Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: The 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.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Residue, Generated, Mutagenesis, Western Blot, Expressing, Transfection, Control, SDS Page, Activation Assay, Construct, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A The first screen was carried out using HEK293T cells transfected with the ERMIT bait/prey, system, the XBP1 splicing reporter, and the siRNA library ( n = 5). The counter screen was carried out using the same experimental setup without the ERMIT bait/prey system ( n = 4). B Volcano plot representing the statistical analysis of the data from the screen, where the y axis represents statistical criteria and the x axis the fold change in luminescence units. C Number of genes associated with the indicated processes, as revealed by functional analysis based on Gene Ontology and Reactome terms, (*): Apoptotic signaling pathway ( P = 0.0083), ER‐associated degradation ( P = 0.0076), and lipid biosynthetic process ( P = 0.0059), respectively; (**): ER stress ( P = 0.0044), Protein folding ( P = 0.003), Calcium homeostasis ( P = 0.0022), N‐glycosylation ( P = 0.0018), respectively. D, E Enrichment of AGR2 homodimer enhancers in functions associated with ER homeostasis (green) and of AGR2 homodimer inhibitors in functions associated with ER homeostasis imbalance (red).

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Transfection, Functional Assay, Glycoproteomics

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A Co‐immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ . *indicates Immunoglobulin heavy and light chains. B Structural association of TMED2 and AGR2. The six TMED2 orientations (pink, dark pink, purple, blue, yellow, and green) best positioned to stabilize the AGR2 dimer (colored tan with N‐termini and dimer interface alpha helix in red). C Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP‐stabilized AGR2 was analyzed under non‐reducing (top blot) or reducing conditions (bottom blot). D = dimeric, M = monomeric AGR2. D Specific interactions between residues in AGR2 dimer (tan) and best oriented TMED2 structure (green). Glu51, Arg74, and Arg97 of TMED2 form ionic interactions with Lys66, Glu59, and Asp97 of AGR2. His28 of TMED2 interacts with Tyr111 of AGR2 through π‐π‐interactions. E Co‐immunoprecipitation of AGR2 wild‐type (WT, left panel) or AGR2 AA mutant (right panel) with TMED2 in HEK293T cells. Immunoprecipitation was performed with mouse anti‐Flag antibody to pull‐down the ectopic protein tag. F Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression upon TMED2 overexpression. Data are representative of four independent experiments. Tubulin (TUB) was used as a loading control. The graph represents average signal normalized on reporter protein expression ±SD. The Mann–Whitney statistical test was used. G HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT as described in Fig E ( n = 3). The graph represents average signal normalized on reporter protein expression ±SD. H Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression in total cell lysate (TCL) upon TMED2 silencing. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control. The graph represents average signal normalized on reporter protein expression ±SD. The Mann–Whitney statistical test was used. I HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT as described in Fig E ( n = 3). The graph represents average signal normalized on reporter protein expression ±SD.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Immunoprecipitation, In Situ, Mutagenesis, Western Blot, Expressing, Over Expression, Control, MANN-WHITNEY, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A The ERMIT‐based validation of the complex formation between AGR2 and LYPD3 or CD59, but not OS9. The monomeric AGR2 E60A was used as bait, while LYPD3, CD59, and OS9 were used as prays ( n = 6). The graph represents average signal normalized on reporter protein expression ±SD. For statistical analyses, the Mann–Whitney test was used. B, C Expression and subcellular localization of CD59‐GFP WT and C94S. D, E Co‐immunoprecipitation of CD59‐GFP WT or CD59 C94S mutant with AGR2 WT (D) or AGR2 AA mutant (E) in HEK293T cells. F–I FACS analysis of CD59 WT (F and G) or CD59 C94S (H and I) total (F and H) and membrane (G and I) expression in HEK293T cells silenced for AGR2 (siAGR2) and TMED2 (siTMED2) when compared to control (siGL2) cells (F and H) or silenced for TMED2 (siTMED2) and overexpressing AGR2 WT or AGR2 AA mutant (G and I). Data are representative of three to four independent experiments. Membranous CD59 expression was detected with anti‐GFP APC. ns: non‐statistically significant; for (F) (*): P = 0.0385, (***): P = 0.0003 (left panel) and P = 0.0002 (right panel), respectively; for (G) (*): P = 0.0334, (**) P = 0.0036, and (***) P = 0.0003; for (H) (*): P = 0.0171 and (**): P = 0.0084; and for (I) (**) P = 0.0086 (left panel) and P = 0.0076 (right panel), respectively. The graph represents average GFP signal ±SD. For statistical analyses, the Mann–Whitney test was used.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Biomarker Discovery, Expressing, MANN-WHITNEY, Immunoprecipitation, Mutagenesis, Membrane, Control

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A Formation of GFP‐LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP‐LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP‐LC3 positive cells for each condition (left panel). (**): P = 0.0032. The graph represents average western blot normalized signal ±SD. For statistical analyses, the Mann–Whitney test was used. B Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 μM chloroquine for 2 h. Actin (ACT) served as a loading control. (*): P = 0.0498. The graph represents average western blot normalized signal ±SD. For statistical analyses, the Mann–Whitney test was used. n = 4. C Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 and 10 μM chloroquine treatment. Actin (ACT) served as a loading control. D HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT as described in Fig E in the presence of gradual amounts of chloroquine. The graph represents average signal ±SD. For statistical analyses, the Mann–Whitney test was used, n = 4. E Western blot analysis of secreted AGR2 upon TMED2 overexpression. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. F Representative pictures of extracellular vesicles (EVs) heterogeneity purified from conditioned media of HEK293T cells transfected with control (ev) or TMED2 plasmid and analyzed by cryo‐electron microscopy. Right upper panel: Western blot analysis of CD63 in total medium (left) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells (middle) and Western blot analysis of AGR2 levels (right) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells. G Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. H Quantification of extracellular AGR2 level (eAGR2) in cells silenced for TMED2 (siTMED2), compared to control (siGL2). Data are representative of three independent experiments and are presented as extracellular‐to‐intracellular AGR2 ratio. (**): P = 0.0029. The graph represents average western blot normalized signal ±SD. For statistical analyses, the Mann–Whitney test was used. I Secretion of AGR2 wild‐type (wt), E60A, and ∆45 mutant as determined by Western blot. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Confocal Microscopy, Staining, Control, Western Blot, MANN-WHITNEY, Expressing, Inhibition, Transfection, Plasmid Preparation, Over Expression, Purification, Cryo-Electron Microscopy, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A Representative histological H&E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice. B Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice. C Semi‐quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT (blank bars) and TMED2 hypomorph mice (black bars) ( n = 6). The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used. D Freshly isolated PBMCs were placed in Boyden chambers toward media conditioned by cells overexpressing AGR2 WT, E60A, ∆45, or AGR2 AA mutants and incubated for 24 h. Migrating cells were then characterized and quantified by flow cytometry using FCS/SSC parameters or CD14 monocyte marker. (**): P = 0.0016. The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used. E, F Freshly isolated PBMCs were placed in Boyden chambers toward media conditioned by cells overexpressing TMED2 and either AGR2 WT (E) or AGR2 AA mutant (F) and incubated for 24 h. Migrating cells were then characterized and quantified as indicated in Fig D. For (E) (*): P = 0.0461 (AGR2WT/TMED2) and 0.018 (AGR2WT/TMED2 + blocking Ab) respectively; (**): P = 0.0053 and (***): P = 0.0048; for (F) (***): P = 0.0007. The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used. G Freshly isolated PBMCs were placed in Boyden chambers toward media conditioned by cells silenced for TMED2 and incubated for 24 h. Migrating cells were then characterized and quantified as indicated in Fig D. (*): P = 0.0149. The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used. H Freshly isolated PBMCs were placed in Boyden chambers toward decreased doses of recombinant AGR2 and incubated for 24 h. CCL2 cytokine was used as positive control for monocyte migration. ns: non‐statistically significant, (*): P = 0.0171, (**): P = 0.0084, (***): P = 0.0003. The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used. I Impact of AGR2 blocking antibodies on AGR2‐mediated monocytes migration was tested using Boyden chambers as described above. The concentrations of recombinant human AGR2 were of 200 ng/ml, and decreasing amounts of antibodies were used from 20 μg to 1 μg. The non‐relevant antibody (Isotype) was used at the maximal dose of 20 μg. Data are representative of three independent experiments. ns: non‐statistically significant, (*): P = 0.0165 (Ab = 20 μg/ml), 0.0223 (Ab = 10 μg/ml) and 0.0493 (Ab = 5 μg/ml), respectively. The graph represents average IHC signal ±SD. For statistical analyses, the Mann–Whitney test was used.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Staining, Expressing, MANN-WHITNEY, Isolation, Incubation, Flow Cytometry, Marker, Mutagenesis, Blocking Assay, Recombinant, Positive Control, Migration

Journal: EMBO Molecular Medicine

Article Title: Control of anterior GR adient 2 ( AGR 2) dimerization links endoplasmic reticulum proteostasis to inflammation

doi: 10.15252/emmm.201810120

Figure Lengend Snippet: A Selective enrichment of AGR2 modulators in colonic Crohn's disease (CD). Percentage of tested genes with altered expression in colonic biopsies from patients with ulcerative colitis (UC, n = 8) and colonic CD (CC, n = 15) and in ileal biopsies from patients with ileo‐colonic CD (IC, n = 9). t ‐test was used. B Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non‐inflamed colonic mucosa from patients with Crohn's Disease (CD) versus healthy controls (C) (dChip software t ‐test). Gene expression (blue: fold decrease; red: fold increase). C TMED2 mRNA expression in healthy controls (CT), colonic Crohn's disease (CC), and ulcerative colitis (UC). ANOVA was used. D Representative immunohistological analysis of TMED2 in non‐inflamed colonic biopsies from healthy controls and patients with active CD. Scale bars: 100 μm. E Representative immunohistological analysis of CD163 (scavenger receptor present in macrophages), AGR2, and TMED2 in non‐inflamed colonic biopsies from healthy controls and patients with active or quiescent CD. F Semi‐quantitative analysis of CD163, AGR2, and TMED2 immunostaining in colonic mucosa from healthy controls (C) and patients with quiescent (Q) or active (A) CD. Three random sections from each patient of the four groups were scored. Final IHC scores (A × B range to 0–9) combine the percentage of positively labeled cells. A: % of IHC positive labeled cells—0 (0%), 1 (< 30%), 2 (30–60%), 3 (> 60%), and the intensity of the reaction product. B: 0 (no reaction), 1 (weak), 2 (mild), 3 (strong) in most of the examined fields. All values are mean ± SEM. n = 12, ANOVA was used as a statistical test. G Schematic representation of the role of AGR2 dimer alteration in intestinal epithelial cells under basal conditions or in Crohn's disease. In normal cells, the ratio of dimeric versus monomeric AGR2 defines proteostasis boundaries in the ER, and those are altered in diseased conditions through the alteration of the expression of AGR2 dimer regulators, thereby leading to pro‐inflammatory signals comprising the release of AGR2 in the extracellular milieu and to the induction of uncontrolled autophagy and the subsequent alteration of protein secretion.

Article Snippet: Primary antibodies against CD163 (AbCam, ab87099), TMED2 (Santa Cruz Biotechnology, sc‐376459), and AGR2 (Novus Biologicals, NBP1‐05936) were used.

Techniques: Expressing, Software, Gene Expression, Immunostaining, Labeling