic reticulum (ER), biological process = protein folding (GO numbers: 0006457, 0071712, 0006986, 0030433, 0034975 and 0061077) and molecular function = protein-disulfide isomerase activity with the help of the GO annotation file for Homo sapiens downloaded from Uniprot August 2022 (
      • Ashburner M.
      • Ball C.A.
      • Blake J.A.
      • Botstein D.
      • Butler H.
      • Cherry J.M.
      • Davis A.P.
      • Dolinski K.
      • Dwight S.S.
      • Eppig J.T.
      • Harris M.A.
      • Hill D.P.
      • Issel-Tarver L.
      • Kasarskis A.
      • Lewis S.
      • Matese J.C.
      • Richardson J.E.
      • Ringwald M.
      • Rubin G.M.
      • Sherlock G.
      Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.
      ). Additionally, all ER proteins were manually scrutinized for possible PDI family members that have not been annotated as such with the suitable GO terms. This further added ERp18, Sep15 and TMX1 to the list.

      Data availability

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Vizcaíno J.A.
      • Csordas A.
      • del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) partner repository with the dataset identifier (will be added in the proof stage).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

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