YAP promotes the activation of NLRP3 inflammasome via blocking K27-linked polyubiquitination of NLRP3

The transcription coactivator YAP plays a vital role in Hippo pathway for organ-size control and tissue homeostasis. Recent studies have demonstrated YAP is closely related to immune disorders and inflammatory diseases, but the underlying mechanisms remain less defined. Here, we find that YAP promotes the activation of NLRP3 inflammasome, an intracellular multi-protein complex that orchestrates host immune responses to infections or sterile injuries. YAP deficiency in myeloid cells significantly attenuates LPS-induced systemic inflammation and monosodium urate (MSU) crystals-induced peritonitis. Mechanistically, YAP physically interacts with NLRP3 and maintains the stability of NLRP3 through blocking the association between NLRP3 and the E3 ligase β-TrCP1, the latter increases the proteasomal degradation of NLRP3 via K27-linked ubiquitination at lys380. Together, these findings establish a role of YAP in the activation of NLRP3 inflammasome, and provide potential therapeutic target to treat the NLRP3 inflammasome-related diseases. YAP is known to play a role both in organ size via the Hippo signalling pathway and in inflammation, though the precise mechanism are unclear. Here, the authors report that YAP promotes activation of the NLRP3 inflammosome through binding interactions.

T he NLRP3 inflammasome is a multiple protein complex that plays critical role in host immune responses to infections or sterile injuries [1][2][3] . It consists of the nucleotide-binding domain, leucine-rich repeat, pyrin domaincontaining protein 3 (NLRP3), the apoptosis-associated speck like protein containing a CARD (ASC) and caspase-1. NLRP3 can be activated by a broad range of microbial components, endogenous danger signals and environmental irritants 1 . Once activated, the NLRP3 inflammasome serves as a platform for the activation of caspase-1, which induces the maturation of interleukin (IL)-1β and IL-18, as well as gasdermin D-mediated pyroptotic cell death [1][2][3] . It is well-known that the deregulated NLRP3 inflammasome drives the progression of many inflammatory, metabolic, degenerative and aging-related diseases, such as atherosclerosis, gout, type 2 diabetes, autoimmune disorders, and Alzheimer's disease [4][5][6][7] . Therefore, NLRP3 inflammasome activity must be tightly regulated to maintain immune homeostasis and avoid detrimental effects. Although the mechanisms by which NLRP3 inflammasome is activated has been studied extensively, its endogenous regulatory networks remain largely unknown.
The Hippo-YAP pathway, which is highly evolutionarily conserved in mammals, is a key regulator to control the organ size 8,9 . This pathway integrates a variety of cellular stress signaling to maintain tissue homeostasis, including mechanical stress, DNA damage and oxidative stress under physiological and pathological conditions 10 . Upon activation of the Hippo pathway, the transcription co-activators YAP and TAZ are phosphorylated by Lats1/2 and retained in the cytoplasm for degradation. When the signaling is turned off, YAP/TAZ are predominantly localized in the nucleus, binding to transcription factors of the TEAD family and other factors to promote gene transcription 8,9 . Recent studies have revealed the role of the Hippo-YAP pathway in innate antibacterial [11][12][13] and antiviral immune responses 14,15 via transcriptional or non-transcriptional activity, providing new insights into the crosstalk between the innate immune system and the Hippo-YAP pathway. Moreover, the elevated expression of YAP is associated with the progression of atherosclerosis 16 , inflammatory bowel disease (IBD) 17 , sepsis 18 , and pancreatitis 19 . Together with the findings that these inflammatory disorders involve the deregulated NLRP3 inflammasome activity, we postulated that YAP might regulates the activation of NLRP3 inflammasome.
In this work, we demonstrate that YAP promotes the activation of NLRP3 inflammasome. Deletion of YAP in myeloid cells significantly attenuates lipopolysaccharide (LPS)-induced systemic inflammation and monosodium urate (MSU) crystals-induced peritonitis. Activation of the Hippo signaling that induces the degradation of YAP markedly decreases the activity of the NLRP3 inflammasome in vitro. Mechanistically, YAP physically interacts with NLRP3 and maintains its stability through blocking the association between NLRP3 and the E3 ligase β-TrCP1, the latter promotes the proteasomal degradation of NLRP3 via K27-linked ubiquitination at lys380. Thus, we uncover a role of YAP in regulation of the NLRP3 inflammasome activation, and provide potential therapeutic target to treat a number of inflammatory disorders, such as atherosclerosis, gout, colitis, and sepsis.

YAP specifically promotes NLRP3 inflammasome activation.
To investigate the role of YAP in the NLRP3 inflammasome activation, we diminished the YAP expression in primary mouse macrophages using small-interfering RNA (siRNA) (Supplementary Fig. 1a). Knockdown of YAP expression markedly suppressed ATP-, nigericin-, or MSU-induced caspase-1 cleavage and release of IL-1β and IL-18, without affecting the tumor necrosis factor alpha (TNF-α) secretion (Fig. 1a, b). We also examined whether YAP specifically regulates the NLRP3 inflammasome activation and found that silencing of YAP expression had no effect on either poly (dA:dT)-induced AIM2 inflammasome or flagellin (FLA) transfection-induced NLRC4 inflammasome activation (Fig. 1a). To confirm this phenomenon, we generated Yap fl/fl lyz2-Cre mice with Yap deletion in myeloid cells 20 . ATP, nigericin or MSU-induced cleavage of caspase-1 and release of IL-1β and IL-18 were markedly suppressed in YAP-deficient (Yap fl/fl lyz2-Cre) macrophages as compared to that of YAP-sufficient (Yap fl/fl ) macrophages (Fig. 1c, d). By contrast, YAP deletion did not alter the release of IL-1β and IL-18 upon poly (dA:dT) or flagellin transfection (Fig. 1c, d). The NLRP3 inflammasome activation induces the oligomerization of ASC, resulting in formation of ASC specks through the pyrin domain [1][2][3] . Accordingly, deletion of YAP markedly inhibited the ASC oligomerization and ASC-speck formation (Fig. 1e, f). NLRP3 is pivotal for caspase-1dependent IL-1β maturation upon non-canonical inflammasome activation 21 . As expected, YAP deficiency suppressed both caspase-1 cleavage and IL-1β release in macrophages transfected with LPS ( Supplementary Fig. 1b, c). Taken together, our data demonstrate that YAP deficiency specially impairs the NLRP3 inflammasome activation.
Cytoplasmic YAP inhibits the proteasomal degradation of NLRP3. Next, we sought to investigate the mechanisms by which YAP promotes the NLRP3 inflammasome activation. It is wellestablished that the activation of NLRP3 inflammasome requires two steps, termed priming and assembly 1 . The priming step involves the stimulation of NLRP3 expression, which is critical for the subsequent inflammasome activation 1,3 . We examined the expression of NLRP3 inflammasome components in mouse macrophages following LPS stimulation and found that YAP deficiency markedly decreased the protein expression of NLRP3 without affecting the expression of caspase-1, pro-IL-1β, and ASC (Fig. 2a). Intriguingly, deletion of YAP failed to affect the mRNA expression of NLRP3 ( Supplementary Fig. 2a), clearly suggest that YAP promotes NLRP3 expression at the post-transcriptional level. In line with the previous findings that NLRP3 degradation impairs the NLRP3 inflammasome activation 1 , YAP deficiency significantly promoted the protein degradation of NLRP3 but not ASC or caspase-1 (Fig. 2b, c). NLRP3 degradation was reversed by the proteasome inhibitor MG-132, but not the lysosome inhibitor chloroquine or the autophagy inhibitor 3-MA ( Fig. 2d-f). suggesting that YAP inhibits the proteasomal degradation of NLRP3.
Previous studies 22,23 have reported that substitution of YAP residue Ser112 with Ala (S112A) generates a nuclear protein with constitutive transcriptional activity ( Supplementary Fig. 2b). Using genetically modified HEK293T cells expressing the NLRP3 inflammasome components, we observed that overexpression of wild-type but not S112A mutant YAP markedly increased the IL-1β release (Fig. 2g) and promoted the NLRP3 expression in a dose-dependent manner (Fig. 2h), while the S112A YAP mutant had no such effect (Fig. 2g, h). Moreover, YAP predominantly localized in the cytoplasm of macrophages upon LPS stimulation ( Supplementary Fig. 2c, d). These results suggest that cytoplasmic YAP is critical for maintaining the stability of NLRP3.
Hippo signaling suppresses the NLRP3 inflammasome activation in a YAP-dependent manner. The Hippo pathway, activated by various cellular stresses, is known to induce the degradation of YAP 8,9 . We then asked whether cellular stressesinduced Hippo signaling could affect the NLRP3 inflammasome activation, and subjected cells to serum starvation or high cell ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22987-3 confluence, both of which are well-known cellular stress conditions that activate the Hippo signaling 24,25 , this led to YAP Ser127 phosphorylation and YAP degradation, indicative of the activation of Hippo signaling ( Supplementary Fig. 3a, b). Serum starvation or high cell confluence markedly inhibited the NLRP3 inflammasome-induced release of IL-1β and IL-18 in WT but not YAP-deficient macrophages, without affecting the secretion of TNF-α (Fig. 3a, b). Upon activation of the Hippo signaling, Lats1/ 2 mediates YAP phosphorylation, leading to its polyubiquitination and degradation 24,25 . To further confirm that the Hippo signaling regulates the activation of NLRP3 inflammasome through YAP, we next investigated the role of Lats1/2 in activation of the NLRP3 inflammasome. Silencing of Lats1/2 expression in macrophages prevented the decrease of YAP expression ( Supplementary Fig. 3c, d), and enhanced NLRP3 agonistsinduced IL-1β and IL-18 secretion during serum starvation (Fig. 3c). The coactivator TAZ is a downstream effector of Hippo signaling 8,9 . However, in contrast to the lung tissues, macrophages did not express detectable TAZ expression in the presence or the absence of LPS stimulation ( Supplementary  Fig. 3e, f). Taken together, our findings indicated that the Hippo signaling suppresses the NLRP3 inflammasome activation in a YAP-dependent manner.
YAP suppresses K27-linked ubiquitination of NLRP3. Protein ubiquitination is a crucial step in the ubiquitin-proteasome degradation pathway 26 . We next determined whether YAP participates in the ubiquitination of NLRP3. We found that NLRP3 ubiquitination was markedly decreased in HEK293T cells overexpressing YAP (Fig. 4a). By contrast, deletion of YAP in myeloid cells increased the NLRP3 ubiquitination (Fig. 4b). Since different types of polyubiquitin linkages mediate distinct biological functions, we expressed Myc-tagged NLRP3 in HEK293T cells together with a series of ubiquitin mutants (K6, K11, K27, K29, K33, K48, and K63), all of which contained only one indicated lysine available for poly-linkage. Notably, over-expressing YAP markedly decreased K27-linked polyubiquitination of NLRP3 but had no appreciable effect on the NLRP3 ubiquitination of other types of linkages (Fig. 4c). When we replaced K27 with an arginine (K27R), YAP was no longer able to decrease the polyubiquitination of NLRP3 (Fig. 4d). Taken together, these data suggest that YAP selectively suppresses the K27-linked ubiquitination of NLRP3.
YAP suppresses β-TrCP1-mediated K27-linked ubiquitination of NLRP3. Since YAP could block the ubiquitination of NLRP3, we speculated that there may exist a YAP-associated enzyme that mediates NLRP3 ubiquitination. The 14-3-3 protein has been reported to bind to phosphorylated YAP, sequestering it in the cytoplasm 8,9 . Previous studies have suggested that 14-3-3σ promotes the ubiquitination of c-myc and COP1 27,28 , prompting us Cytoplasmic YAP inhibits the proteasomal degradation of NLRP3. a Immunoblot analysis of extracts from Yap fl/fl lyz2-Cre or Yap fl/fl mouse peritoneal macrophages, then stimulated for indicated times with LPS. b, c Immunoblot analysis of extracts from Yap fl/fl lyz2-Cre or Yap fl/fl mouse peritoneal macrophages stimulated with LPS for 4 h, and then treated for various times with cycloheximide (CHX) (b). NLRP3, ASC and Caspase-1 expression levels were quantitated by measuring band intensities using "ImageJ" software. The values were normalized to actin (c) (mean ± SD, two-way ANOVA with Bonferroni test, Yap fl/fl lyz2-Cre vs. Yap fl/fl , left panel: **P = 0.0054, ***P = 0.0002, ***P = 0.0001 in sequence; n = 3 independent experiments). d-f Immunoblot analysis of extracts from Yap fl/fl lyz2-Cre or Yap fl/fl mouse peritoneal macrophages stimulated with LPS for 4 h, and then treated with different doses of MG-132 (d), and chloroquine (e), and 3-MA (f) for 8 h. g ELISA of IL-1β in supernatants from HEK293T cells transfected with NLRP3, ASC, pro-caspase-1, pro-IL-1β, YAP-WT or YAP-S112A plasmids and stimulated with 10 μM nigericin (mean ± SD, one-way ANOVA with Bonferroni test, the third and fourth group vs. the second group ***P = 0.0001, ns > 0.9999; n = 3 independent experiments). h Immunoblot analysis of lysates from HEK293T cells transfected with NLRP3, YAP-WT, or YAP-S112A plasmids. Similar results were obtained from three independent experiments. Source data are provided as a Source Data file.  to examine the role of 14-3-3σ in NLRP3 ubiquitination. However, expressing 14-3-3σ did not enhance the ubiquitination of NLRP3 ( Supplementary Fig. 4a).
YAP disrupts the interaction between NLRP3 and β-TrCP1 through competing with β-TrCP1 to bind NLRP3. To explore the mechanism by which YAP blocks β-TrCP1-mediated K27linked ubiquitination of NLRP3, we determined whether YAP affects the physical interaction between β-TrCP1 and NLRP3. Coexpression of YAP decreased the interaction between β-TrCP1 and NLRP3 (Fig. 5d), whereas YAP deficiency increased the capacity of β-TrCP1 to bind NLRP3 (Fig. 5e), indicating that YAP disrupts the interaction between β-TrCP1 and NLRP3. Three possible explanations might explain the underlying mechanisms. First, YAP may affect the serine phosphorylation of NLRP3, leading to the decreased binding between NLRP3 and β-TrCP1. Second, YAP might directly interact with β-TrCP1 and thereby mask the NLRP3 binding site of β-TrCP1. Third, YAP might directly interact with NLRP3 and thereby mask the β-TrCP1 binding site of NLRP3.
To test the first possibility, we overexpressed YAP in iBMDMs and assessed NLRP3 serine phosphorylation. LPS stimulation led to the increase of NLRP3 serine phosphorylation, which was not affected by YAP overexpression, excluding the first possibility ( Supplementary Fig. 5a). To test the second possibility, we overexpressed YAP-S366A mutant (in humans, the homologous mutant is YAP-S381A), which lost the β-TrCP1 binding capacity 31 (Supplementary Fig. 5b). However, the S366A substitution in YAP did not affected the NLRP3 and β-TrCP1 binding ( Supplementary Fig. 5c), suggesting that YAP interrupts the NLRP3 and β-TrCP1 interaction independent of its direct binding to β-TrCP1. Next, we tested the third possibility and observed an association between YAP and NLRP3 (Supplementary Fig. 5d). This phenomenon was confirmed by using Proximity Ligation Assay (PLA), which could visualize the protein-protein interactions in vivo ( Supplementary Fig. 5e, f). As shown by co-immunoprecipitation assay, we found that the Cterminal transactivation domain (residues 151-488) rather than the N-terminal TEAD binding domain of YAP physically interacted with NLRP3 ( Supplementary Fig. 5g, h). Coexpression of wild-type YAP or its C-terminal transactivation domain but not the N-terminal TEAD-binding domain interrupted the interaction between NLRP3 and β-TrCP1 (Supplementary Fig. 5i), indicating that the NLRP3-YAP binding inhibits the NLRP3-β-TrCP1 interaction. Further, we observed that both YAP and β-TrCP1 bound to the same domain of NLRP3 (NACHT and LRR domain) ( Supplementary Fig. 5j, k, l). Taken together, these results demonstrate that YAP disrupts the interaction between NLRP3 and β-TrCP1 through competing with β-TrCP1 to bind NLRP3.
β-TrCP1 inhibits NLRP3 inflammasome activation. To determine whether β-TrCP1 might inhibit the NLRP3 inflammasome activation, we knocked down the expression of β-TrCP1 in primary macrophages by using siRNA. Silencing of β-TrCP1 expression in primary macrophages markedly enhanced the release of IL-1β or IL-18, but did not affect the TNF-α secretion, upon stimulation by NLRP3 agonists (Fig. 6a). Accordingly, silencing of β-TrCP1 expression markedly decreased the NLRP3 expression (Fig. 6b). Moreover, upon silencing of β-TrCP1 in YAP-deficient macrophages from Yap fl/fl lyz2-Cre mice, the decline in IL-1β or IL-18 secretion and NLRP3 expression was rescued as compared to that of in macrophages from control Yap fl/fl mice (Fig. 6c, d), which further confirmed that YAP could suppress β-TrCP1-mediated NLRP3 degradation. These results clearly suggest that β-TrCP1 inhibits NLRP3 inflammasome activation.
Lys380 in NLRP3 is essential for its ubiquitination and degradation. Next, we sought to identify the β-TrCP1-mediated ubiquitination site of NLRP3. HEK293T cells were transfected with plasmids encoding a series of truncation mutants of NLRP3 (▵PYD, ▵NACHT and▵LRR) (Supplementary Fig. 5j) and HAubiquitin-K27, with or without exogenous β-TrCP1 expression. Notably, deletion of the NACHT domain prevented NLRP3 ubiquitination upon β-TrCP1 co-expression ( Supplementary   Fig. 6a), suggesting that β-TrCP1 mediates the ubiquitination of the NACHT domain of NLRP3. We further analyzed the most conserved lysine residues within the NACHT domain among species and constructed plasmids encoding mutated NLRP3 with individual lysine residue replaced by arginine (K/R) (Supplementary Fig. 6b). The K380R substitution rather than other point mutations abolished β-TrCP1-mediated K27-linked ubiquitination of NLRP3 (Fig. 7a). We next tested whether K27-linked ubiquitin chains on K380 might function as a degradation signal for NLRP3, and observed a significantly slower degradation rate of the K380R NLRP3 mutant as compared to that of wild-type NLRP3 (Fig. 7b). Further, co-expression of β-TrCP1 failed to promote the degradation of the K380R NLRP3 mutant (Fig. 7c).
To further address the functional importance of K380 ubiquitination, we reconstituted the NLRP3 inflammasome in HEK293T cells and observed that the expression of K380R mutant enhanced IL-1β secretion as compared to that of wildtype NLRP3 (Fig. 7d). To confirm this phenomenon in immune cells, we induced the expression of WT NLRP3 or its K380R mutant in NLRP3 −/− iBMDMs using lentivirus, and then stimulated the LPS-primed iBMDMs with ATP or nigericin. We found that expression of the K380R mutant significantly increased the release of IL-1β but not TNF-α secretion as compared to that of cells expressing WT NLRP3 (Fig. 7e, f). Taken together, these data indicate that K380 of NLRP3 is an important site for the K27 ubiquitination and the degradation of NLRP3.

YAP promotes NLRP3 inflammasome activation in vivo.
Finally, we investigated the role of YAP in NLRP3 inflammasome activation in vivo. Intraperitoneal (i.p.) injection of LPS induces IL-1β secretion and neutrophil infiltration in a NLRP3-dependent manner 35,36 . Notably, the secretion of IL-1β but not TNF was significantly reduced in Yap fl/fl lyz2-Cre mice, as compared to that of in control Yap fl/fl mice (Fig. 8a). Moreover, deletion of YAP in myeloid cells markedly attenuated endoxemia-induced lung injury, as evaluated by histopathology (Fig. 8b). To confirm the role of YAP in regulating NLRP3 inflammasome activation in vivo, we injected i.p. the mice with monosodium urate (MSU), which induces neutrophil infiltration and peritonitis in a NLRP3 dependent manner 35,36 . We observed that deletion of YAP in myeloid cells significantly inhibited MSU-induced IL-1β release and neutrophil infiltration as compared to that of in control Yap fl/fl mice (Fig. 8c, d and Supplementary Fig. 7). Collectively, these findings demonstrated that YAP promotes NLRP3 inflammasome activation in vivo.

Discussion
The Hippo pathway participates in the innate immune responses in multiple aspects, including antibacterial immune response, antiviral immune response and macrophage polarization 17,37,38 . Accumulating evidence suggest that up-regulation of YAP expression is associated with the development of inflammatory diseases, such as atherosclerosis 16 , inflammatory bowel disease (IBD) 17 , sepsis 18 and pancreatitis 19 . However, the underlying mechanisms by which YAP regulates inflammation and promotes the pathogenesis of inflammatory diseases are not fully understood. In this study, we demonstrated that myeloid YAP deficiency specifically impairs NLRP3 inflammasome activation both in vitro and in vivo. In addition, serum starvation-or high cell confluence-induced Hippo signaling activation that induces the degradation of YAP markedly decreased the activity of the NLRP3 inflammasome. Mechanistically, YAP maintains the stability of NLRP3 by direct binding to NLRP3, thereby blocking the accessibility of E3 ligase β-TrCP1, the latter promotes the proteasomal degradation of NLRP3 via K27-linked ubiquitination at K380 (Fig. 8e). Thus, our study provides a mechanistic insight into the regulatory roles of YAP in inflammation.
Accumulated evidence reveal that altered NLRP3 expression is associated with the pathogenesis of several inflammatory disorders, including atherosclerosis 39,40 , type-2 diabetes (T2D) 41 , and autoimmune diseases 42 . Our study suggests that YAP may be a potential therapeutic target to regulate NLRP3 protein expression in many inflammatory diseases. This is consistent with a recent report 43 showing elevated YAP expression in atherosclerotic plaques, which are enriched in NLRP3 expression 40,41 . It was also found that YAP protein levels correlate with plasma IL-1β levels in patients with ST-segment elevated myocardial infarction (STEMI) 43 , suggesting that YAP could be a potential target for the treatment of atherosclerosis. Serum starvation and high cell confluence, the well-known cellular stresses that activate Hippo signaling, could inhibit the NLRP3 inflammasome activation in a YAP-dependent manner. Given that other cellular stresses, including matrix stiffness, mechanical stress, and longrange hormonal signals, are able to activate the Hippo-YAP pathway, we propose that these stress signaling might also regulate the NLRP3 inflammasome activation in the lung, a highly mechanical organ 44 .
Ubiquitination of NLRP3 plays an important role in the regulation of inflammasome 1,45 . To date, NLRP3 has been reported to be ubiquitinated with both K48 and K63 linkages. A series of enzymes that are responsible for K48-linked ubiquitination of NLRP3 have been identified, including PAI2 46 , MARCH7 35 , FBXL2 47 , Trim31 48 , and Cbl-b 49 . All of these bind to NLRP3 and mediate the degradation of NLRP3 either by the autophagylysosomal pathway or the ubiquitin-proteasome pathway. BRCC3-mediated deubiquitination of NLRP3 is required for NLRP3 inflammasome assembly and activation 50 , in which the ubiquitination of NLRP3 consists of both K48 and K63 linkages. In our study, we found that NLRP3 can be ubiquitinated with K27 linkage, leading to subsequent NLRP3 degradation in the ubiquitin-proteasome pathway. This finding provides insights into the regulation of NLRP3 degradation. Although most studies have shown that K48-linked ubiquitination is the signal that mediates proteasomal degradation 51,52 , our work provides compelling evidence that K27-linked ubiquitination could also lead to the proteasomal degradation of target proteins [53][54][55] .
Moreover, we identified that Lys380 is the K27-linked ubiquitination site by screening the most-conserved lysine residues in the NACHT domain of NLRP3. When we replaced lysine with arginine, the degradation of NLRP3 was inhibited. Accordingly, cells expressing the NLRP3 K380R mutant released more IL-1β as compared to cells expressing WT NLRP3. Thus, our study β-TrCP1, one of the best-characterized mammalian F-box proteins of SCF ligase, has been reported to be involved in the ubiquitination of well-established substrates, such as YAP, βcatenin, IκB, caspase-3 34,56 . All of these studies have suggested that β-TrCP1-mediated substrate ubiquitination is K48-linked. However, in current study, we did not observe that β-TrCP1mediated K48-linked ubiquitination of NLRP3. Instead, β-TrCP1 promoted K27-linked ubiquitination of NLRP3 and its subsequent degradation. It has been well-documented that β-TrCP1's substrates contain a consensus DSG(X) 2+n S degron 33,34 . The serine residues in this degron could be phosphorylated by specific protein kinases, resulting in β-TrCP1 recognition and subsequent ubiquitination of the substrates. We identified the 890-NSGLTS-895 motif, an "atypical" XSG(X) 2+N S motif in NLRP3, is essential for β-TrCP1 binding. However, the kinase responsible for NLRP3 serine phosphorylation remains unknown. Previous studies have shown that kinases JNK1 36 , PKA 57,58 , PKD 59 , and AKT 60 could mediates serine phosphorylation of NLRP3. However, the reported phosphorylation site was neither serine 891 nor 895. Whether these kinases could also mediate serine 891/895 phosphorylation and whether unrecognized kinases account for NLRP3 phosphorylation, are interesting questions for future studies. Previous studies have suggested that 14-3-3 family members including 14-3-3ε, 14-3-3η, and14-3-3τ, can bind to pyrin and regulate the activation of the pyrin inflammasome 61,62 . Although we found that 14-3-3σ has no effect on the Fig. 7 Lys380 in NLRP3 is essential for its ubiquitination and degradation. a Immunoblot analysis of lysates from HEK293T cells transfected with HAtagged K27-linked ubiquitin (K27-Ub), β-TrCP1 and Myc-NLRP3 or indicated mutant Myc-NLRP3, followed by IP with anti-Myc, probed with anti-HA. b Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, Myc-NLRP3 K380R, and then treated for various times with cycloheximide (CHX, 100 ug/mL) (top). NLRP3 and NLRP3 K380R expression levels were quantitated by measuring band intensities using "ImageJ" software. The values were normalized to actin (bottom) (mean ± SD, two-way ANOVA with Bonferroni test, NLRP3-K380R vs. NLRP3-WT lower panel: *P = 0.0129, *P = 0.0321, **P = 0.0010 in sequence; n = 3 independent experiments). c Immunoblot analysis of lysates from HEK293T cells transfected with Flag-β-TrCP1 and Myc-NLRP3 or Myc-NLRP3 K380R. d ELISA of IL-1β in supernatants from HEK293T cells transfected with ASC, pro-caspase-1, pro-IL-1β, NLRP3 or NLRP3 K380R plasmids and stimulated with 10 μM nigericin (mean ± SD, one-way ANOVA with Bonferroni test, the third group vs. second group ***P < 0.0001; n = 3 independent experiments). e, f NLRP3 −/− iBMDMs were infected with lentivirus expressing mouse WT or NLRP3-K380R, respectively. Immunoblot analysis of indicated proteins (e), then treated these cells with LPS plus ATP or nigericin for detection of IL-1β and TNF-α in supernatants (f) (mean ± SD, two-way ANOVA with Bonferroni test, NLRP3-K380R vs. NLRP3-WT left panel: *P = 0.0475, **P = 0.0037 in sequence, n = 3 independent experiments). Similar results were obtained from three independent experiments. Source data are provided as a Source Data file. ubiquitination of NLRP3, the role of the14-3-3 family in NLRP3 inflammasome activation deserves further investigation.
In this work, our study uncovers a role of YAP in regulation of the NLRP3 inflammasome activation, and provides potential therapeutic target to treat a number of inflammatory disorders, such as atherosclerosis, gout, colitis and sepsis.

Methods
Mice. Yap fl/fl mice and Lyz2-Cre mice on the C57BL/6 background were from Jackson laboratories 20 . Yap fl/fl mice were crossed with Lyz2-Cre mice to obtain Yap fl/fl lyz2-Cre mice. Wild-type C57BL/6 mice (8-10 weeks old) were purchased from Hunan SJA Laboratory Animal Co.Ltd (Changsha, China). All the animals were generated in specific pathogen-free (SPF) levels and daily cycles of 12 h of light at 23°C and 12 h of dark at 21°C. All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Central South University. Cell culture. To obtain mouse primary peritoneal macrophages, mice were injected intraperitoneally with 3% thioglycolate. Three days later, peritoneal exudate cells were harvested and incubated. Two hours later, nonadherent cells were removed and the adherent monolayer cells were used as peritoneal macrophages. HEK293T cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin.
Reconstitution of NLRP3 inflammasome in HEK293T cells. A standard reconstitution system in HEK293T cells was referred 63 . HEK293T cells were seeded into 24-well plates at density of 2 × 10 5 cells per well in complete cell culture medium overnight before transfection. The cells were transfected with plasmids expressing pro-IL-1β-flag (200 ng/well), pro-caspase-1-myc (100 ng/well), ASC-myc (20 ng/ well), NLRP3-myc (200 ng/well) using Linear Polyethylenimine. Twenty-four to 36 h later, replace the medium with 250 μL DMEM cell culture medium, then 10 μM nigericin was added one hour before supernatant collection.
ASC oligomerization and ASC speck formation. Peritoneal macrophages were plated in 6-well plates (2 × 10 6 cells per well), then stimulated with 100 ng/mL LPS for 3 h, followed by stimulation with or without 10 μM nigericin (1 h). The cells were lysed with Triton Buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 0.1 mM PMSF and EDTA-free protease inhibitor cocktail] at 4°C for 10 min, then remove 30 μL as whole cell lysates. The rest of cell lysates were centrifuged at 6000 × g at 4°C for 15 min, the supernatant was collected and pellet was washed twice and re-suspended in 200 μL Triton Buffer, and cross-linked for 30 min at 37°C with 2 mM disuccinimidyl suberate (DSS). The pellets were collected after centrifuged for 15 min at 6000 × g, and dissolved in SDS loading buffer for immunoblot analysis For ASC speck formation, peritoneal macrophages were seeded on chamber slides and allowed to attach overnight. The next day, the cells were treated with indicated stimulators. Then the cells were fixed in 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 for 30 min. Then the slides were blocked with phosphate-buffered saline (PBS) containing 1% Bovine serum albumin (BSA), followed by ASC and DAPI staining.
Proximity ligation assay. Proximity ligation assays were carried out to detect protein-protein interactions in situ by using Duolink reagents (Sigma). Briefly, primary macrophages were cultured on a six-well object glass in RPMI medium 1640. After stimulated with LPS for 4 h or not, cells were fixation in 4% paraformaldehyde and permeabilization with PBS containing 0.1% Triton X-100, then incubated over night with primary antibody pair of different species directed to NLRP3 (1:200), YAP (1:200), β-TrCP1 (1:200). Probe incubation, ligation and amplification reaction were carried out according to the manufacturer instructions. Thus, each individual pair of proteins generated a spot that could be visualized using fluorescent microscopy (Nikon Ti2-U).
Quantitative PCR. Total RNA was extracted by using RNA Fast 200 kit according to the manufacturer's instructions (FASTAGEN). Complementary DNA was synthesized by using TransScript All-in-One First-Strand cDNA Synthesis Super-Mix for qPCR (TransGen Biotech) according to the manufacturer's protocols. Quantitative PCR was performed using SYBR Green (Vazyme Biotech) on a LightCycler 480 (Roche Diagnostics), and data were normalized by the level of βactin expression in each individual sample. The 2 −ΔΔCT method was used to calculate relative expression changes.
Immunoprecipitation and western blot. HEK293T cells after transfection or peritoneal macrophages after stimulation, then lysed in IP buffer, which contained 1% (v/v) Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 50 mM EDTA, 150 mM NaCl, and a protease inhibitor cocktail, pre-cleared cell lysates were then subjected to anti-Flag M2 affinity gel or anti-Myc affinity gel for 2 h or with specific Abs and protein G plus-agarose overnight, then washed four times with IP buffer. Immunoprecipitates were eluted by boiling with 1% (w/v) SDS sample buffer. For ubiquitination analysis, cells were lysed in IP buffer supplemented with 10 mM NEM (Sigma), 1% SDS, protease inhibitor cocktail and PMSF. Obtained cell lysates were subsequently boiled for 5 min at 95°C, diluted to 0.1% SDS with IP buffer, and immunoprecipitated.
For immunoblot analysis, cells were lysed with CLB buffer (CST) supplemented with protease inhibitor cocktail and PMSF, and then protein concentrations in the extracts were measured with a bicinchoninic acid assay (Pierce). Equal amounts of extracts were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then they were transferred onto nitrocellulose membranes for immunoblot analysis.
ELISA assay for cytokines. Levels of IL-1β, TNF-α, and IL-18 collected from cell culture and sera were determined using quantitative ELISA kits (eBioscience or abcam) according to the manufacturer's instructions.
In vivo LPS challenge. Yap fl/fl lyz2-Cre mice and Yap fl/fl littermates were injected intraperitoneally with LPS (20 mg/kg body weight), after 6 h, the mice were killed, and the serum concentrations of IL-1β, and TNF-α were measured by ELISA.
MSU-induced peritonitis in vivo. Yap fl/fl lyz2-Cre mice and Yap fl/fl littermates were i.p. injected with 1 mg MSU (dissolved in 500 μL PBS) for 6 h. Peritoneal cavities were washed with 10 mL ice-cold PBS. The peritoneal lavage fluids were collected and concentrated for ELISA analysis with Amicon Ultra 10 K filter (UFC900308) from Millipore. Peritoneal exudate cells were collected and analyzed by FACS.