Journal: Journal of Medicinal Chemistry

Article Title: Probing Immune Signatures of Conjugated Pattern Recognition Receptor Ligands Identifies Chimeras with Potent Adjuvant and Antitumor Activities

doi: 10.1021/acs.jmedchem.6c00372

Figure Lengend Snippet: Receptor-specific activities of individual PRR ligands and conjugated PRR ligands. HEK-Blue NOD2, TLR2, TLR7, TLR4, or HEK-Lucia RIG-I cells were treated with the compounds (1 or 10 μM as indicated) and the corresponding positive controls (1 μM MDP for NOD2, 100 nM Pam3CSK4 for TLR2, 1 μM imiquimod for TLR7, 100 ng/mL LPS for TLR4, and 100 ng/mL Poly­(I/C) for RIG-I) for 18 h. The activities are shown relative to the vehicle-treated control (0.1% DMSO). Data are mean ± SEM of three independent experiments. EC 50 values were determined in HEK-Blue NOD2, TLR2, and TLR7 cells in at least three independent experiments with eight concentrations (1 nM to 10 μM for NOD2 cells, and 0.1 nM to 1 μM for TLR2 and TLR7 cells).

Article Snippet: HEK-Blue NOD2 (Cat. code: hkb-hnod2), TLR2 (Cat. code: hkb-htlr2), TLR4 (Cat. code: hkb-htlr4), and TLR7 (Cat. code: hkb-htlr7) cell lines (Invivogen, San Diego, CA) are derived from HEK293 cells by cotransfection of hNOD2, hTLR2, hTLR4, or hTLR7 genes, respectively, and a nuclear factor-κB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.

Techniques: Control

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Schematic of the SMaRT framework , which resolves macrophage continuum states into inflammatory CoLAMs (iCoLAMs, “accelerators”) and non-inflammatory CoLAMs (niCoLAMs, “brakes”), and the validation of niColAMs as specialized populations that sense and clear microbes via a NOD2- and CCDC88A (GIV) dependent pathway. B. Ingenuity Pathway Analysis (IPA) of lamina propria myeloid cells from refractory IBD patients highlights host-and microbe-driven upstream pathways ; pathways most relevant to this study are bolded. See also Supplementary Figure S1 for expression of key niColAM genes linking cAMP–CREB signaling to NOD2. C. Immunoblot (IB) of thioglycolate-elicited peritoneal macrophages (TGPMs) from WT and CCDC88A (GIV)-KO mice. D. Experimental workflow for integrated multi-omic analyses: WT and GIV-KO TGPMs were infected with AIEC LF82 and harvested at 0, 10, and 45 minutes for phosphoproteomics. E-F. Phosphoproteomic analyses reveal dynamic, GIV-dependent regulation of cAMP and MAPK signaling. Venn diagram (G) highlights intersecting iColAM and niColAM genes that are phosphomodulated dynamically upon AIEC LF82 infection in WT TGPMs. Heatmap ( F ) depicts differentially phosphomodulated proteins within cAMP and MAPK pathways in WT and KO macrophages across infection timepoints (row-wise Z-scores; red = upregulated, blue = downregulated).

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Biomarker Discovery, Expressing, Western Blot, Infection, Phospho-proteomics

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A . Schematic summarizing canonical LPS•TLR4•GIV signaling : in the basal state, GIV binding to TLR4 prevents receptor dimerization and signaling; upon LPS stimulation, GIV dissociates, allowing TLR4 dimerization and activation of proinflammatory pathways. B . Schematic summarizing MDP-induced NOD2•GIV signaling : resting NOD2 is ADP-bound and autoinhibited; MDP triggers ADP–ATP exchange, conformational ‘opening’ of the LRR module, and NOD2 dimerization with recruitment of downstream partners. C. Conceptual framework highlighting this study’s focus on microbial impact via the GIV•Gαi ┤cAMP◊PKA axis, which regulates cAMP and PKA suppression in IBD and lists the knowledge gaps (“?”). D. Coimmunoprecipitation (Co-IP) of HA-tagged NOD2 and GIV-FLAG from HEK293 cells (±ATPγS pre-incubation, ±MDP stimulation). Bound complexes (top) and lysates (bottom) were immunoblotted (IB) for HA/myc-tagged NOD2 and GIV. E- F. Densitometry of representative immunoblots (top) and schematic summary (bottom) show that NOD2•GIV disassembly coincides with NOD2 dimer formation. G . Study design of single vs sequential PLA assay. H . Representative confocal images of ligated GIV●Gαi and GIV●NOD2 complexes in THP1-derived macrophages challenged with MDP (0–6 hours) GIV●NOD2 complexes (in Red PLA) top panel ; GIV●Gαi complexes (in Green PLA) bottom panel . J. Representative confocal images of GIV●Gαi and GIV●NOD2 complexes in THP1-derived macrophages challenged with MDP (0–3hours) GIV●NOD2 complexes (in Red PLA) and GIV●Gαi complexes (in Green PLA) I . Quantification of PLA signals from 0-3 hours MDP stimulation, ∼20–30 random fields (n = 4–5 repeats). K . Quantification of PLA signals from 0-6 hours MDP stimulation, ∼20–30 random fields (n = 4–5 repeats). One-way ANOVA with Tukey’s post-test; p-values shown above bars; p ≤ 0.05 considered significant. Statistics : All results are displayed as mean ± SEM (n = 3 biological replicates). Significance was tested using two-way/one-way ANOVA followed by Tukey’s test for multiple comparisons. p -value ≤ 0.05 is considered as significant.

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Binding Assay, Activation Assay, Co-Immunoprecipitation Assay, Incubation, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Dual-color PLA reveals concurrent GIV interactions with Gαi (green) and NOD2 (red) in PMA-differentiated THP-1 macrophages stimulated with MDP (10 µg/ml, 1 hour). Yellow signals indicate sites of spatial overlap. Inset : Representative foci. 3D surface reconstruction and line-scan analysis (right) confirm co-localization. Nuclei in blue. Scale bars, 10 µm. B. Immunogold electron microscopy of primary murine peritoneal macrophages exposed to E. coli LF82 (MOI 30, 60 minutes). Grids stained for NOD2 (12 nm, red) and GIV (18 nm, blue). Left : montage showing flipped hemifusomes (fHFs) budding into MVBs; inset magnifications and schematic (below) depict sequential MVB formation in WT cells. Right : Quantification of mature v immature phagophores, as determined by the presence or absence of HFs, fHFs or MVBs (see also Supplementary Figure S5B for electron micrographs from GIV-KO cells). C. HA-tagged NOD2 immunoprecipitated from HEK cells following MDP stimulation (1 hour) or nucleotides (ADP, ATP, or ATPγS for 30 minutes after permeabilization). Immunoblots detect associated GIV and NOD2. D. GST pulldown of recombinant His-GIV-CT with GST-NOD2-LRR at varying pH (5.5, 6.5, 7.4) ± MDP (20 µM). Bound His-GIV detected by immunoblotting (IB); GST loading confirmed by Ponceau S. E. Mechanistic model: NOD2 remains inactive at steady state (1). MDP sensing triggers NOD2 nucleotide exchange, dimerization, and GIV association (2). Acidic pH during phagophore maturation stabilizes the NOD2•GIV complexes (3). Steps 2- and 3 suppress GIV●Gαi complexes enabling cAMP surge. Oligomerization of NOD2 drives NOD2•GIV dissociation, promoting GIV•Gi complex formation and NOD2 inactivation (4).

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Electron Microscopy, Staining, Immunoprecipitation, Western Blot, Recombinant

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Homology model of the 10th LRR of human NOD2 (green; based on rabbit NOD2, PDB:5IRN ) docked with the GEM motif of GIV (black; PDB:6MHF). A’. Magnified view highlights critical binding residue L998, whereas F996 appears dispensable. L998 appears critical for binding, whereas F996 appears dispensable. A”. Crystal structure of GIV’s GEM motif (black; PDB:6MHF ) bound to Gαi (SwII region); R208 is critical for the interaction. B . Sequence alignment of human NOD1, NOD2, Gαi (SwII), and rabbit Gαi (PDB:5IRN). Black arrows mark residues prioritized for mutagenesis; red asterisks (*) and double asterisks (**) indicate strategies for designing NOD2 chimeras using NOD1 or Gαi sequences. C-D. Pulldown assays using GST or GST–GIV with lysates of HEK293 cells expressing WT or mutant NOD2 (panel C). Bound HA-NOD2 was visualized by immunoblot with anti-HA mAb (IB). Quantification of three independent biological repeats is shown in D. E-F. GST pulldown assays of recombinant His–GIV-CT or its F1685A mutant (deficient in binding to Gαi and predicted in A as deficient in binding NOD2) with GST–NOD2-LRR. Bound proteins were detected by immunoblot; equal loading confirmed by Ponceau S (GST) and immunoblotting (IB; anti-His mAb). Quantification from three independent repeats of the assay in panel F. G-I. GST pulldown assays of recombinant His–GIV-CT, His–Gαi WT, or W211A mutant (deficient in GIV-binding ) with GST–NOD2-LRR (panel E). Bound proteins were detected by immunoblot; equal input confirmed by IB (panel F). Quantification from three repeats shown in I. J. MDP-dependent suppression of FSK-induced cAMP levels in HeLa cells expressing WT or NOD2 L998 mutant, assessed using BRET-based CAMYEL biosensor (Bar graph of AUC). See also Supplementary Figure S5(A-B) for assay schematic and the timeline of Forskolin (FSK) treatment and additional visualization of readouts. See also Supplementary Figure S5(C-D) for 0-60 minutes MDP-dependent suppression of FSK-induced cAMP levels in HeLa cells expressing WT or NOD2 L998 mutant. K. Schematic of the NOD2●GIV●Gαi interface in cAMP control. ( Top) Upon sensing MDP (early ), WT NOD2 engages GIV first and then relays the signal to activate Gαi and blunt cAMP surges ( late) ; ( Bottom) whereas the NOD2 L998A mutant fails to engage GIV or relay the signal to Gαi proteins, resulting in sustained cAMP accumulation Statistics : All results are displayed as mean ± SEM (n = 3 biological replicates). Significance was tested using two-way/one-way ANOVA followed by Tukey’s test for multiple comparisons. p -value ≤ 0.05 is considered as significant.

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Binding Assay, Residue, Sequencing, Mutagenesis, Expressing, Western Blot, Recombinant, Control

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Schematic of perturbagens used to dissect the cAMP cascade and GIV’s G protein regulatory function in phagolysosomal fusion. B . ELISA-based cAMP levels in WT and GIV-KO TGPMs infected with AIEC LF82, for 5 or 15 minutes. C. Quantification of normalized phospho-PKA substrate levels, normalized to β-Actin and expressed as fold-change over t₀ (time 0 minutes, baseline), from immunoblots from Supplementary Figure S6A . Lysates were prepared from WT and GIV-KO TGPMs infected with AIEC LF82 for 0–45 minutes following MDP pre-treatment (10 µg/ml). Bar graph quantification corresponds to Supplementary Figure S6B. See also, Supplementary Figure S6 ( F-G ) for immunoblots and quantification of normalized phospho-PKA substrate levels of HeLa WT and KO lysates stimulated with MDP for 0-45minutes. D-E. Viable bacterial counts in PMA-differentiated WT and GIV-KO THP-1 macrophages, untreated (−) or pre-treated (+) with PKA inhibitors (H89, 30 µM; PKI 14–22, 10 µM; BLU2864, 30 µM) or EPAC inhibitor (ESI-09, 10 µM) for 30 minutes prior to AIEC LF82 infection (1 hour, MOI 1:50). F. Percent change in viable bacterial counts in PMA-differentiated WT and GIV-KO THP-1 macrophages, untreated (−) or pre-treated (+) with 8-Br-cAMP (10 mM) or Fsk (20 µM) for 30 minutes prior to AIEC LF82 infection (1 hour, MOI 1:50). G. Domain architecture of TAT-GIV constructs . The TAT transduction domain (TAT-PTD) fused to His- and HA-tags, linked to the C-terminal GIV segment (aa 1660–1870). Key binding sites are indicated for NOD2 and Gαi (blue) and TLR4 (red). WT TAT-GIV-CT (top) and the CT-F1685A mutant (bottom) are shown. See also Supplementary Fig. 6D -E for purification and uptake assays. H. Workflow of gentamicin protection assay in WT and GIV-KO TGPMs pretreated with WT or F1685A TAT-GIV CT mutant (100 nM, 30 minutes), followed by AIEC LF82 infection (MOI 1:50, 1 hour). I. Viable bacterial counts in WT and GIV-KO TGPMs pretreated with WT or the F1685A TAT-GIV CT prior to AIEC LF82 infection. J. BRET-based cAMP assay in HeLa cells. Left : Workflow: cells transfected with 400 nM WT or F1685A TAT-GIV CT, followed by MDP treatment (10 µg/ml). Right : Normalized cAMP levels in WT and GIV-KO HeLa cells, ±WT or F1685A TAT-GIV CT, measured using CAMYEL biosensor. Statistics : Data are mean ± SEM (n = 3 biological replicates). Significance was tested by one-/two-way ANOVA with Tukey’s post-test; p ≤ 0.05 was considered significant.

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Construct, Transduction, Binding Assay, Mutagenesis, Purification, cAMP Assay, Transfection

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: B. The NOD2●GIV●Gαi module functions as a ‘toggle switch’ controlling host cAMP signaling in response to microbial cues. Left : The LRR domain of NOD2 binds the GEM motif of GIV, sequestering GIV from Gαi and transiently limiting inhibition of the AC→cAMP→PKA axis. This allows an early cAMP surge, known to restrain NFκB-dependent inflammation . Right : Subsequently, dissociation of NOD2●GIV complexes restore GIV●Gαi interactions at phagophore membranes, suppressing cAMP→CREB→PKA signaling to favor phagolysosome fusion. C. Comparison of dead versus live bacteria reveals that suppression of macrophage cAMP requires live microbes, which contribute to the initial cAMP surge via diverse mechanisms summarized in the schematic. D. Multilayered networks connect specific gut microbes (brown) and their cAMP-modulating microbial genes (green) to host proteins (PRRs, signaling molecules), highlighting disease-specific host pathways regulating cellular cAMP. Strong feedback inhibitory loops (red) are orchestrated by proteins that are localized to phagophore membranes (* asterisk). E. Multilayered networks linking gut microbes (brown) to host genes reveal both canonical GPCR-dependent (shown before ) and non-canonical GIV-dependent (this work) G protein signaling pathways enriched for disease-specific host pathways in IBD.

Article Snippet: The sections were incubated with mouse anti-NOD2 antibody (Santa Cruz, sc-56168, 1:50 dilution) and rabbit anti-GIV antibody (Millipore sigma, ABT80; 1:50 dilution) followed by secondary antibodies 18 nm colloidal gold of donkey anti-rabbit IgG and 12 nm gold of donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Inhibition, Comparison, Bacteria, Protein-Protein interactions

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Schematic of the SMaRT framework , which resolves macrophage continuum states into inflammatory CoLAMs (iCoLAMs, “accelerators”) and non-inflammatory CoLAMs (niCoLAMs, “brakes”), and the validation of niColAMs as specialized populations that sense and clear microbes via a NOD2- and CCDC88A (GIV) dependent pathway. B. Ingenuity Pathway Analysis (IPA) of lamina propria myeloid cells from refractory IBD patients highlights host-and microbe-driven upstream pathways ; pathways most relevant to this study are bolded. See also Supplementary Figure S1 for expression of key niColAM genes linking cAMP–CREB signaling to NOD2. C. Immunoblot (IB) of thioglycolate-elicited peritoneal macrophages (TGPMs) from WT and CCDC88A (GIV)-KO mice. D. Experimental workflow for integrated multi-omic analyses: WT and GIV-KO TGPMs were infected with AIEC LF82 and harvested at 0, 10, and 45 minutes for phosphoproteomics. E-F. Phosphoproteomic analyses reveal dynamic, GIV-dependent regulation of cAMP and MAPK signaling. Venn diagram (G) highlights intersecting iColAM and niColAM genes that are phosphomodulated dynamically upon AIEC LF82 infection in WT TGPMs. Heatmap ( F ) depicts differentially phosphomodulated proteins within cAMP and MAPK pathways in WT and KO macrophages across infection timepoints (row-wise Z-scores; red = upregulated, blue = downregulated).

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Biomarker Discovery, Expressing, Western Blot, Infection, Phospho-proteomics

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A . Schematic summarizing canonical LPS•TLR4•GIV signaling : in the basal state, GIV binding to TLR4 prevents receptor dimerization and signaling; upon LPS stimulation, GIV dissociates, allowing TLR4 dimerization and activation of proinflammatory pathways. B . Schematic summarizing MDP-induced NOD2•GIV signaling : resting NOD2 is ADP-bound and autoinhibited; MDP triggers ADP–ATP exchange, conformational ‘opening’ of the LRR module, and NOD2 dimerization with recruitment of downstream partners. C. Conceptual framework highlighting this study’s focus on microbial impact via the GIV•Gαi ┤cAMP◊PKA axis, which regulates cAMP and PKA suppression in IBD and lists the knowledge gaps (“?”). D. Coimmunoprecipitation (Co-IP) of HA-tagged NOD2 and GIV-FLAG from HEK293 cells (±ATPγS pre-incubation, ±MDP stimulation). Bound complexes (top) and lysates (bottom) were immunoblotted (IB) for HA/myc-tagged NOD2 and GIV. E- F. Densitometry of representative immunoblots (top) and schematic summary (bottom) show that NOD2•GIV disassembly coincides with NOD2 dimer formation. G . Study design of single vs sequential PLA assay. H . Representative confocal images of ligated GIV●Gαi and GIV●NOD2 complexes in THP1-derived macrophages challenged with MDP (0–6 hours) GIV●NOD2 complexes (in Red PLA) top panel ; GIV●Gαi complexes (in Green PLA) bottom panel . J. Representative confocal images of GIV●Gαi and GIV●NOD2 complexes in THP1-derived macrophages challenged with MDP (0–3hours) GIV●NOD2 complexes (in Red PLA) and GIV●Gαi complexes (in Green PLA) I . Quantification of PLA signals from 0-3 hours MDP stimulation, ∼20–30 random fields (n = 4–5 repeats). K . Quantification of PLA signals from 0-6 hours MDP stimulation, ∼20–30 random fields (n = 4–5 repeats). One-way ANOVA with Tukey’s post-test; p-values shown above bars; p ≤ 0.05 considered significant. Statistics : All results are displayed as mean ± SEM (n = 3 biological replicates). Significance was tested using two-way/one-way ANOVA followed by Tukey’s test for multiple comparisons. p -value ≤ 0.05 is considered as significant.

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Binding Assay, Activation Assay, Co-Immunoprecipitation Assay, Incubation, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Dual-color PLA reveals concurrent GIV interactions with Gαi (green) and NOD2 (red) in PMA-differentiated THP-1 macrophages stimulated with MDP (10 µg/ml, 1 hour). Yellow signals indicate sites of spatial overlap. Inset : Representative foci. 3D surface reconstruction and line-scan analysis (right) confirm co-localization. Nuclei in blue. Scale bars, 10 µm. B. Immunogold electron microscopy of primary murine peritoneal macrophages exposed to E. coli LF82 (MOI 30, 60 minutes). Grids stained for NOD2 (12 nm, red) and GIV (18 nm, blue). Left : montage showing flipped hemifusomes (fHFs) budding into MVBs; inset magnifications and schematic (below) depict sequential MVB formation in WT cells. Right : Quantification of mature v immature phagophores, as determined by the presence or absence of HFs, fHFs or MVBs (see also Supplementary Figure S5B for electron micrographs from GIV-KO cells). C. HA-tagged NOD2 immunoprecipitated from HEK cells following MDP stimulation (1 hour) or nucleotides (ADP, ATP, or ATPγS for 30 minutes after permeabilization). Immunoblots detect associated GIV and NOD2. D. GST pulldown of recombinant His-GIV-CT with GST-NOD2-LRR at varying pH (5.5, 6.5, 7.4) ± MDP (20 µM). Bound His-GIV detected by immunoblotting (IB); GST loading confirmed by Ponceau S. E. Mechanistic model: NOD2 remains inactive at steady state (1). MDP sensing triggers NOD2 nucleotide exchange, dimerization, and GIV association (2). Acidic pH during phagophore maturation stabilizes the NOD2•GIV complexes (3). Steps 2- and 3 suppress GIV●Gαi complexes enabling cAMP surge. Oligomerization of NOD2 drives NOD2•GIV dissociation, promoting GIV•Gi complex formation and NOD2 inactivation (4).

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Electron Microscopy, Staining, Immunoprecipitation, Western Blot, Recombinant

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Homology model of the 10th LRR of human NOD2 (green; based on rabbit NOD2, PDB:5IRN ) docked with the GEM motif of GIV (black; PDB:6MHF). A’. Magnified view highlights critical binding residue L998, whereas F996 appears dispensable. L998 appears critical for binding, whereas F996 appears dispensable. A”. Crystal structure of GIV’s GEM motif (black; PDB:6MHF ) bound to Gαi (SwII region); R208 is critical for the interaction. B . Sequence alignment of human NOD1, NOD2, Gαi (SwII), and rabbit Gαi (PDB:5IRN). Black arrows mark residues prioritized for mutagenesis; red asterisks (*) and double asterisks (**) indicate strategies for designing NOD2 chimeras using NOD1 or Gαi sequences. C-D. Pulldown assays using GST or GST–GIV with lysates of HEK293 cells expressing WT or mutant NOD2 (panel C). Bound HA-NOD2 was visualized by immunoblot with anti-HA mAb (IB). Quantification of three independent biological repeats is shown in D. E-F. GST pulldown assays of recombinant His–GIV-CT or its F1685A mutant (deficient in binding to Gαi and predicted in A as deficient in binding NOD2) with GST–NOD2-LRR. Bound proteins were detected by immunoblot; equal loading confirmed by Ponceau S (GST) and immunoblotting (IB; anti-His mAb). Quantification from three independent repeats of the assay in panel F. G-I. GST pulldown assays of recombinant His–GIV-CT, His–Gαi WT, or W211A mutant (deficient in GIV-binding ) with GST–NOD2-LRR (panel E). Bound proteins were detected by immunoblot; equal input confirmed by IB (panel F). Quantification from three repeats shown in I. J. MDP-dependent suppression of FSK-induced cAMP levels in HeLa cells expressing WT or NOD2 L998 mutant, assessed using BRET-based CAMYEL biosensor (Bar graph of AUC). See also Supplementary Figure S5(A-B) for assay schematic and the timeline of Forskolin (FSK) treatment and additional visualization of readouts. See also Supplementary Figure S5(C-D) for 0-60 minutes MDP-dependent suppression of FSK-induced cAMP levels in HeLa cells expressing WT or NOD2 L998 mutant. K. Schematic of the NOD2●GIV●Gαi interface in cAMP control. ( Top) Upon sensing MDP (early ), WT NOD2 engages GIV first and then relays the signal to activate Gαi and blunt cAMP surges ( late) ; ( Bottom) whereas the NOD2 L998A mutant fails to engage GIV or relay the signal to Gαi proteins, resulting in sustained cAMP accumulation Statistics : All results are displayed as mean ± SEM (n = 3 biological replicates). Significance was tested using two-way/one-way ANOVA followed by Tukey’s test for multiple comparisons. p -value ≤ 0.05 is considered as significant.

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Binding Assay, Residue, Sequencing, Mutagenesis, Expressing, Western Blot, Recombinant, Control

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: A. Schematic of perturbagens used to dissect the cAMP cascade and GIV’s G protein regulatory function in phagolysosomal fusion. B . ELISA-based cAMP levels in WT and GIV-KO TGPMs infected with AIEC LF82, for 5 or 15 minutes. C. Quantification of normalized phospho-PKA substrate levels, normalized to β-Actin and expressed as fold-change over t₀ (time 0 minutes, baseline), from immunoblots from Supplementary Figure S6A . Lysates were prepared from WT and GIV-KO TGPMs infected with AIEC LF82 for 0–45 minutes following MDP pre-treatment (10 µg/ml). Bar graph quantification corresponds to Supplementary Figure S6B. See also, Supplementary Figure S6 ( F-G ) for immunoblots and quantification of normalized phospho-PKA substrate levels of HeLa WT and KO lysates stimulated with MDP for 0-45minutes. D-E. Viable bacterial counts in PMA-differentiated WT and GIV-KO THP-1 macrophages, untreated (−) or pre-treated (+) with PKA inhibitors (H89, 30 µM; PKI 14–22, 10 µM; BLU2864, 30 µM) or EPAC inhibitor (ESI-09, 10 µM) for 30 minutes prior to AIEC LF82 infection (1 hour, MOI 1:50). F. Percent change in viable bacterial counts in PMA-differentiated WT and GIV-KO THP-1 macrophages, untreated (−) or pre-treated (+) with 8-Br-cAMP (10 mM) or Fsk (20 µM) for 30 minutes prior to AIEC LF82 infection (1 hour, MOI 1:50). G. Domain architecture of TAT-GIV constructs . The TAT transduction domain (TAT-PTD) fused to His- and HA-tags, linked to the C-terminal GIV segment (aa 1660–1870). Key binding sites are indicated for NOD2 and Gαi (blue) and TLR4 (red). WT TAT-GIV-CT (top) and the CT-F1685A mutant (bottom) are shown. See also Supplementary Fig. 6D -E for purification and uptake assays. H. Workflow of gentamicin protection assay in WT and GIV-KO TGPMs pretreated with WT or F1685A TAT-GIV CT mutant (100 nM, 30 minutes), followed by AIEC LF82 infection (MOI 1:50, 1 hour). I. Viable bacterial counts in WT and GIV-KO TGPMs pretreated with WT or the F1685A TAT-GIV CT prior to AIEC LF82 infection. J. BRET-based cAMP assay in HeLa cells. Left : Workflow: cells transfected with 400 nM WT or F1685A TAT-GIV CT, followed by MDP treatment (10 µg/ml). Right : Normalized cAMP levels in WT and GIV-KO HeLa cells, ±WT or F1685A TAT-GIV CT, measured using CAMYEL biosensor. Statistics : Data are mean ± SEM (n = 3 biological replicates). Significance was tested by one-/two-way ANOVA with Tukey’s post-test; p ≤ 0.05 was considered significant.

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Construct, Transduction, Binding Assay, Mutagenesis, Purification, cAMP Assay, Transfection

Journal: bioRxiv

Article Title: A NOD2-Encoded Toggle Switch Resolves the Host–Microbe Battle Over Cyclic AMP Control

doi: 10.64898/2026.03.29.715116

Figure Lengend Snippet: B. The NOD2●GIV●Gαi module functions as a ‘toggle switch’ controlling host cAMP signaling in response to microbial cues. Left : The LRR domain of NOD2 binds the GEM motif of GIV, sequestering GIV from Gαi and transiently limiting inhibition of the AC→cAMP→PKA axis. This allows an early cAMP surge, known to restrain NFκB-dependent inflammation . Right : Subsequently, dissociation of NOD2●GIV complexes restore GIV●Gαi interactions at phagophore membranes, suppressing cAMP→CREB→PKA signaling to favor phagolysosome fusion. C. Comparison of dead versus live bacteria reveals that suppression of macrophage cAMP requires live microbes, which contribute to the initial cAMP surge via diverse mechanisms summarized in the schematic. D. Multilayered networks connect specific gut microbes (brown) and their cAMP-modulating microbial genes (green) to host proteins (PRRs, signaling molecules), highlighting disease-specific host pathways regulating cellular cAMP. Strong feedback inhibitory loops (red) are orchestrated by proteins that are localized to phagophore membranes (* asterisk). E. Multilayered networks linking gut microbes (brown) to host genes reveal both canonical GPCR-dependent (shown before ) and non-canonical GIV-dependent (this work) G protein signaling pathways enriched for disease-specific host pathways in IBD.

Article Snippet: For single PLA assay, coverslips were incubated overnight at 4°C with primary antibodies against GIV (rabbit, Millipore; ABT80, 1:150 dilution) and NOD2 (mouse, Santa Cruz, sc-56168,1:50 dilution), followed by incubation with species-specific PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidified chamber.

Techniques: Inhibition, Comparison, Bacteria, Protein-Protein interactions

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: TLR8 agonists remodel the tumor immune microenvironment through PF4-dependent T cell recruitment and ancillary mechanisms

doi: 10.1007/s00262-026-04329-8

Figure Lengend Snippet: Dynamics of immune cell subsets in response to agonist stimulation. A Schematic of the experimental workflow. Immune profiling by flow cytometry was performed 48 h after a single intratumoral injection of PRR agonists. B Percentage of CD45 + leukocytes among live cells following intratumoral administration of different agonists: TLR3 agonist poly IC (25 µg/tumor), TLR7 agonist imiquimod hydrochloride (25 µg/tumor), TLR8 agonist TL8-506 (10 µg/tumor), TLR9 agonist CpG ODN 2395 (50 µg/tumor), STING agonist ADU-S100 ammonium salt (25 µg/tumor), NOD1 agonist Tri-DAP (10 µg/tumor), and NOD2 agonist murabutide (5 µg/tumor). C Proportion of cDCs within the CD45 + population. D Percentage of ZsGreen + cells among DCs. E Proportion of macrophages within the CD45 + population. F Percentage of ZsGreen + cells among macrophages. G Proportion of CD8 + T cells within the CD45 + compartment. H Proportion of cCD4 + T cells within the CD45 + compartment. I Proportion of Tregs within the CD45 + compartment, pooled from two independent experiments. J Ratio of cCD4 + T cells to Tregs. (K) Ratio of CD8 + T cells to Tregs. Data represent the mean ± SEM ( n = 8 mice per group). Statistical comparisons were performed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: When tumors reached approximately 200 mm 3 , PRR agonists were administered via intratumoral injection at the following doses: TLR3 agonist poly(I:C) (Sigma-Aldrich, #42,424–50-0; 25 μg/tumor); TLR7 agonist imiquimod hydrochloride (MedChemExpress, #HY-B0180A; 25 μg/tumor); TLR8 agonist TL8-506 (InvivoGen, #tlrl-tl8506; 10 μg/tumor); TLR8 agonist motolimod (MedChemExpress, #HY-13773; 50 μg/tumor); TLR9 agonist CpG ODN 2395 (Class C) (InvivoGen, #tlrl-2395–1; 50 μg/tumor); STING agonist ADU-S100 ammonium salt (MedChemExpress, #HY-12885B; 25 μg/tumor); NOD1 agonist Tri-DAP (InvivoGen, #tlrl-tdap; 10 μg/tumor); and NOD2 agonist murabutide (InvivoGen, #tlrl-mbt; 5 μg/tumor).

Techniques: Flow Cytometry, Injection