Journal: bioRxiv

Article Title: A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

doi: 10.1101/2022.03.22.485305

Figure Lengend Snippet: ( A ) Domain organization of CARD8. CARD8 undergoes autoproteolysis between the ZU5 and UPA subdomains. The size in kilodaltons (kDa) of each fragment is indicated. ( B ) Lysates of the indicated cell lines were analyzed by immunoblotting. ( C ) HEK 239T cells were transfected with plasmids encoding the indicated FLAG-tagged constructs (left). Lysates were analyzed by immunoblotting (right). ( D ) Edman degradation analysis revealed residues F150 to Y156 (colored red) as the N-terminus CARD8 p44 . F150 is shown on the CARD8 structure predicted by Alpha-Fold . ( D ) CARD8 −/− THP-1 cells stably expressing CARD8 WT, S297A, or β1-149 were treated with compounds for 5 hours before LDH release and immunoblotting analyses. ( F ) HEK 293T CASP1+GSDMD cells were transfected with plasmids encoding the indicated plasmids. After 24 h, cells were treated with dTAG-13 for 5 h before LDH release and immunoblot analyses. Data in ( E ) (n=3) and ( F ) (n=3) are means ± standard deviation (SD) of replicates. **** p < 0.0001, by Student’s two-sided t -test. NS, not significant. All data, including immunoblots, are representative of three or more independent experiments.

Article Snippet: Antibodies used include: GSDMD rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), Cleaved-GSDMD NT rabbit monoclonal Ab (Abcam, Ab215203), PARP rabbit polyclonal Ab (Cell Signaling Tech, 9542), GAPDH rabbit monoclonal Ab (Cell Signaling Tech, 14C10), DPP9 rabbit polyclonal (Abcam, ab42080), HA rabbit monoclonal Ab (Cell Signaling Tech, C29F4), HA mouse monoclonal Ab (Cell Signaling Tech, 6E2), CARD8 CT rabbit polyclonal Ab (Abcam, ab24186), CARD8 NT rabbit polyclonal Ab (Abcam, ab194585), GFP rabbit polyclonal Ab (Cell Signaling Tech, 2555), V5 mouse monoclonal Ab (Sigma Aldrich, V8012), FLAG mouse monoclonal Ab (Sigma Aldrich, F1804), 20S Proteasome β5 mouse monoclonal (Santa Cruz Biotech, sc-393931), Ubiquitin rabbit polyclonal (Cell Signaling Tech, 3933), IRDye 800CW donkey anti-rabbit (LICOR, 925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072).

Techniques: Western Blot, Transfection, Construct, Stable Transfection, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

doi: 10.1101/2022.03.22.485305

Figure Lengend Snippet: ( A ) HEK 293T cells were transiently transfected with C-terminally FLAG-tagged CARD8 FL S297A and incubated for 24 h. In the last 6 hours, the cells were treated as indicated before lysates were harvested. FLAG-tagged proteins were then enriched on anti-FLAG beads, and the eluates were analyzed by immunoblotting. It should be noted that the signals detected on the anti-ubiquitin immunoblots in the BORT-treated samples were likely due to ubiquitinated substrates that bound non-specifically to the anti-FLAG beads, as no laddering pattern of CARD8 (in both the anti-UBQ and FLAG immunoblots) was observed. ( B ) HEK 293T cells were transiently transfected with the indicated plasmid constructs prior to immunoblotting analyses. It should be noted that the laddering of the CT protein is likely due to incomplete dissociation of its oligomers by the reducing agent and boiling procedure. ( C ) HEK 293T cells were transiently transfected with the indicated constructs. Lysates were harvested and HA-tagged proteins were enriched. The input and elution fractions were analyzed by immunoblotting. The CARD8 E274R mutation renders its inability to bind to DPP8/9. ( D ) HEK 293T CASP1+GSDMD cells were transfected with plasmids encoding the indicated plasmids. After 24 h, cells were treated with dTAG-13 for 5 h before immunoblotting analyses. ( E ) CARD8 −/− THP-1 cells stably expressing CARD8 FL K10R were treated with the indicated compounds prior to LDH release and immunoblotting analyses. ( F ) A tet-on plasmid encoding CARD8 was transfected into HEK 293T cells. After 20 h, cells were co-treated with the indicated drugs for 24 h before lysates were analyzed by immunoblotting. All immunoblots are representative of 3 or more independent experiments.

Article Snippet: Antibodies used include: GSDMD rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), Cleaved-GSDMD NT rabbit monoclonal Ab (Abcam, Ab215203), PARP rabbit polyclonal Ab (Cell Signaling Tech, 9542), GAPDH rabbit monoclonal Ab (Cell Signaling Tech, 14C10), DPP9 rabbit polyclonal (Abcam, ab42080), HA rabbit monoclonal Ab (Cell Signaling Tech, C29F4), HA mouse monoclonal Ab (Cell Signaling Tech, 6E2), CARD8 CT rabbit polyclonal Ab (Abcam, ab24186), CARD8 NT rabbit polyclonal Ab (Abcam, ab194585), GFP rabbit polyclonal Ab (Cell Signaling Tech, 2555), V5 mouse monoclonal Ab (Sigma Aldrich, V8012), FLAG mouse monoclonal Ab (Sigma Aldrich, F1804), 20S Proteasome β5 mouse monoclonal (Santa Cruz Biotech, sc-393931), Ubiquitin rabbit polyclonal (Cell Signaling Tech, 3933), IRDye 800CW donkey anti-rabbit (LICOR, 925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072).

Techniques: Transfection, Incubation, Western Blot, Ubiquitin Proteomics, Plasmid Preparation, Construct, Mutagenesis, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

doi: 10.1101/2022.03.22.485305

Figure Lengend Snippet: ( A ) CARD8 −/− THP-1 stably expressing the indicated constructs were treated with the compounds for 8 hours before LDH release and immunoblotting analyses. PARP (poly ADP-ribose polymerase 1) cleavage to its p89 fragment was assessed as an indicator of apoptosis. ( B ) CARD8 −/− THP-1 cells stably expressing the indicated constructs were treated with the indicated compounds for 24 h before LDH release and immunoblotting analyses. Data in ( A ) and ( B ) are means ± standard deviation (SD) of three replicates. **** p < 0.0001 by Student’s two-sided t -test. NS, not significant. ( C ) Lysates of CARD8 −/− THP-1 cells stably expressing the indicated constructs were analyzed by immunoblotting. ( D ) HEK 293T CASP1+GSDMD cells were transiently transfected with plasmids encoding the WT and K26R CT fragments and incubated for 16 h prior to immunoblotting analyses. All data, including immunoblots, are representative of three or more independent experiments. ( E ) Proposed model for the regulation of CARD8 by the 20S proteasome. In unstressed cells, the 20S proteasome removes the disordered region to generate CARD8 p44 , which acts as an inhibitor in the DPP8/9 ternary complex. In VbP-stressed cells, the 20S degrades the entire CARD8 NT fragment, possibly due to ZU5 domain misfolding, releasing CARD8 CT from autoinhibition. Increased degradation of the full NT fragment enables the CT fragment to overcome the ternary complex.

Article Snippet: Antibodies used include: GSDMD rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), Cleaved-GSDMD NT rabbit monoclonal Ab (Abcam, Ab215203), PARP rabbit polyclonal Ab (Cell Signaling Tech, 9542), GAPDH rabbit monoclonal Ab (Cell Signaling Tech, 14C10), DPP9 rabbit polyclonal (Abcam, ab42080), HA rabbit monoclonal Ab (Cell Signaling Tech, C29F4), HA mouse monoclonal Ab (Cell Signaling Tech, 6E2), CARD8 CT rabbit polyclonal Ab (Abcam, ab24186), CARD8 NT rabbit polyclonal Ab (Abcam, ab194585), GFP rabbit polyclonal Ab (Cell Signaling Tech, 2555), V5 mouse monoclonal Ab (Sigma Aldrich, V8012), FLAG mouse monoclonal Ab (Sigma Aldrich, F1804), 20S Proteasome β5 mouse monoclonal (Santa Cruz Biotech, sc-393931), Ubiquitin rabbit polyclonal (Cell Signaling Tech, 3933), IRDye 800CW donkey anti-rabbit (LICOR, 925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072).

Techniques: Stable Transfection, Expressing, Construct, Western Blot, Standard Deviation, Transfection, Incubation

Journal: Arthritis research & therapy

Article Title: Endoplasmic reticulum aminopeptidase 2 regulates CD4 + T cells pyroptosis in rheumatoid arthritis.

doi: 10.1186/s13075-024-03271-3

Figure Lengend Snippet: Fig. 1 Pyroptosis of CD4+ T cells is increased in RA patients. A Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression in CD4+ T cells from NCs and RA patients. B Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in A (n = 3). C Concentrations of secreted IL-1β measured by ELISAs (n = 5). D CD4+ T cell death was quantified for control and RA CD4+ T cells on day 3 after stimulation by flow cytometric analysis of Annexin V+ 7AAD+ CD4+ T cells (n = 5). Representative flow plots of Annexin V and 7AAD staining of CD4+T cells and a histogram of the percentage of Annexin V+ 7AAD+ CD4+T cells are shown. E CD4+ T cell death was quantified by measuring LDH release (n = 5). F Cleaved Caspase-1 was identified by FLICA staining and flow cytometric analysis. Representative scatterplots of FLICA+ CD4+ T cells in the controls and RA patients are shown. G NLRP3, Caspase-1, GSDMD, and ASC transcripts compared in NCs and RA CD4 + T cells. All data are presented as the mean ± SD. Statistical significance was determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis; ASC, apoptosis-associated speck-like protein containing a CARD; NLRP3: NOD-like receptor protein 3; CASP1, Caspase-1; GSDMD-N, N-terminal pore-forming domain of GSDMD

Article Snippet: Antibodies were against ASC/TMS1 (Proteintech, 10,500- 1-AP), NLRP3 (Proteintech, 19,771–1-AP), GLI1 (Proteintech, 66,905–1-Ig), Caspase-1 (ab207802) and ERAP2 (Abcam, ab69037) from Abcam (Cambridge, USA), GSDMD (NBP2-33,422) from Novus (CO, USA), SHH (sc-166685) and SMO (Santa Cruz, sc-365112) from Santa Cruz (TX, USA).

Techniques: Western Blot, Expressing, Control, Staining

Journal: Arthritis research & therapy

Article Title: Endoplasmic reticulum aminopeptidase 2 regulates CD4 + T cells pyroptosis in rheumatoid arthritis.

doi: 10.1186/s13075-024-03271-3

Figure Lengend Snippet: Fig. 2 ERAP2 regulates pyroptosis in CD4+ T cells. NC and RA CD4+ T cells were stimulated for 72 h. Then, NC CD4+ T cells were infected with control shRNA virus (NC-shcontrol), and RA CD4+ T cells were infected with control shRNA virus or ERAP2 shRNA virus (RA-shcontrol/ RA-shERAP2). A, B ERAP2 expression in isolated CD4+ T cells is shown in representative immunoblots (left) and plots of the relative band density for NCs (n = 5) and RA patients (n = 5) with normalization to β-actin expression (right). C ERAP2 transcript levels in NC and RA CD4+ T cells (n = 5). D, E Flow cytometric analysis of CD4+ T cells treated as indicated and stained with Annexin V/7AAD (n = 3). Representative flow plots of Annexin V and 7AAD staining of CD4+ T cells and a histogram of the percentage of Annexin V+ 7AAD+ CD4+ T cells are shown, and representative scatterplots of FLICA+ cells identified by flow cytometric analysis (n = 3). F Concentrations of secreted IL-1β measured by ELISAs (n = 5). G, H Pyroptotic cells were identified by Hoechst 33,342/PI staining; the nuclei were stained blue with Hoechst 33,342, while pyroptotic cells were stained red with PI (n = 3). Scale bars: 50 μm. I T-cell death was quantified by measuring LDH release (n = 5). J Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). K Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in J (n = 3). All data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparisons test for multigroup comparisons or Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Article Snippet: Antibodies were against ASC/TMS1 (Proteintech, 10,500- 1-AP), NLRP3 (Proteintech, 19,771–1-AP), GLI1 (Proteintech, 66,905–1-Ig), Caspase-1 (ab207802) and ERAP2 (Abcam, ab69037) from Abcam (Cambridge, USA), GSDMD (NBP2-33,422) from Novus (CO, USA), SHH (sc-166685) and SMO (Santa Cruz, sc-365112) from Santa Cruz (TX, USA).

Techniques: Infection, Control, shRNA, Virus, Expressing, Isolation, Western Blot, Staining

Journal: Arthritis research & therapy

Article Title: Endoplasmic reticulum aminopeptidase 2 regulates CD4 + T cells pyroptosis in rheumatoid arthritis.

doi: 10.1186/s13075-024-03271-3

Figure Lengend Snippet: Fig. 3 Overexpression of ERAP2 promotes CD4+ T cell pyroptosis. Normal CD4+ T cells were stimulated for 72 h. Then, CD4+ T cells were infected with control lentivirus or lentivirus encoding ERAP2 (LV-NC/ LV-ERAP2). A Flow cytometric analysis of CD4+ T cells treated as indicated and stained with Annexin V/7AAD (n = 3). Representative flow plots of Annexin V and 7-AAD staining of CD4+T cells and a histogram of the percentage of Annexin V+ 7AAD+ CD4+T cells are shown. B Concentrations of secreted IL-1β measured by ELISAs (n = 5). C Representative scatterplots of FLICA+ cells identified by flow cytometric analysis (n = 3). D T-cell death was quantified by measuring LDH release (n = 5). E Pyroptotic cells were identified by Hoechst 33,342/PI staining; the nuclei were stained blue with Hoechst 33,342, while pyroptotic cells were stained red with PI (n = 3). Scale bars: 50 μm. F Representative immunoblots showing ASC, NLRP3, cleaved Caspase-1, and GSDMD-N expression (n = 3). G Quantification of ASC, NLRP3, cleaved Caspase-1, and GSDMD-N protein expression shown in F (n = 3). All data are presented as the mean ± SD. Statistical significance was determined by Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Article Snippet: Antibodies were against ASC/TMS1 (Proteintech, 10,500- 1-AP), NLRP3 (Proteintech, 19,771–1-AP), GLI1 (Proteintech, 66,905–1-Ig), Caspase-1 (ab207802) and ERAP2 (Abcam, ab69037) from Abcam (Cambridge, USA), GSDMD (NBP2-33,422) from Novus (CO, USA), SHH (sc-166685) and SMO (Santa Cruz, sc-365112) from Santa Cruz (TX, USA).

Techniques: Over Expression, Infection, Control, Staining, Western Blot, Expressing

Journal: Arthritis research & therapy

Article Title: Endoplasmic reticulum aminopeptidase 2 regulates CD4 + T cells pyroptosis in rheumatoid arthritis.

doi: 10.1186/s13075-024-03271-3

Figure Lengend Snippet: Fig. 5 ERAP2 induces pyroptosis in RA CD4 + T cells by inhibiting the Hedgehog signaling pathway. CD4+ T cells were stimulated for 72 h. Then, normal CD4+ T cells were infected with control lentivirus or lentivirus encoding ERAP2 (LV-NC/ LV-ERAP2). RA CD4 + T cells were infected with control shRNA virus or ERAP2 shRNA virus (RA-shcontrol/ RA-shERAP2). A, B SHH, SMO, and GLI1 expression in isolated CD4 + T cells is shown in representative immunoblots (left) and plots of the relative band density for NCs (n = 3) and RA patients (n = 3) with normalization to β-actin expression (right). C, D ERAP2, SHH, SMO, and GLI1 expression in isolated CD4 + T cells with overexpression or knockdown is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). E, F GLI1, ASC, NLRP3, cleaved Caspase-1 and GSDMD-N expression in isolated CD4 + T cells with GANT58 treatment (20 µm) is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). G, H GLI1, ASC, NLRP3, cleaved Caspase-1 and GSDMD-N expression in isolated CD4 + T cells with Purmorphamine treatment (20 µm) is shown in representative immunoblots (left) and plots of the relative band density with normalization to β-actin expression (right). All data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple-comparisons test for multigroup comparisons or Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, normal control; RA, rheumatoid arthritis

Article Snippet: Antibodies were against ASC/TMS1 (Proteintech, 10,500- 1-AP), NLRP3 (Proteintech, 19,771–1-AP), GLI1 (Proteintech, 66,905–1-Ig), Caspase-1 (ab207802) and ERAP2 (Abcam, ab69037) from Abcam (Cambridge, USA), GSDMD (NBP2-33,422) from Novus (CO, USA), SHH (sc-166685) and SMO (Santa Cruz, sc-365112) from Santa Cruz (TX, USA).

Techniques: Infection, Control, shRNA, Virus, Expressing, Isolation, Western Blot, Over Expression, Knockdown