USP18 Antagonizes Pyroptosis by Facilitating Selective Autophagic Degradation of Gasdermin D

As a key executioner of pyroptosis, Gasdermin D (GSDMD) plays a crucial role in host defense and emerges as an essential therapeutic target in the treatment of inflammatory diseases. So far, the understanding of the mechanisms that regulate the protein level of GSDMD to prevent detrimental effects and maintain homeostasis is currently limited. Here, we unveil that ubiquitin-specific peptidase 18 (USP18) works as a negative regulator of pyroptosis by targeting GSDMD for degradation and preventing excessive innate immune responses. Mechanically, USP18 recruits E3 ubiquitin ligase mind bomb homolog 2 (MIB2) to catalyze ubiquitination on GSDMD at lysine (K) 168, which acts as a recognition signal for the selective autophagic degradation of GSDMD. We further confirm the alleviating effect of USP18 on LPS-triggered inflammation in vivo. Collectively, our study demonstrates the role of USP18 in regulating GSDMD-mediated pyroptosis and reveals a previously unknown mechanism by which GSDMD protein level is rigorously controlled by selective autophagy.

In (B and C), data are represented as mean values ± SEM, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.
Quantitative analysis of relative protein level of GSDMD was shown in (H and J), respectively.(K and L) HEK293T cells were transfected with Flag-GSDMD, together with Myc-EV or Myc-USP18 for 18 h, then treated with Earle's balanced salt solution (EBSS) as indicated time points.Cell lysates were collected for immunoblot analysis (K).Quantitative analysis of relative protein level of GSDMD was shown in (L).(M to P) Immunoblot analysis of wild type (WT), BECN1-knockout (KO, M), or ATG5-KO (O) HEK293T cells transfected with Flag-GSDMD for 18 h, then treated with CHX (100 μg/ml) as indicated time points.Quantitative analysis of relative protein level of GSDMD was shown in (N and P), respectively.(Q and R) HEK293T cells were transfected with control siRNA or USP18 siRNA for 36 h, then the cells were transfected with HA-GSDMD, together with Flag-empty vector (EV), Flag-p62, Flag-OPTN, Flag-NDP52, Flag-NBR1 or Flag-TOLLIP for 24 h.Cell lysates were collected for immunoprecipitation (IP) and immunoblot analysis (Q).Quantitative analysis of relative immunoprecipitated level of HA-p62, HA-OPTN, HA-NDP52, or HA-NBR1 was shown in (R).(S and T) THP-1-derived macrophages were transfected with siCtrl or siUSP18 (#1, #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment.Cell lysates were collected for immunoprecipitation (IP) and immunoblot analysis (S).Quantitative analysis of relative immunoprecipitated level of p62, OPTN, or NDP52 was shown in (T).(U) Quantitative analysis of relative protein level of GSDMD in Fig. 2Q.In (A, C, E, G, I    Myc-USP18 or Myc-USP18 C64A for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.

Cell lysates were collected for immunoprecipitation and immunoblot analysis. (E)
HEK293T cells were transfected with siCtrl or siUSP18 (#1, #2) for 48 h, then transfected with HA-GSDMD and Flag-MIB2 for 18 h, followed by Baf A1 (0.2 μM) treatment for 6 h.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(F) WT (sgCtrl) and USP18-KO (sgUSP18) HEK293T cells were transfected with HA-GSDMD and Flag-MIB2 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(G) THP-1-derived macrophages were transfected with siCtrl or siUSP18 for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment.Mock, untreated.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(H) HEK293T cells were transfected with siCtrl or siUSP18 for 36 h, then transfected with HA-GSDMD and Flag-MIB2 for 24 h.Cell lysates were collected for immunoblot analysis.(I) HEK293T cells were transfected with siCtrl or siUSP18 for 36 h, then transfected with HA-Ub and Flag-GSDMD, together with Myc-EV or Myc-MIB2 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.Cell lysates were collected for immunoprecipitation and immunoblot analysis.

Fig. S1 .
Fig. S1.USP18 knockdown promotes pyroptosis.(A to C) THP-1-derived macrophages were transfected with control small interfering RNA (siCtrl) or two , K, O, Q and S), data are representative of three independent experiments with similar results.In (B, D, F, H, J, L, N, P, R, T and U), quantitative analysis of indicated protein levels was determined by Image Lab software, data are represented as mean values ± SD, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.

Fig. S3 .
Fig. S3.USP18 promotes the ubiquitination of GSDMD at K168 and facilitatesthe subsequent degradation of GSDMD.(A) HEK293T cells were transfected with HA-ubiquitin (HA-Ub) and Flag-GSDMD, together with Myc-empty vector (EV) or Myc-USP18 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.Cell lysates were collected for immunoprecipitation (IP) and immunoblot analysis.WCL, whole cell lysates.(B) THP-1-derived macrophages were transfected with siCtrl or siUSP18 (#1, #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(C) HEK293T cells were transfected with Flag-GSDMD and HA-K6-Ub, HA-K11-Ub, HA-K27-Ub, or HA-K29-Ub, together with Myc-empty vector (EV) or Myc-USP18 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(D to G) HEK293T cells were transfected with HA-Ub and Flag-GSDMD WT or its mutants, together with Myc-EV or Myc-USP18 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h.Cell lysates were collected for immunoprecipitation and immunoblot analysis.(H) Immunoblot analysis of HEK293T cells transfected with Flag-GSDMD or its mutants, together with HA-EV or HA-USP18.In (A to H), data are representative of three independent experiments with similar results, quantification of the indicated protein levels was determined by Image Lab software, data are represented as mean values ± SD, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.

Fig. S5 .
Fig. S5.USP18 recruits MIB2 to promote GSDMD degradation.(A) Immunoprecipitation (IP) and immunoblot analysis of HEK293T cells transfected with HA-MIB2 and Flag-USP18.(B) Immunoprecipitation and immunoblot analysis of HEK293T cells transfected with HA-USP18 and Flag-MIB2.(C) HEK293T cells were transfected with HA-GSDMD and Flag-MIB2, together with wild type (WT) derived macrophages were transfected with siUSP18 plus siCtrl or siUSP18 plus siMIB2 for 48 h.Cell lysates were collected for immunoblot analysis.(K) HEK293T cells were transfected with siUSP18 plus siCtrl or siUSP18 plus siMIB2 for 36 h, then transfected with HA-GSDMD and Myc-CASP1 or Myc-CASP4 for 24 h.Cell supernatants were collected for LDH release assay.In (A to J), data are representative of three independent experiments with similar results.In (C to J), quantification of the indicated protein levels was determined by Image Lab software, data are represented as mean values ± SD, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.In (K), data are represented as mean values ± SEM, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.

Fig. S6 .
Fig. S6.USP18 and MIB2 can directly promote the degradation of N-terminal form of GSDMD.(A-B) HEK293T cells were transfected with Flag-EV, Flag-GSDMD, or Flag-GSDMD-N for 24 h, respectively.Cell lysates were collected for immunoblot analysis (A), and cell supernatants were harvested for LDH (lactate dehydrogenase) assay (B).(C) HEK293T cells were transfected with Flag-WT GSDMD-N, Flag-K168R GSDMD-N, Flag-I104N GSDMD-N, Flag-I104N/K168R GSDMD-N for 24 h, respectively.Cell lysates were collected for immunoblot analysis.(D and E) HEK293T cells were transfected with HA-USP18 (D) or HA-MIB2 (E), together with Flag-EV, Flag-GSDMD, or Flag-I104N GSDMD, respectively.Cell lysates were collected for immunoprecipitation (IP) and immunoblot analysis.WCL, whole cell lysates.(F and G) HEK293T cells were transfected with Flag-GSDMD or Flag-I104N GSDMD, together with HA-USP18 (F) or HA-MIB2 (G), respectively.Cell lysates were collected for immunoblot analysis.(H and I) HEK293T cells were transfected with Flag-I104N GSDMD-N or Flag-I104N/K168R GSDMD-N, together with HA-USP18 (H) or HA-MIB2 (I), respectively.Cell lysates were collected for immunoblot analysis.In (A and C to I), data are representative of three independent experiments with similar results.In (D to I), quantification of the indicated protein levels was determined by Image Lab software, data are represented as mean values ± SD, P values were determined by unpaired two-tailed Student's t test of n=3 independent biological experiments.In (B), data are represented as mean values ± SEM of n=3 independent biological experiments.