Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: ( A ) HeLa cells stably expressed GFP-ZNFX1 and mCherry-NLRP3 were treated with nigericin for the indicated times, fixed, and stained with anti-TGN46 antibody. Scale bar, 10 μm. ( B ) Quantification of the percentage of cells showing NLRP3 translocation from 100 cells in ( A ). n = 4 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. ( C ) WT, ZNFX1 KO, ZNFX1 rescue, and ZNFX1 OE THP-1-derived macrophages were fixed and stained with anti-TGN46 and anti-NLRP3 antibodies, followed by secondary antibodies conjugated with Alex fluor-647 and Alex fluor-568. Scale bar, 10 μm. ( D ) Quantification of the percentage of cells showing NLRP3 puncta on TGN46+ vesicles from 100 cells in ( C ). n = 4 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. ( E ) ZNFX1 KO HeLa cells were co-expressed with mMaroon1-ASC, mCherry-NLRP3, and GFP-ZNFX1. Fluorescence imaging was captured under a 63X objective using Leica SP8 laser scanning microscopy. Scale bar, 10 μm. ( F ) Quantification of cells containing colocalized proteins in ( E ). n = 4 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. ( G ) ZNFX1 rescue THP-1-derived macrophages were treated with the indicated inflammasome agonists (Nig, 45 min; polydA:dT, 12 h; MDP, 16 h; salm, 16 h). Cells were fixed, and GFP-ZNFX-1 and ASC specks were detected with GFP-based fluorescence and anti-ASC-based immunofluorescence, respectively. Inset shows ASC specks and GFP-ZNFX1. Scale bar, 10 μm. ( H ) Quantification of cells with ASC specks containing GFP-ZNFX1 in ( G ) from 100 cells. n = 3 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. .

Article Snippet: Cells were incubated with the following primary antibodies overnight at 4 °C: anti-NLRP3 (AdipoGen, 1:250), anti-ASC, anti-TGN46, anti-TGN38 (Novus Biologicals, 1:100), and anti-ZNFX1(Abcam, 1:250).

Techniques: Stable Transfection, Staining, Translocation Assay, Derivative Assay, Fluorescence, Imaging, Laser-Scanning Microscopy, Immunofluorescence

Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: ( A ) Predicted domains and identified variants in the ZNFX1 amino acid sequence. ( B ) The ZNFX1 open reading frame containing three nonsynonymous patient mutations was reintroduced to ZNFX1 KO THP-1 cells through lentivirus-mediated delivery. Cells were primed with LPS and stimulated with nigericin for 1 h. Proteins in the culture medium supernatant and cell lysate were detected using the indicated antibodies. ( C ) Quantification of caspase-1 P20 level in ( B ). n = 3 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. For each biological replicate, band intensity was measured using ImageJ. The WT control was set to 1, and the ratio for ZNFX1 KO cells was calculated by dividing their intensity by the corresponding WT control intensity. ( D ) WT, ZNFX1 RE, and ZNFX1 mutants THP-1 derived macrophages were fixed and stained with anti-TGN46 and anti-NLRP3 antibodies, followed by Alex fluor-647 and Alex fluor-568 conjugated secondary antibodies. Scale bar, 10 μm. ( E ) Quantification of the percentage of cells with NLRP3’s translocation to the TGN from 100 cells in ( D ). n = 4 biological replicates, mean ± s.d., two-sided Student’s t -test. ( F ) ZNFX1 KO THP-1 derived macrophages rescued with WT or pathogenic alleles of ZNFX1 were immunoprecipitated with FLAG-M2 beads. Proteins in input cell lysate and immunoprecipitate were detected using indicated antibodies. ( G , H ) Cells with the indicated genotype were primed with LPS and treated with nigericin, with or without MCC950 pretreatment. NLRP3 inflammation activation was measured by immunoblot in ( G ) and ELISA in ( H ). n = 4 biological replicates for ( H ), mean ± s.d., Student’s t -test, two-tailed. ( I ) Working model for ZNFX1 in inhibiting NLRP3 inflammasome. In the resting stage, ZNFX1 directly interacts with smaller NLRP3 species to sequester NLRP3 in the cytoplasm, preventing its translocation to TGN vesicles to form cage-like structures. NLRP3 agonists disrupt the NLRP3-ZNFX1 interaction, promoting NLRP3 translocation to the TGN and initiating NLRP3 inflammasome activation. After activation, ZNFX1 is likely recruited to the mature NLRP3 inflammasome, where caspase-1 cleaves ZNFX1 to fragments, further releasing NLRP3 and forming a feed-forward loop. ZNFX1 with missense human pathogenic lesions cannot interact with NLRP3, presumably due to conformational changes, leading to hyperinflammation-related diseases when stimulated by NLRP3 activators. .

Article Snippet: Cells were incubated with the following primary antibodies overnight at 4 °C: anti-NLRP3 (AdipoGen, 1:250), anti-ASC, anti-TGN46, anti-TGN38 (Novus Biologicals, 1:100), and anti-ZNFX1(Abcam, 1:250).

Techniques: Sequencing, Control, Derivative Assay, Staining, Translocation Assay, Immunoprecipitation, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: ( A , B ) WT ZNFX1 or ZNFX1 containing Walker A or Walker B motif mutations were introduced back to ZNFX1 KO THP-1 cells by lentivirus-mediated delivery. Cells were primed with LPS and stimulated with nigericin. Proteins from medium supernatant or cell lysate were examined with immunoblot ( A ) and ELISA ( B ). n = 4 biological replicates, mean ± s.d., Student’s t -test, two-tailed. ( C ) WT, ZNFX1 KO, or ZNFX1 KO cells complemented with ZNFX1 harboring helicase mutations were fixed and stained with anti-TGN46 and anti-NLRP3 antibodies, followed by Alex fluor-647 and Alex fluor-568 conjugated secondary antibodies, respectively. Scale bar, 10 μm. ( D ) Quantification of the percentage of cells with NLRP3’s TGN translocation from 100 cells in ( C ). n = 3, mean ± s.d., two-sided Student’s t -test. ( E ) Coimmunoprecipitation analysis of NLRP3 with WT or helicase mutants of ZNFX1. Proteins in input cell lysate, as well as precipitation, were detected with indicated antibodies. ( F , G ) Cells with indicated genotype were primed with LPS and treated with nigericin, with or without MCC950 pretreatment, NLRP3 inflammation activation was measured by ELISA in ( F ) and immunoblot in ( G ). n = 4 biological replicates, mean ± s.d., Student’s t -test, two-tailed. .

Article Snippet: Cells were incubated with the following primary antibodies overnight at 4 °C: anti-NLRP3 (AdipoGen, 1:250), anti-ASC, anti-TGN46, anti-TGN38 (Novus Biologicals, 1:100), and anti-ZNFX1(Abcam, 1:250).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Translocation Assay, Activation Assay

Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cells were incubated with the following primary antibodies overnight at 4 °C: anti-NLRP3 (AdipoGen, 1:250), anti-ASC, anti-TGN46, anti-TGN38 (Novus Biologicals, 1:100), and anti-ZNFX1(Abcam, 1:250).

Techniques: Mouse Assay, Recombinant, Mutagenesis, Sequencing, SYBR Green Assay, Modification, Magnetic Beads, Protease Inhibitor, Membrane, Enzyme-linked Immunosorbent Assay, Software