Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation
2019; Springer Nature; Volume: 11; Issue: 6 Linguagem: Inglês
10.15252/emmm.201810120
ISSN1757-4684
AutoresMarion Maurel, Joanna Obacz, Tony Avril, Yong‐Ping Ding, Olga Papadodima, Xavier Tréton, Fanny Daniel, Eleftherios Pilalis, Johanna Hörberg, Wenyang Hou, Marie‐Claude Beauchamp, Julien Tourneur‐Marsille, Dominique Cazals‐Hatem, Lucia Sommerová, Afshin Samali, Jan Tavernier, Roman Hrstka, Aurélien Dupont, Delphine Fessart, Frédéric Delom, Martín E. Fernández-Zapico, Gregor Jansen, Leif A. Eriksson, David Y. Thomas, Loydie A. Jerome‐Majewska, Ted R. Hupp, Aristotelis Chatziioannou, Éric Chevet, Éric Ogier‐Denis,
Tópico(s)Pancreatic function and diabetes
ResumoResearch Article30 April 2019Open Access Transparent process Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation Marion Maurel Marion Maurel INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France VIB Department of Medical Protein Research, UGent, Gent, Belgium Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Joanna Obacz Joanna Obacz INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Tony Avril Tony Avril INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Yong-Ping Ding Yong-Ping Ding INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Olga Papadodima Olga Papadodima Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Search for more papers by this author Xavier Treton Xavier Treton INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Fanny Daniel Fanny Daniel INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Eleftherios Pilalis Eleftherios Pilalis Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece International Centre for Cancer Vaccine Science, Gdansk, Poland Search for more papers by this author Johanna Hörberg Johanna Hörberg Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author Wenyang Hou Wenyang Hou Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Marie-Claude Beauchamp Marie-Claude Beauchamp Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Julien Tourneur-Marsille Julien Tourneur-Marsille INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Dominique Cazals-Hatem Dominique Cazals-Hatem INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Lucia Sommerova Lucia Sommerova Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Search for more papers by this author Afshin Samali Afshin Samali orcid.org/0000-0002-8610-8375 Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Jan Tavernier Jan Tavernier VIB Department of Medical Protein Research, UGent, Gent, Belgium Search for more papers by this author Roman Hrstka Roman Hrstka Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Search for more papers by this author Aurélien Dupont Aurélien Dupont Microscopy Rennes Imaging Centre, and Biosit, UMS3480 CNRS, University of Rennes 1, Rennes Cédex, France Search for more papers by this author Delphine Fessart Delphine Fessart University of Bordeaux, Bordeaux, France Search for more papers by this author Frédéric Delom Frédéric Delom University of Bordeaux, Bordeaux, France Search for more papers by this author Martin E Fernandez-Zapico Martin E Fernandez-Zapico Division of Oncology Research, Department of Oncology, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Gregor Jansen Gregor Jansen orcid.org/0000-0002-0289-0022 Biochemistry Department, McGill University Life Sciences Complex, Montréal, QC, Canada Search for more papers by this author Leif A Eriksson Leif A Eriksson Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author David Y Thomas David Y Thomas Biochemistry Department, McGill University Life Sciences Complex, Montréal, QC, Canada Search for more papers by this author Loydie Jerome-Majewska Loydie Jerome-Majewska Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Ted Hupp Ted Hupp International Centre for Cancer Vaccine Science, Gdansk, Poland Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edimburgh, UK Search for more papers by this author Aristotelis Chatziioannou Corresponding Author Aristotelis Chatziioannou [email protected] orcid.org/0000-0003-2078-0844 Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Eric Ogier-Denis Corresponding Author Eric Ogier-Denis [email protected] orcid.org/0000-0002-0057-7593 INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Marion Maurel Marion Maurel INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France VIB Department of Medical Protein Research, UGent, Gent, Belgium Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Joanna Obacz Joanna Obacz INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Tony Avril Tony Avril INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Yong-Ping Ding Yong-Ping Ding INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Olga Papadodima Olga Papadodima Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece Search for more papers by this author Xavier Treton Xavier Treton INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Fanny Daniel Fanny Daniel INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Eleftherios Pilalis Eleftherios Pilalis Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece International Centre for Cancer Vaccine Science, Gdansk, Poland Search for more papers by this author Johanna Hörberg Johanna Hörberg Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author Wenyang Hou Wenyang Hou Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Marie-Claude Beauchamp Marie-Claude Beauchamp Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Julien Tourneur-Marsille Julien Tourneur-Marsille INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Dominique Cazals-Hatem Dominique Cazals-Hatem INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Lucia Sommerova Lucia Sommerova Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Search for more papers by this author Afshin Samali Afshin Samali orcid.org/0000-0002-8610-8375 Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland Search for more papers by this author Jan Tavernier Jan Tavernier VIB Department of Medical Protein Research, UGent, Gent, Belgium Search for more papers by this author Roman Hrstka Roman Hrstka Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Search for more papers by this author Aurélien Dupont Aurélien Dupont Microscopy Rennes Imaging Centre, and Biosit, UMS3480 CNRS, University of Rennes 1, Rennes Cédex, France Search for more papers by this author Delphine Fessart Delphine Fessart University of Bordeaux, Bordeaux, France Search for more papers by this author Frédéric Delom Frédéric Delom University of Bordeaux, Bordeaux, France Search for more papers by this author Martin E Fernandez-Zapico Martin E Fernandez-Zapico Division of Oncology Research, Department of Oncology, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Gregor Jansen Gregor Jansen orcid.org/0000-0002-0289-0022 Biochemistry Department, McGill University Life Sciences Complex, Montréal, QC, Canada Search for more papers by this author Leif A Eriksson Leif A Eriksson Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden Search for more papers by this author David Y Thomas David Y Thomas Biochemistry Department, McGill University Life Sciences Complex, Montréal, QC, Canada Search for more papers by this author Loydie Jerome-Majewska Loydie Jerome-Majewska Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada Search for more papers by this author Ted Hupp Ted Hupp International Centre for Cancer Vaccine Science, Gdansk, Poland Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edimburgh, UK Search for more papers by this author Aristotelis Chatziioannou Corresponding Author Aristotelis Chatziioannou [email protected] orcid.org/0000-0003-2078-0844 Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece e-NIOS PC, Kallithea-Athens, Greece Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet [email protected] orcid.org/0000-0001-5855-4522 INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Eric Ogier-Denis Corresponding Author Eric Ogier-Denis [email protected] orcid.org/0000-0002-0057-7593 INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France Université Paris-Diderot Sorbonne Paris-Cité, Paris, France APHP Beaujon Hospital Clichy la Garenne, Paris, France Search for more papers by this author Author Information Marion Maurel1,2,3,4,‡, Joanna Obacz1,2,‡, Tony Avril1,2,‡, Yong-Ping Ding5,6,7, Olga Papadodima8, Xavier Treton5,6,7, Fanny Daniel5,6,7, Eleftherios Pilalis8,9, Johanna Hörberg10, Wenyang Hou11, Marie-Claude Beauchamp11, Julien Tourneur-Marsille5,6,7, Dominique Cazals-Hatem5,6,7, Lucia Sommerova12, Afshin Samali4, Jan Tavernier3, Roman Hrstka12, Aurélien Dupont13, Delphine Fessart14, Frédéric Delom14, Martin E Fernandez-Zapico15, Gregor Jansen16, Leif A Eriksson10, David Y Thomas16, Loydie Jerome-Majewska11, Ted Hupp9,12,17, Aristotelis Chatziioannou *,8,18, Eric Chevet *,1,2 and Eric Ogier-Denis *,5,6,7 1INSERM U1242, “Chemistry, Oncogenesis Stress Signaling”, University of Rennes, Rennes, France 2Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France 3VIB Department of Medical Protein Research, UGent, Gent, Belgium 4Apoptosis Research Centre, School of Natural Sciences, NUI Galway, Galway, Ireland 5INSERM, UMR1149, Team «Gut Inflammation», Research Centre of Inflammation, Paris, France 6Université Paris-Diderot Sorbonne Paris-Cité, Paris, France 7APHP Beaujon Hospital Clichy la Garenne, Paris, France 8Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, Athens, Greece 9International Centre for Cancer Vaccine Science, Gdansk, Poland 10Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, Sweden 11Departments of Anatomy and Cell Biology, Human Genetics, and Pediatrics, McGill University, Montreal, QC, Canada 12Regional Centre for Applied Molecular Oncology (RECAMO), Brno, Czech Republic 13Microscopy Rennes Imaging Centre, and Biosit, UMS3480 CNRS, University of Rennes 1, Rennes Cédex, France 14University of Bordeaux, Bordeaux, France 15Division of Oncology Research, Department of Oncology, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA 16Biochemistry Department, McGill University Life Sciences Complex, Montréal, QC, Canada 17Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine, Edinburgh University, Edimburgh, UK 18e-NIOS PC, Kallithea-Athens, Greece ‡These authors contributed equally to this work *Corresponding author. Tel: +30 2107273751; E-mail: [email protected] *Corresponding author. Tel: +33 223237258; E-mail: [email protected] *Corresponding author. Tel: +33 157277307; E-mail: eric.[email protected] EMBO Mol Med (2019)11:e10120https://doi.org/10.15252/emmm.201810120 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein–protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses. Synopsis This study provides molecular insights into the anterior gradient 2 (AGR2) mode of action in the endoplasmic reticulum (ER) with an emphasis on the regulation of AGR2 dimers. Dysregulation of AGR2 dimer formation leads to inflammation as seen in inflammatory bowel disease (IBD) and Crohn's disease. AGR2 is a chaperone involved in the regulation of protein quality control in the ER. We have developed and used a novel cellular protein-protein interaction screen to identify cellular regulators of AGR2 dimerization. We identify specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. Modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. In IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses. Introduction The regulation of protein homeostasis (proteostasis) in the endoplasmic reticulum (ER) has recently emerged as an important pathophysiological mechanism involved in the development of different diseases (Hetz et al, 2015). The capacity of the ER to cope with the protein misfolding burden is controlled by the kinetics and thermodynamics of folding and misfolding (also called proteostasis boundary), which are themselves linked to the ER proteostasis network capacity (Powers et al, 2009). The ER ensures proper folding of newly synthesized proteins through the coordinated action of ER-resident molecular chaperones, folding catalysts, quality control, and degradation mechanisms. Anterior gradient 2 (AGR2), a folding catalyst, binds to nascent protein chains, and it is required for the maintenance of ER homeostasis (Persson et al, 2005; Higa et al, 2011; Chevet et al, 2013). Loss of AGR2 has been associated with intestinal inflammation (Park et al, 2009; Zhao et al, 2010), and several studies have demonstrated that unresolved ER stress leads to spontaneous intestinal inflammation (Kaser et al, 2013). Although anterior gradient proteins, including AGR2, were identified more than a decade ago, their precise biological functions remain ill-defined. AGR2 was first defined as an ER-resident foldase (Chevet et al, 2013) and was also shown to exhibit extracellular activities (Fessart et al, 2016), and all of these functions most likely depend on protein–protein interactions. Previously, a few AGR2 interacting partners have been identified (Maslon et al, 2010; Yu et al, 2013) and fewer have been validated as genuine AGR2 binding partners in the ER. Moreover, biochemical approaches have shown that AGR2 forms dimers (Ryu et al, 2012; Patel et al, 2013). As such, this justifies an in-depth study to characterize the protein–protein interaction-dependent regulatory mechanisms controlling AGR2 dimerization and functions. In mammals, AGR2 is generally present in mucus secreting epithelial cells and is highly expressed in Paneth and goblet intestinal cells, with the highest levels in the ileum and colon (Komiya et al, 1999; Chang et al, 2008a,b). In goblet cells, AGR2 forms mixed disulfide bonds with Mucin 2 (MUC2), allowing for its correct folding and secretion (Park et al, 2009; Zhao et al, 2010). MUC2 is an essential component of the gastrointestinal mucus covering the epithelial surface of gastrointestinal tract to confer the first line of defense against commensal bacteria. Knockout of AGR2 inhibits MUC2 secretion by intestinal cells, thereby decreasing the amount of intestinal mucus and leading to a spontaneous granulomatous ileocolitis, closely resembling human inflammatory bowel disease (IBD; Zhao et al, 2010). Accordingly, decreased AGR2 expression and some of AGR2 variants were identified as risk factors in IBD (Zheng et al, 2006). However, despite the strong link between AGR2 and the etiology of IBD, the molecular mechanism by which AGR2 regulates its activity and contributes to the development of IBD still remains elusive. To unveil the molecular functions of AGR2 and its pathophysiological roles, we have developed the ER Mammalian protein–protein Interaction Trap (ERMIT), which allows us to specifically detect AGR2 protein–protein interactions in the ER. We found that upon ER proteostasis alteration AGR2 dimer was disrupted and a siRNA screen using ERMIT identified TMED2 as a major regulator of AGR2 dimerization. Moreover, we demonstrated that variations in TMED2 expression resulted in AGR2 secretion and pro-inflammatory phenotypes in vitro, in mouse models and in patients’ samples. Hence, we propose that ER proteostasis-mediated control of AGR2 dimerization, which might depend on TMED2, promotes AGR2 release in the extracellular environment thereby enhancing monocyte recruitment and pro-inflammatory phenotypes. Results AGR2 forms stress-regulated homodimer in the endoplasmic reticulum Structural studies showed that AGR2 forms dimers through residues E60 (Fig 1A) and C81 (Ryu et al, 2012; Patel et al, 2013), respectively. Results involving E60 in AGR2 dimerization were confirmed using molecular dynamics (Fig 1B). The dimeric versus monomeric equilibrium of AGR2 was also investigated using molecular modeling approaches. Indeed, the reduced dimer stability of the E60A mutant was verified by performing 200 ns molecular dynamics simulations of wild-type and mutant dimers (Appendix Fig S1A–F). E60 of each monomer stabilizes the dimer by forming salt bridges to K64 of the other monomer. The WT system remains stable throughout the simulation, whereas the E60A mutant form rapidly dissociates, as identified in increased RMSD, radius of gyration, and distance measurements, concomitant with loss of interaction energy. These results indicate that AGR2 might exist under both monomeric and homodimeric forms. Figure 1. AGR2 dimerization with ERMIT assay, principles, and validation of the methodThe ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. A. Upper panel: NMR structure of the non-covalent dimer of AGR2 (PDB ID: 2LNS). The dimer domain is highlighted with a yellow circle (residue 54–70 of each monomer). Monomer A is colored green and monomer B in pink. The figure was generated in Chimera. Lower panel: a close-up illustration of the dimer domain, showing that the dimer is stabilized through two salt bridges between E60 and K64 of each monomer. B. Molecular modeling showing root-mean-square deviation (RMSD) plot for the MD simulation of the AGR2 WT and E60A mutant. The interaction energies are defined as the sum of the short-range Coulomb interactions and the short-range Lennard-Jones potential between monomer A and B. C, D. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS–PAGE and immunoblotted using anti-AGR2, anti-ERK1, or anti-calnexin (CANX) antibodies (for loading control). E. Principles of the ERMIT assay. A wild-type IRE1 bait is used to report for dimerization, whereas a kinase catalytic mutant (K599A) is used as a control to prove that signal observed with the wild-type form is due to IRE1 activation. F. AGR2 dimerization was monitored with ERMIT [wild-type (WT)]. The luminescence signal was abrogated when using the kinase dead (KD) constructs and by the constructs exhibiting mutations in the dimerization domain [E60A or C81S or double mutant (DM)]. The graph represents average signal normalized on reporter protein expression ± SD [n = 5; **: WT/KD (P = 0.002), E60/WT (P = 0.0034), E60/KD (P = 0.001), C81/KD (P = 0.004), DM/WT (P = 0.0021), and DM/KD (P = 0.0017), respectively; *: C81/WT (P = 0.0098)]. The Mann–Whitney statistical test was used. G. Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4). The graph represents average signal normalized on reporter protein expression ±SD. Download figure Download PowerPoint To validate the dimerization of AGR2 in our cellular models, cells were transfected with a previously validated siRNA against AGR2 (Higa et al, 2011) and its corresponding control siRNA. Cells were then treated with the chemical cross-linker DSP. Cross-linked proteins were resolved on either non-reducing (top blot; Fig 1C) or reducing (middle blot; Fig 1C) conditions and analyzed by Western blot. Data included in Fig 1C revealed that AGR2 exists predominantly as homodimers. Since AGR2 is also involved in protein quality control in the ER (Higa et al, 2011), we evaluated the impact of ER homeostasis disruption on AGR2 dimerization. DSP-mediated protein cross-linking of tunicamycin-treated cells revealed that AGR2 homodimers disappeared upon ER stress induced by tunicamycin, whereas total AGR2 expression levels did not change significantly (Fig 1D). To further dissect the mechanisms by which AGR2 dimerizes, we developed the ERMIT assay (Fig 1E). ERMIT is a mammalian two-hybrid method, adapted from the existing ER-MYTH yeast assay (Jansen et al, 2012) and based on the functional complementation of the IRE1 signaling pathway. IRE1 is normally maintained in an inactive state by its association with the molecular chaperone BiP. Upon accumulation of misfolded proteins in the ER, initiating ER stress, IRE1 competes with those proteins for binding to BiP. When activated, IRE1 cleaves XBP1 mRNA at two consensus sites to initiate an unconventional splicing reaction. This spliced mRNA leads to the generation of a functional XBP1 transcription factor (Hetz et al, 2015). In the ERMIT assay, the luminal domain of IRE1 was replaced by different bait proteins (Fig 1E), and independently of ER stress, bait and prey interactions lead to IRE1 activation and subsequent XBP1 splicing. This splicing is monitored by a XBP1 splicing luciferase reporter system (Hetz et al, 2015). To determine whether AGR2 dimerizes in the ER, we replaced the luminal domain of IRE1 with AGR2 wild-type (WT), or two AGR2 dimerization inactive mutants [E60A, C81A, or the E60A/C81A double mutant (DM)]. The transmembrane and WT or kinase dead (KD) cytosolic domains of IRE1 were used as positive controls. These AGR2-IRE1 chimeric constructs were transfected into HEK293T cells, and their expression and localization to the ER were verified by Western blot (Appendix Fig S1G) and immunofluorescence microscopy (Appendix Fig S1H). ERMIT signals produced by HEK293T cells transfected with the different AGR2 baits were then quantified (Fig 1F). As IRE1 overexpression induces its auto-activation (Hetz et al, 2015), the ERMIT assay was optimized using low quantities of the transfected plasmids to ensure that no IRE1 auto-activation was detectable. In confirmation of the validity of the activation assay, all the IRE1 KD baits reduced the luminescence signal by more than 90% (Fig 1F), thus confirming that the signal observed was not due to the activation of endogenous IRE1. The AGR2-WT bait produced the highest signal indicating that the dimerization of AGR2 occurred in the ER. The C81A mutant showed a 25% decrease in the signal, relative to AGR2-WT, whereas the E60A or the DM reduced the signal by about 80%. This demonstrates that AGR2 dimerizes in the ER and that the E60 residue plays a key role in this in vivo interaction whereas the C81 does not. Moreover, ER stress induced by DTT treatment showed a dose-dependent dissociation of AGR2 homodimers as assessed by the decrease in luminescence observed for all the constructs tested (Fig 1G). The same result was observed when ER stress was induced by thapsigargin or tunicamycin (Appendix Fig S1I). An IC50 was then calculated for each of the ER stressors (Appendix Fig S1J). Stress-related AGR2 functions in the ER were also evaluated using 35S-methionine pulse-chase followed by AGR2 immunoprecipitation to investigate the dynamics of AGR2 binding to other partners. Five AGR2 binding partners were visualized using this method in HeLa cells (bands 1–5, Appendix Fig S2A). Interestingly, the kinetics of association of these proteins with AGR2 differed between basal and ER stress conditions. The association of the proteins corresponding to
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