1. Volume 69, Issue 4, Pages 551-565.e7 (February 2018)
Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling 
Tatiana Goncharov, Stefanie Hedayati, Melinda M. Mulvihill, Anita Izrael-Tomasevic, Kerry Zobel, Surinder Jeet, Anna V. Fedorova, Celine Eidenschenk, Jason deVoss, Kebing Yu, Andrey S. Shaw, Donald S. Kirkpatrick, Wayne J. Fairbrother, Kurt Deshayes, Domagoj Vucic 
Molecular Cell 
Volume 69, Issue 4, Pages e7 (February 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 551-565.e7DOI: (10.1016/j.molcel.2018.01.016)
Copyright © 2018 Elsevier Inc. Terms and Conditions

3. Figure 1
XIAP-Selective Antagonists Do Not Cause c-IAP1 Degradation or Induce Cell Death
(A) XIAP-selective antagonists preferentially bind XIAP BIR2 domain. SPR assays were performed using recombinant BIR2 and BIR3 domains of XIAP and c-IAP1 with indicated compounds.
(B) Bivalent XIAP antagonists bind XIAP BIR2-BIR3 domain protein. Due to the bivalent nature of the compounds, affinities can only be estimated (see STAR Methods).
(C) XIAP-selective antagonists do not cause c-IAP1 degradation. THP1 cells were treated with indicated compounds (all 1 μM) for 20 hr, and lysates were examined by western blotting with indicated antibodies.
(D) XIAP-targeting homodimer compound XB2d89 causes proteasomal degradation of XIAP. THP1 cells were left untreated or treated with XB2d89 for 20 hr in the presence or absence of indicated proteasomal (MG132), lysosomal (CA-074), or autophagy inhibitors (3-MA+BFA). Cell lysates prepared using Triton X-100 (T) or SDS (S) containing buffers were examined by western blotting with indicated antibodies.
(E) NOD2 does not affect XIAP or c-IAP1 stability. 293T cells transiently expressing vector or NOD2 construct were treated with indicated compounds for 20 hr, and cellular lysates were examined by western blotting with indicated antibodies.
(F) XIAP-selective antagonists do not induce cell death. EVSA T cells were treated with increasing doses of indicated compounds for 24 hr, and cell viability was assessed by CellTiter-Glo assay.
(G) HT1080 cells were treated with BV6 or XB2d89 in the absence or presence of TNF (5 ng/mL) and agonistic DR5 (50 ng/mL) antibody for 24 hr. Cell viability was assessed as in (F). Results represent mean and SD from three independent experiments.
See also Figure S1.
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4. Figure 2
XIAP-Selective Antagonists Disrupt Physical Interaction between XIAP and RIP2 and Inhibit NOD2-Mediated Cytokine Upregulation
(A) XIAP BIR2 domain binds RIP2 kinase domain. XIAP BIR2 domain was immobilized on an SPR chip, and RIP2 kinase domain was injected at concentrations ranging from 150 to 2,500 nM. The binding affinity of 590 nM was determined using a kinetic fit with a simple 1:1 binding model (orange).
(B) XIAP selective antagonists block XIAP BIR2-RIP2 kinase interaction. XIAP BIR2, BIR3, or BIR2-3 was immobilized on an SPR chip and tested for binding to RIP2 kinase domain (1 μM) in the presence of absence of indicated antagonists (2 μM).
(C) HEK-Blue-hNOD2 cells were treated overnight with MDP (0.2 μg/mL) in the presence of indicated XIAP antagonists (1 μM), and NOD2 activation was assessed by a blue reporter assay.
(D) 239T cells were transiently transfected with NF-κB luciferase reporter plasmids and vector for 20 hr and left untreated or treated with TNF (10 ng/mL) for 5 hr. NF-κB activity was assessed using dual-luciferase reporter assay.
(E) Primary bone-marrow-derived macrophages (BMDMs) were treated with L18-MDP in the absence or presence of various IAP antagonists (1 μM) for 6 hr. Levels of indicated cytokines were determined by Luminex. Quantitative data are presented as the mean and SD from three independent repeats.
(F) Mice were treated with indicated amounts of XB2m54 for 1 hr prior to treatment with L18-MDP for 4 hr. Levels of indicated cytokines were determined as in (E).
See also Figure S2.
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5. Figure 3
XIAP-Selective Antagonists Block NOD2-Mediated Signaling and RIP2 Ubiquitination
(A and B) Freshly isolated human peripheral blood mononuclear cells from two donors and THP1 cells were treated with MDP in the absence or presence of indicated IAP antagonists (1 μM) for 16 hr. Levels of TNF, interferon γ (IFNγ) (A) and IL-6 (B) were determined by Luminex. Quantitative data are presented as the mean and SD from three independent repeats.
(C) THP1 cells were treated with MDP in the presence of indicated antagonists (1 μM). Cellular lysates were examined by western blotting with indicated antibodies.
(D) THP1 cells were treated with MDP for indicated times followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with indicated ubiquitin-chain-specific antibodies, and precipitates and lysates were examined by western blotting using indicated antibodies.
(E) THP1 cells were treated with MDP for 30 min in the presence or absence of XB2d89 (1 μM; added 1 hr prior) followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with indicated ubiquitin-chain-specific antibodies, and precipitates and lysates were examined by western blotting using indicated antibodies.
See also Figure S3.
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6. Figure 4 Analyses of NOD2-Mediated Ubiquitination
(A) Experiment design scheme. THP1 cells were treated with MDP in the absence or presence of XIAP antagonist XB2d89 (1 μM; added 1 hr prior). Ubiquitin remnants were enriched using anti-K-ε-GG antibody, identified in LC-MS/MS, and quantified using peak area under curve (AUC). Two duplicate injections were acquired for each condition.
(B) Volcano plot of global protein ubiquitination profiling showing p value versus log2 ratio changes between MDP 30 min and control.
(C) Linear mixed-effect model (LiME) plot of RIP2 showing ubiquitination changes under various treatment conditions. Black lines correspond to AUC abundance from confidently quantified K-GG peptide features. Protein ubiquitination change generated by LiME is shown in red.
(D) Extracted ion chromatograms of RIP2 K376 and K538 features show remarkable difference in their responses to XB2d89 treatment after NOD2 signaling activation by MDP treatment. Each condition is color-coded according to (A), and XICs from duplicate injections are plotted. Peak alignment among these runs was performed based on global retention time shift.
(E) XB2d89 prevents RIP2 ubiquitination at K538 and K410 during NOD2 activation. Site level changes were calculated between conditions with or without 1 hr XB2d89 pre-incubation, in addition to MDP for 30 min. Two biological replicate analyses were acquired and plotted. p value was calculated based on the observations made in both replicates.
(F and G) RIP2 with mutations in ubiquitination sites is deficient in mediating NOD2 signaling. THP1 cells expressing WT or K410R/K538R RIP2 or RIP2 KO cells were treated with MDP for 6 hr (F) or 30 min (G). Supernatants of untreated and treated cells were examined for IL-8 levels using IL-8 ELISA (F). Following 30-min treatment with MDP, cells were lysed in 6 M urea buffer and lysates immunoprecipitated with K63 or linear ubiquitin-chain-specific antibodies. Precipitates and lysates were examined by western blotting using indicated antibodies (G). Quantitative data are presented as the mean and SD from three independent repeats.
See also Figure S4 and Tables S1 and S2.
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7. Figure 5
RIP2 Kinase Inhibition Blocks NOD2 Signaling and RIP2 Ubiquitination
(A) RIP2 inhibitor GSK583 efficiently blocked MDP-induced signaling cytokine production in vivo. Mice were treated with indicated amounts of GSK583 for 1 hr prior to treatment with L18-MDP for 4 hr. Levels of indicated cytokines were determined by Luminex.
(B) THP1 cells were treated with MDP for 30 min in the presence or absence of GSK583 (1 μM; added 45 min prior) followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with indicated ubiquitin-chain-specific antibodies, and precipitates and lysates were examined by western blotting using indicated antibodies.
(C) GSK583 blocks XIAP BIR2-RIP2 kinase interaction. XIAP BIR2, BIR3, or BIR2-3 were immobilized on an SPR chip and tested for binding to RIP2 kinase domain in the presence or absence of GSK583 (1 μM).
(D) Combination of XB2m54 and GSK583 inhibits NOD2-mediated cytokine production in vivo. Mice were treated with XB2m54 (100 mg/kg) and GSK583 (30 mg/kg) for 1 hr prior to treatment with L18-MDP for 4 hr. Levels of indicated cytokines were determined by Luminex.
(E) Combination of XB2m54 and GSK583 inhibits NOD2-mediated signaling and RIP2 ubiquitination. THP1 cells were treated with MDP for 30 min in the presence or absence of XB2m54 and GSK583 (0.5 μM each) followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with indicated ubiquitin-chain-specific antibodies, and precipitates and lysates were examined by western blotting using indicated antibodies.
See also Figure S5.
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8. Figure 6
Kinase Inhibitors that Disrupt XIAP:RIP2 Binding Block NOD2 Signaling and RIP2 Ubiquitination
(A) RIP2 kinase inhibitors show varying ability to block the XIAP BIR2:RIP2 interaction. XIAP BIR2 was immobilized on an SPR chip and tested for binding to RIP2 kinase domain (1 μM) in the presence or absence of the indicated RIP2 inhibitors (0.125, 0.5, and 2 μM). Binding levels were normalized using a DMSO control to set the XIAP BIR2:RIP2 response level to 100% (3 black lines indicate 3 replicates).
(B) HEK-Blue-hNOD2 cells were treated overnight with MDP in the presence of indicated kinase inhibitors (1 μM), and NOD2 activation was assessed by a blue reporter assay. Quantitative data are presented as the mean and SD from three independent repeats.
(C) THP1 cells were treated with MDP for 30 min in the presence or absence of indicated kinase inhibitors (1 μM; added 1 hr prior) followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with K63 and linear ubiquitin chain antibodies, and precipitates and lysates were examined by western blotting using indicated antibodies.
See also Figure S6.
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9. Figure 7 RIP2 Kinase Activity Is Not Needed for NOD2 Signaling
(A) HEK-Blue-hNOD2 RIP2−/− cells stably expressing GFP, WT RIP2, or D146N RIP2 were treated overnight with MDP (0.2 μg/mL) in the presence or absence of GSK583 or ponatinib (2.5 μM), and NOD2 activation was assessed by a blue reporter assay. Quantitative data are presented as the mean and SD from three independent repeats.
(B) HEK-Blue-hNOD2 RIP2−/− cells stably expressing GFP, WT RIP2, or D146N RIP2 were treated with MDP for 30 min in the presence or absence of GSK583 (1 μM; 45 min prior) followed by lysis in 6 M urea buffer. Lysates were immunoprecipitated with K63 ubiquitin chain antibody, and precipitates and lysates were examined by western blotting using indicated antibodies. Asterisk indicates non-specific band.
(C) His-tagged RIP2 WT or D146N or GST-RIP1 kinase domain proteins (250 nM) were incubated with MBP-XIAP or MBP (250 nM) immobilized on amylose beads for 45 min. Inputs and pull-downs were visualized by staining with SimplyBlue Safe Stain.
(D) HEK-Blue-hNOD2 RIP2−/− cells stably expressing GFP, WT, or D146N RIP2 were left untreated or treated with MDP in the presence or absence of GSK583 (1 μM; 45 min prior) and immunoprecipitated with anti-XIAP antibody following cell lysis. Proteins in total cell lysates and immunoprecipitates were analyzed by western blotting with indicated antibodies. Asterisk indicates non-specific band.
(E) XIAP BIR2 domain binds D146N RIP2. XIAP BIR2 domain was immobilized on an SPR chip, and WT or D146N RIP2 proteins were injected at concentrations ranging from 150 to 2,500 nM. Due to the instability of D146N RIP2, 5% glycerol was added to the running buffer. The Kd of 2.2 and 4.4 μM for WT and D146N RIP2 proteins was determined using a 1:1 binding affinity fit.
(F) Mass spectrometry analysis of WT and D146N RIP2 proteins. An envelope of peaks for the WT protein corresponds to the addition of multiple phosphate groups (80 amu), which are not present in case of D146N mutant. For D146N RIP2 mutant, the deconvoluted mass corresponds to acetylated apo-protein (+42 amu).
See also Figure S7.
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