nod like receptor family pyrin domain  (Proteintech)

Journal: bioRxiv

Article Title: The Yersinia pestis virulence effector YopM binds to a key regulatory site on the human pyrin death domain to inhibit inflammasome activation and effector-triggered immunity

doi: 10.64898/2026.02.25.707193

Figure Lengend Snippet: A) Domain arrangements of RSK, PRK, and monomeric human and mouse pyrin and regions targeted for binding by YopM. The C-terminus of YopM binds the N-terminal kinase domain of RSK, and the concave surface of YopM LRR domain binds the kinase domain of PRK and the PYDs of human and mouse pyrin, the latter weakly (dashed arrow). Color shad ing is used to denote amino acid sequence difference between human and murine PYDs. HR1=homology region 1; C2=C2-like domain; B=B-box; CC=coiled coil. B) Anti-parallel dimer of inactive pyrin resulting from coiled-coil interactions. C. Model of YopM hijacking and sequentially activating by phosphorylation RSK and PRK and different targeting mechanisms for human and mouse pyrin. Individual kinase domains of RSK and PRK and monomeric pyrin proteins are shown for simplicity. Phosphorylation of two serine residues in the pyrin linker results in binding of a 14-3-3 dimer and the formation of a hairpin that facilitates sequestration of the PYD by the B-box. To inactivate human pyrin YopM releases PRK prior to PYD binding and using hijacked RSK to phosphorylate the linker. Role of the released PRK is unknown. To inactivate mouse pyrin YopM uses hijacked PRK to target and phosphorylate the linker. Role of the B-box in negative regulation of mouse pyrin is unknown.

Article Snippet: Primary antibodies used in this study include rabbit-anti-mouse phospho-serine 241 monoclonal antibody (1:1,000 dilution, Ab201784; Abcam), rabbit anti-human pyrin monoclonal (1:1,000, Cell Signaling #58525 and #40649), rabbit-anti-mouse/human polyclonal β-actin (1:1,000 # 4967; Cell Signaling), rabbit anti- Y. pestis YopM polyclonal (1:10,000 dilution, antibody provided by Susan Straley) and mouse anti-HIS tag monoclonal (1:5,000 dilution, # MMS-156P BioLegend).

Techniques: Binding Assay, Sequencing, Phospho-proteomics

Journal: bioRxiv

Article Title: The Yersinia pestis virulence effector YopM binds to a key regulatory site on the human pyrin death domain to inhibit inflammasome activation and effector-triggered immunity

doi: 10.64898/2026.02.25.707193

Figure Lengend Snippet: A,B) The indicated YopM proteins were N-terminally tagged with T25, and hPYDs were C-terminally tagged with T18. Plasmids encoding the fused partners, ZIP positive controls or empty vector (EV) controls were co-transformed into E. coli to test for BACTH interactions as in . Significance determined by one-way ANOVA and Tukey’s post hoc test in comparison to PestA ( A ) or KIM ( B ) with hPYD. C,D) THP-1 cells were left uninfected (UI) or infected for 4 h at MOI 30 with Y. pseudotuberculosis yopJ C172A Δ yopM complemented with empty vector (EV) or the indicated YopM isoforms or variants. C) Immunoblot analysis of THP-1 lysates with the indicated antibodies. Band intensity signals show the ratio of phospho 241/total pyrin. Results are representative of two independent experiments. D) ELISA of IL-1β released from THP-1 cells. Results are averages and standard deviations with data pooled from at least 3 independent experiments. Significance determined by one-way ANOVA and Tukey’s post hoc test. Comparisons of each group to EV are shown. ****p<0.0001; ***p<0.001; ns, not significant.

Article Snippet: Primary antibodies used in this study include rabbit-anti-mouse phospho-serine 241 monoclonal antibody (1:1,000 dilution, Ab201784; Abcam), rabbit anti-human pyrin monoclonal (1:1,000, Cell Signaling #58525 and #40649), rabbit-anti-mouse/human polyclonal β-actin (1:1,000 # 4967; Cell Signaling), rabbit anti- Y. pestis YopM polyclonal (1:10,000 dilution, antibody provided by Susan Straley) and mouse anti-HIS tag monoclonal (1:5,000 dilution, # MMS-156P BioLegend).

Techniques: Plasmid Preparation, Transformation Assay, Comparison, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: NLRP10 engages oxidized DNA through a Schiff-base mechanism and dissociates from NLRP3 upon inflammasome activation

doi: 10.1038/s42003-025-09501-x

Figure Lengend Snippet: A Mitochondrial and cytosolic fractions were isolated from WT or NLRP3 KO iBMDMs treated with or without ATP. Amplification of 5698 and 591 bp fragments in both fractions are run on agarose gels. B Western blot analysis of mitochondrial and cytosolic fractions isolated from THP-1 cells, probed for NLRP3 on a PVDF membrane. β-actin serves as a loading control for the cytosolic fraction. VDAC confirms the absence of mitochondrial contamination in the cytosol, and GAPDH confirms the lack of cytosolic contamination in the mitochondria. C Quantification of NLRP3 in the mitochondria and cytosol with or without treatment with LPS and nigericin. Error bars: mean ± SEM, analyzed with two-way ANOVA. N = 3, ∗ p = 0.0256, ∗∗∗∗ p < 0.0001.

Article Snippet: Fractions were analyzed on a total protein NuPAGE 4–12% Bis-Tris gel run at 200 V for 30 min. Fractions were further analyzed with PVDF membrane western blots blocked in 2.5% BSA and probed with NLRP3 pyrin targeting (Adipogen) or an NLRP10 NACHT targeting antibody (Cell Signaling).

Techniques: Isolation, Amplification, Western Blot, Membrane, Control

Journal: Communications Biology

Article Title: NLRP10 engages oxidized DNA through a Schiff-base mechanism and dissociates from NLRP3 upon inflammasome activation

doi: 10.1038/s42003-025-09501-x

Figure Lengend Snippet: A NLRP3 helix one 3-17 is amphipathic with hydrophobic areas in orange and hydrophilic areas in blue (PDB 7PZC). B NLRP3 pyrin domain AlphaFold3 model shows an amphipathic first helix 1-16 with a predicted DeepLoc score of 0.6678. C NLRP3 SWISS-Model based on hOGG1 bound to oxidized DNA helix one 2-11 shows regions of hydrophobicity and hydrophilicity. DeepLoc prediction software of N-terminal residues 1–11 predicts association with mitochondria with a score of 0.8659.

Article Snippet: Fractions were analyzed on a total protein NuPAGE 4–12% Bis-Tris gel run at 200 V for 30 min. Fractions were further analyzed with PVDF membrane western blots blocked in 2.5% BSA and probed with NLRP3 pyrin targeting (Adipogen) or an NLRP10 NACHT targeting antibody (Cell Signaling).

Techniques: Software

Journal: Communications Biology

Article Title: NLRP10 engages oxidized DNA through a Schiff-base mechanism and dissociates from NLRP3 upon inflammasome activation

doi: 10.1038/s42003-025-09501-x

Figure Lengend Snippet: A IL-1β western blots of LPS-primed and nigericin-activated THP-1 cells treated with increasing doses of TH5487. Ponceau S. stain serves as a loading control. Representative pro-IL-1β western blot. Quantification of IL-1β western blot data. Error bars: mean ± SEM, analyzed with one-way ANOVA. N = 3, ∗∗∗∗ p < 0.0001, ∗∗∗ p = 0.0003, 0.0008, ∗ p = 0.0154. B IL-1β western blots of LPS-primed and nigericin-activated THP-1 cells treated with increasing doses of SU0268. Ponceau S. stain serves as a loading control. Representative pro-IL-1β western blot. Quantification of IL-1β western blot data. Error bars: mean ± SEM, analyzed with one-way ANOVA. N = 3, ∗∗ p = 0.0026, ∗ p = 0.0174. C NLRP3 IP pulldown of NLRP10 in LPS-primed nigericin-activated THP-1 cells. D Quantification of Western blot data. Error bars: mean ± SEM, analyzed with one-way ANOVA. N = 3, ∗∗∗∗ p < 0.0001. E Coomassie SDS gels following affinity chromatography for NLRP10 (left) and size exclusion chromatography for NLRP3 (right). F NLRP3 IP pulldown of NLRP10 with purified protein. G Quantification of Western blot data. Mean ± SEM, one-way ANOVA. N = 3, ∗ p = 0.0129, ∗∗∗ p = 0.0007.

Article Snippet: Fractions were analyzed on a total protein NuPAGE 4–12% Bis-Tris gel run at 200 V for 30 min. Fractions were further analyzed with PVDF membrane western blots blocked in 2.5% BSA and probed with NLRP3 pyrin targeting (Adipogen) or an NLRP10 NACHT targeting antibody (Cell Signaling).

Techniques: Western Blot, Staining, Control, Affinity Chromatography, Size-exclusion Chromatography, Purification