NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation

Abstract

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

Keywords: CP: Immunology; NLRP3; ZDHHC7; endotoxic shock; inflammasome; palmitoylation; peritonitis; trans-Golgi network localization.

Figure 1.. NLRP3 is S-palmitoylated by ZDHHC7 in macrophages

(A) Palmitoylation of NLRP3 in BMDMs by Alk14 labeling and click chemistry assay. The NLRP3 palmitoylation level was quantified and normalized to NLRP3 protein levels in the input samples. (B) NLRP3 palmitoylation in PMA-differentiated THP-1 cells treated with Alk14, LPS, and nigericin. (C) Relative mRNA of Zdhhc genes in BMDMs determined by quantitative real-time qPCR; the mRNA level was normalized to β-actin. Data are represented as mean ± SEM. (D) Representative confocal images of NLRP3 and ZDHHC7 in LPS-primed BMDMs; the nucleus was stained with DAPI. Scale bar: 5 μm. (E) THP-1 cells were differentiated with PMA and then treated with LPS and nigericin. Cell lysates were collected for IP to detect endogenous NLRP3-ZDHHC7 association. (F) Palmitoylation of FLAG-NLRP3 in HEK293Tcells with ZDHHC7 expression was detected using Alk14 labeling and in-gel fluorescence. Hydroxylamine (HAM) treatment was used to confirm S-palmitoylation, which is HAM sensitive; the NLRP3 palmitoylation level was normalized to NLRP3 protein levels in the IP samples. (G) NLRP3 palmitoylation in WT and Zdhhc7 KO BMDMs, determined with Alk14 labeling. Cells were primed with LPS and activated with ATP as indicated. (H) Palmitoylation of NLRP3 was determined in WT and ZDHHC7 KO THP-1 cells. See also Figures S1–S4.

Figure 2.. NLRP3 is S-palmitoylated on Cys126 by ZDHHC7

(A) Schematic of NLRP3 domains, showing the location of Cys126 and sequence alignment of NLRP3 from different species. (B) Palmitoylation of mouse NLRP3 WT and cysteine-to-serine mutants (CS) in HEK293T cells, determined with Alk14 labeling. (C and D) Palmitoylation of the mouse (C) and human (D) NLRP3 WT and cysteine-to-serine mutant (C126S of mouse NLRP3, C130S of human NLRP3) in HEK293T cells. (E) The generation of the CRISPR-edited NLRP3 C126A mutant (top) and sequencing verification (bottom). (F) Palmitoylation of NLRP3 in WT and C126ABMDMs by Alk14 labeling. (G) Palmitoylation of mouse NLRP3 WT and C126S that were reconstituted stably in NLRP3-deleted THP-1 cells. (H) Palmitoylation of human NLRP3 WT and C130S that were reconstituted stably in NLRP3-deleted THP-1 cells.

Figure 3.. ZDHHC7-mediated NLRP3 Cys126 palmitoylation is important for NLRP3 inflammasome activation in macrophages

(A and B) Immunoblot analysis of NLRP3, caspase-1, GSDMD, cleaved caspase-1 (p20), and cleaved GSDMD (NT) in whole-cell lysate (WCL) of NLRP3 WT, C126A, and KO (N3-KO) BMDMs that were primed with LPS and activated with nigericin (Nig, A) or ATP (B). Cells were lysed with 2.5% SDS lysis buffer. (C and D) Levels of IL-1β (C) and IL-18 (D) in the cell culture medium of BMDMs in (A). (E) Immunoblot analysis in WCL of NLRP3 WT and C126A BMDMs that had Zdhhc7 knocked down (siZd7), primed with LPS, and activated with Nig. (F) Immunoblot analysis in WCL of NLRP3 WT and C126A (CA) BMDMs that were primed with LPS and treated with DMSO or 2-BP before activation with Nig. (G) Immunoblot analysis in NLRP3-deleted THP-1 cells that were reconstituted with WT or C126S (CS) NLRP3 and had ZDHHC7 knocked down (siZD7). Data with error bars are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired Student’s t test. See also Figure S5.

Figure 4.. NLRP3 Cys126 palmitoylation promotes NLRP3 localization on the TGN

(A) Representative confocal images of GFP-tagged human NLRP3 WT or C130S expressed in HEK293T cells that were treated with DMSO or 2-BP. NLRP3-GFP is shown in green, the TGN was stained with anti-58K Golgi (red), and the nucleus was stained with DAPI (blue). Scale bar: 5 μm. (B) Quantification of images in (A) by Pearson’s correlation coefficient using ImageJ. (C) Confocal images of the WT and CS mutant of mouse NLRP3-FLAG in HEK293T cells that were treated with Nig for the indicated times. NLRP3 was stained with anti-FLAG (green), the TGN was stained with anti-TGN38 (red), and the nucleus was stained with DAPI (blue). Scale bar: 5 μm. (D) Quantification of images in (C) by Pearson’s correlation coefficient using ImageJ. (E) Representative confocal images for endogenous NLRP3 in WT and Zdhhc7 KO (Zd7-KO) BMDMs. NLRP3 was stained green, the TGN was stained with anti-58K Golgi (red), and the nucleus was stained with DAPI (blue). Scale bar: 5 μm. (F) Quantification of images in (E) by Pearson’s correlation coefficient. Data with error bars are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired Student’s t test. See also Figure S6.

Figure 5.. Cys126 palmitoylation promotes ASC recruitment and oligomerization after inflammasome activation

(A) Immunoblot analysis of NLRP3 interaction with ASC expressed in HEK293T cells by coIP with DMSO or 2-BP treatment for 12 h. (B) NLRP3 interaction with ASC expressed in WT or ZDHHC7 KO (ZD7-KO) HEK293T cells detected by coIP. (C and D) NLRP3 interaction with ASC in WT and Zdhhc7 KO BMDMs (C) or WT and Nlrp3 C126A BMDMs (D). Cells were primed with LPS and activated with Nig. IP was performed with anti-NLRP3 (anti-N3), and immunoblotting was done with anti-ASC and anti-NLRP3. (E–G) Immunoblot analysis of insoluble ASC and its oligomerization in ZDHHC7 WT or KO BMDMs (E), in BMDMs that were treated with DMSO or 2-BP (F), and in NLRP3 WT, C126A, or N3-KO BMDMs (G). Disuccinimidyl suberate (DSS) was used to cross-link ASC in BMDMs activated with ATP or Nig as indicated. See also Figure S6.

Figure 6.. ZDHHC7-promoted Cys126 palmitoylation affects NLRP3 inflammasome activation in vivo

(A and B) Mouse serum IL-1β and IL-18 (A) and IL-6 and TNF-α (B), measured by ELISA for WT mice with LPS-induced endotoxic shock after administration of DMSO or 2-BP (LPS, n = 5 or 4; PBS, n = 4). (C) Survival data of mice in response to LPS challenge after administration of DMSO or 2-BP (n = 6). The survival curve was statistically analyzed with log rank (Mantel-Cox) test. (D and E) Mouse serum IL-1β and IL-18 (D) and TNF-α (E), measured by ELISA for ZDHHC7 WT and KO (Zd7-KO) mice with endotoxic shock (n = 9). Mice were administered LPS for 8 h, and serum was collected for ELISA. (F) Serum IL-1β and HMGB1, measured by ELISA, for NLRP3 WT and C126A mice with endotoxic shock (n = 5). (G and H) Numbers of peritoneal exudate cells (PECs) and infiltrated neutrophils and macrophages in peritoneal lavage fluid (G) and ELISA of IL-1β in the serum (H) of ZDHHC7 WT and KO (Zd7-KO) mice that were injected intraperitoneally (i.p.) with LPS and MSU (n = 5). Data with error bars are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired Student’s t test. See also Figure S7.

Figure 7.. ZDHHC7 promotes NLRP3 activation, while ZDHHC12 terminates inflammasome signaling

(A) Palmitoylation of NLRP3 in ZDHHC7-deleted (sgZD7), ZDHHC12-deleted (sgZD12), or control (sgCtrl) THP-1 cells differentiated with PMA, primed with LPS, and activated with Nig for 1 h. (B) Palmitoylation of NLRP3 in WT or NLRP3-KO THP-1 cells reconstituted with NLRP3 WT or cysteine-to-serine (CS) mutants. (C) Palmitoylation of NLRP3 WT or cysteine-to-alanine (CA) mutants expressed in HEK293T cells with ZDHHC12 (ZD12) co-expression. (D) Palmitoylation of NLRP3 WT or CA mutants in HEK293T cells with ZDHHC7 (ZD7) co-expression. (E) Immunoblot analysis of the WCL of sgZD7, sgZD12, or sgCtrl THP-1 cells differentiated with PMA, primed with LPS, and activated with Nig for 1 h. (F) ELISA of human IL-1β in the cell culture medium of THP-1 cells in (E). (G) Immunoblot analysis of NLRP3 and cleaved GSDMD (NT) in the WCL of WT or NLRP3-KO THP-1 cells that were reconstituted with the NLRP3 WT or CS mutant, differentiated with PMA, primed with LPS, and activated with Nig for 1 h. (H) ELISA of human IL-1β in the cell culture medium in (G). Data with error bars are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by unpaired Student’s t test.