A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death*

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser533 phosphorylation.

NOD-like receptor family CARD-containing 4 (NLRC4) 4 is a cytoplasmic immune receptor involved in innate immune response. It induces caspase-1-mediated inflammation and pyroptosis in response to bacterial infection or upon detection of bacterial flagellin, type III secretion system rod, or needle proteins, recognition of which is mediated by various NAIP paralogs (1)(2)(3)(4). NLRC4 has a distinct N-terminal caspase activation and recruitment domain (CARD), a nucleotide binding domain (NBD), and a leucine-rich repeat (LRR) domain that forms an autoinhibitory loop and is involved in intramolecular interactions with amino acid residues of NBD (5). Detection of an antigen triggers NLRC4 complexation, which results in activation of caspase-1 via structural changes such as reorientation of the autoinhibitory LRR domain away from NBD (5)(6)(7)(8); an activated NLRC4 undergoes oligomerization through intermolecular interaction and recruits a number of other proteins including procaspase-1 to form a multiprotein complex known as inflammasome. The multiprotein complex formed by NLRC4 facilitates the proximity-dependent autocatalysis of caspase-1. Studies using murine proteins have shown that other NLR family members, NAIPs determine specificity of NLRC4 toward an antigen and also enable inflammasome formation (2,4,9). Protein modification such as phosphorylation at NLRC4-Ser 533 has been shown to be crucial in NLRC4 inflammasome activation (10,11); however, molecular events that follow NLRC4-Ser 533 phosphorylation are poorly understood.
More recently, NLRC4 was shown to be expressed in nonhematopoietic gut cells (12,13) and in lung epithelial cells (14). Given that alveolar epithelium of lung is repeatedly exposed to airborne pathogenic or inflammatory agents, the role of NLRC4 in protection against agents invading lung epithelium needs to be investigated. Although most studies have focused on caspase-1-mediated functions of NLRC4, we have earlier shown that in human alveolar epithelial adenocarcinoma cells, NLRC4 engages caspase-8 to induce apoptotic cell death. In these cells, cell death is independent of caspase-1 activation. The proteasomal component SUG1 directly interacts with NLRC4, leading to its ubiquitination and formation of a caspase-8-containing multiprotein complex resulting in caspase-8 activation and apoptotic cell death. An artificial deletion mutant expressing amino acids 1-561 of NLRC4 (⌬LRR-NLRC4) forms cytoplasmic aggregates and is constitutively active in inducing caspase-8-dependent cell death (15). Importantly, ⌬LRR-NLRC4 fails to induce cell death upon knockdown of SUG1 or co-expression of K196M-SUG1, a dominant negative variant that lacks ATPase activity, indicating a crucial role for cellular SUG1 in NLRC4-mediated caspase-8-dependent cell death signaling. Molecular mechanisms involved in multiprotein complex formation by NLRC4 for caspase-8 activation may differ from those involved in caspase-1 activation and must be investigated.
Mutations in NLRC4 have been implicated in various autoimmune genetic disorders; a missense mutation T337S is known to cause recurrent macrophage activation syndrome and autoinflammation (16). Another mutation V341A results in enterocolitis and nearly fatal episodes of autoinflammation (17). More recently, the missense mutation H443P has been shown to result in hypersensitivity of the heterozygotes toward cold exposure and has been implicated to cause familial cold autoinflammatory syndrome (FCAS) in a non-consanguineous Japanese family (18). These three mutants cause constitutive caspase-1 activation and IL-1␤ production. However, it is likely that these mutants are altered in some other properties that lead to different disease phenotypes.
To study function of NLRC4 and consequences of its mutations on caspase-8 activation, we used the human alveolar epithelial adenocarcinoma cell line (A549), which represents a model for studies on lung biology. We provide evidence for the role of FADD in caspase-8 activation by an NLR family cytosolic receptor NLRC4. A FCAS causing mutant of NLRC4, H443P, otherwise known to mediate caspase-1-dependent inflammation, also induces caspase-8-dependent and caspase-1-independent apoptosis in A549 cells. This mutant shows enhanced interaction with SUG1, which mediates caspase-8 activation and cell death. We also identify the requirement of NLRC4-Ser 533 phosphorylation for caspase-8-mediated apoptotic cell death and show that NLRC4-H443P is not dependent on this phosphorylation for caspase-8 activation and induction of cell death. Thus our results show that the H443P mutant is altered in several properties in addition to constitutive caspase-1 activation reported previously.

Results
NLRC4-H443P Constitutively Activates Caspase-8-dependent Cell Death Signaling-Missense mutations H443P, T337S, and V341A in NLRC4 result in constitutive activation of NLRC4 triggering caspase-1-mediated inflammatory response and have been implicated in various sterile autoinflammatory diseases (16 -18). Positions of these mutations are indicated in Fig. 1A. NLRC4 mediates caspase-8-dependent apoptotic cell death independent of caspase-1 in lung alveolar epithelial cells (15). To test whether the missense mutations H443P, T337S, and V341A in NLRC4 result in caspase-8 activation, GFP fusion proteins of WT-NLRC4, NLRC4-H443P, NLRC4-T337S, and NLRC4-V341A were transiently expressed in A549 cells for 20 h, and the cells were examined for subcellular localization of overexpressed protein and features of cell death such as nuclear fragmentation, cytoplasmic shrinkage, and membrane blebbing. We observed that all the mutants show diffused cytoplasmic distribution (supplemental Fig. S1A), a pattern similar to that of WT-NLRC4. NLRC4-H443P induced apoptotic cell death in a significantly greater number of expressing cells compared with WT-NLRC4, NLRC4-T337S, and NLRC4-V341A (Fig. 1B). Cell death induced by H443P mutant was further confirmed using an apoptosis assay kit, which detects phosphatidylserine and membrane integrity as apoptotic markers. We observed that H443P mutant indeed induced apoptosis in a significantly higher number of expressing cells compared with WT-NLRC4 or T337S or V341A mutants (Fig. 1C). Western blotting analysis of whole cell lysates of A549 cells expressing NLRC4 and NLRC4-H443P showed significantly higher caspase-8 activation in NLRC4-H443P-expressing cells compared with cells expressing WT-NLRC4 (Fig. 1D) as detected by abundance of p43/p41 cleaved fragment of pro-caspase-8, an indicator of active caspase-8. We also confirmed that expression of all three mutants constitutively activates caspase-1 ( Fig.  1E) as described earlier (18). To determine the roles of caspase-8 and caspase-1 in cell death induced by NLRC4-H443P, we co-expressed catalytically inactive C360Acaspase-8 (mCaspase-8) or catalytically inactive mCaspase-1 along with NLRC4-H443P in A549 cells for 20 h and quantitated cell death. Co-expression of mCaspase-8 showed a significant reduction in cell death induced by NLRC4-H443P, whereas co-expression of mCaspase-1 had no significant effect (Fig. 1F). Levels of overexpressed proteins indicated in the Western blot showed that reduced cell death was not due to reduced expression of NLRC4-H443P in mCaspase-8-expressing cells. These observations suggest that NLRC4-H443P induces apoptotic cell death mediated by caspase-8 and independent of caspase-1. We have earlier shown that caspase-8 activation by NLRC4 is not restricted to only one cell line and that ⌬LRR-NLRC4 could activate caspase-8 in MCF-7 cells, as well as in human macrophage THP-1 cells (15). Unlike THP1 cells, A549 or HEK293 cells do not show detectable levels of NLRC4 and caspase-1 (supplemental Fig. S1C). To further exclude that effects of NLRC4-H443P mutant are restricted to A549 cells, we examined levels of apoptosis in HEK293 cells expressing WT-NLRC4 or H443P mutant by morphological criteria, as well as annexin-V/7-AAD staining. Indeed, NLRC4-H443P induced significantly higher apoptotic cell death compared with WT-NLRC4 (Fig. 1G).
Because SUG1 interacts with NBD and H443P mutation is in the winged helix domain (WHD) of NLRC4 (supplemental Fig. S2A), we therefore wished to determine the domain requirement for caspase-8 activation by NLRC4. Using various deletion constructs of NLRC4, we observed that CARD, as well as NBD (including WHD) are required for caspase-8 activation and induction of cell death (supplemental Fig. S2, B and C). Truncated variants having CARD alone, NBD and WHD alone, or the SUG1-interacting region alone do not induce cell death.
NLRC4-H443P Shows Altered Interaction with SUG1 and Ubiquitinated Proteins-SUG1 has been shown to physically interact with NLRC4, and a region spanning amino acids 91-253 in NBD of NLRC4 ( Fig. 2A) is sufficient for SUG1 interaction (15). Given that NLRC4-H443P-induced cell death is mediated by SUG1, we tested whether NLRC4-H443P shows any altered interaction with endogenous SUG1. GFP-tagged NLRC4-H443P and WT-NLRC4 were overexpressed in HEK293T cells and whole cell lysates subjected to immunoprecipitation using GFP antibody. Western blotting analysis of the precipitates showed that higher levels of endogenous SUG1 coprecipitated with NLRC4-H443P than with WT-NLRC4 (Fig. 3, A and B). We also examined the level of ubiquitinated proteins in the immunoprecipitates and found that compared with WT-NLRC4, the H443P mutant immunoprecipitates showed a significantly higher level of cellular ubiquitinated proteins (Fig. 3, A and C). These results suggest that compared with WT-NLRC4, the H443P mutant shows stronger interaction with endogenous SUG1 and ubiquitinated proteins. The ubiquitination of the H443P mutant was also significantly higher compared with WT-NLRC4, as determined by quantitation of the corresponding band in the ubiquitin blots (Fig. 3, A and D). We also observed that, unlike NLRC4-H443P, the V341A mutant neither showed enhanced interaction with SUG1 nor higher ubiquitination of coprecipitated cellular proteins (Fig. 3E).
Ubiquitination Is Required for NLRC4-H443P-induced Cell Death-Because H443P mutant-induced cell death is dependent on SUG1 and SUG1 is known to mediate ubiquitination of proteins including NLRC4 (15), it is likely that NLRC4-H443Pinduced cell death is dependent on ubiquitination. We examined this possibility by using K48R and K63R mutants of ubiquitin that block polyubiquitination by inhibiting Lys 48 -linked and Lys 63 -linked chain elongation, respectively (22). Co-expression of K48R-ubiquitin strongly inhibited NLRC4-H443Pinduced cell death, whereas wild type ubiquitin had no significant effect (Fig. 3F). Co-expression of K63R-ubiquitin also inhibited cell death, although it was less effective than K48R mutant (Fig. 3F). These results suggest that Lys 48 -linked polyubiquitination and to a lesser extent Lys 63 -linked polyubiquitination mediates NLRC4-H443P-induced cell death.
FADD Is Required for NLRC4-mediated Cell Death and Caspase-8 Activation-FADD is well known for its role in Fasmediated cell death signaling (23). FADD and caspase-8 have been shown to play crucial role in many cell death and inflammation pathways (11, 24 -28). We tested whether FADD is involved in cell death signaling mediated by NLRC4. dnFADD is a deletion construct that lacks death effector domain (DED) and acts as a dominant negative for functional FADD (Fig. 4A). A truncated variant of NLRC4 which lacks the LRR domain ( Fig. 4A) is constitutively active in inducing caspase-8 activation and cell death (15). We overexpressed the two constitutively active forms, ⌬LRR-NLRC4 and NLRC4-H443P, with or without dnFADD in A549 cells and examined cells for apoptotic features. We found that dnFADD significantly inhibited cell death induced by NLRC4-H443P (Fig. 4B) and ⌬LRR-NLRC4 (Fig. 4E). Expression level of proteins were checked by Western blotting (Fig. 4, D and F) to confirm that the difference in ex-tent of cell death was not due to differences in expression of NLRC4-H443P or ⌬LRR-NLRC4. As expected, co-expression of dnFADD with NLRC4-H443P reduced the number of Cl.Casp-8-positive cells (Fig. 4C). Reduced Cl.Casp-8 was also seen in the lysates of cells co-expressing ⌬LRR-NLRC4 with dnFADD ( Fig. 4F). Earlier, our work has shown that co-expression of SUG1 with WT-NLRC4 enables it to initiate caspase-8dependent apoptotic cell death. We found that FADD is also required for NLRC4-SUG1-mediated cell death because co-expressed dnFADD could rescue apoptotic cell death (Fig. 4G) induced by co-expressed NLRC4 and SUG1.
To further investigate the role of FADD in cell death signaling induced by NLRC4, we examined whether FADD is part of the multimolecular complex formed by NLRC4 or ⌬LRR-NLRC4. Because overexpression of FADD induced extensive cell death, HA-tagged dnFADD was overexpressed in HEK293T cells along with GFP fusion proteins of NLRC4 or ⌬LRR-NLRC4 for 24 h; whole cell lysates were subjected to immunoprecipitation using HA antibody and analyzed for the presence of NLRC4 or ⌬LRR-NLRC4 using GFP antibody. We observed that WT-NLRC4, as well as the constitutively active ⌬LRR-NLRC4, was present in immunoprecipitates of dnFADD (Fig. 4H).
To determine the role of SUG1 and FADD in NLRC4-S533D-induced cell death, we co-expressed HA-tagged K196M-SUG1 and dnFADD along with NLRC4-S533D in A549 cells for 24 h. Quantitation of apoptosis revealed that co-expressed dnFADD can reduce NLRC4-S533D-induced cell death, but K196M-SUG1 does not have any effect on NLRC4-S533D-mediated cell death signaling (Fig. 5H). Immunoprecipitation assay showed that, unlike NLRC4-H443P, NLRC4-S533D does not show stronger affinity for cellular SUG1 compared with WT-NLRC4; however, we observed higher ubiquitination of NLRC4-S533D as well as coprecipitated cellular proteins compared with WT-NLRC4 (Fig. 5I).
NLRC4-H443P Induces Apoptosis Independent of Phosphorylation at Ser 533 -We also checked for the role of Ser 533 phosphorylation in NLRC4-H443P-induced cell death. The loss of the Ser 533 phosphorylation site in NLRC4-H443P did not have any effect on its ability to constitutively induce caspase-8-mediated cell death because a phosphodefective double mutant NLRC4-H443P/S533A showed apoptosis in 29.52 Ϯ 3.181.90% expressing cells compared with 28.67 Ϯ 1.430.30% cell death induced by NLRC4-H443P (Fig. 6A). Also, Cl.Casp-8 levels were found to be similar in whole cell lysates of cells expressing NLRC4-H443P or NLRC4-H443P/S533A mutants (Fig. 6B), indicating redundancy of Ser 533 phosphorylation for NLRC4-H443P-induced apoptotic signaling.

FADD Is Required, but Ser 533 Phosphorylation Is Dispensable for NLRC4-ubiquitin-induced Cell Death and Caspase-8
Activation-NLRC4 modified by addition of two ubiquitin molecules at its C-terminal end (Fig. 7A) can recruit caspase-8 in to a multimolecular complex, resulting in caspase-8 activation and cell death (15). We tested involvement of FADD in NLRC4-Ub2-induced caspase-8 activation. A549 cells expressing NLRC4-Ub2 with or without dnFADD were examined for apoptotic features. We observed that dnFADD co-expression significantly inhibits NLRC4-Ub2-induced cell death (Fig. 7B). As expected, Western blotting analysis of Cl.Casp-8 levels in whole cell lysates also revealed compromised caspase-8 activation upon dnFADD co-expression (Fig. 7C). These results indicated that FADD has a crucial role in NLRC4-Ub2-mediated cell death signaling. We also tested dependence of NLRC4-Ub2mediated apoptotic signaling on Ser 533 phosphorylation. A549 cells were examined for apoptotic features as well as caspase-8 activation upon transient expression of NLRC4-Ub2 and phosphodefective mutant NLRC4-Ub2-S533A for 24 h. It was observed that NLRC4-Ub2 remains constitutively active despite the loss of the Ser 533 phosphorylation site (Fig. 7, D and  E). To test whether caspase-8 forms a molecular complex with NLRC4 or NLRC4 modified with two ubiquitin moieties, mCaspase-8 was transiently expressed along with WT-NLRC4 or NLRC4-Ub2 in HEK293 cells, and whole cell lysates were subjected to immunoprecipitation using caspase-8 antibody. Western blotting analysis of the immunoprecipitates showed that both WT-NLRC4 and NLRC4-Ub2 form a complex with mCaspase-8. Interaction of mCaspase-8 with GFP-NLRC4-Ub2 was stronger than with NLRC4 because relatively more protein coprecipitated (Fig. 7F).

Discussion
In this study, we have used disease associated mutants of NLRC4 (H443P, T337S, and V341A) to examine their effect on apoptotic signaling in human lung epithelial cells. We identify a novel function for NLRC4-H443P to induce constitutive caspase-8 activation and cell death, in addition to its ability to mediate caspase-1 activation during inflammatory response. NLRC4, a component of inflammasome, has primarily been shown to aid in sensing of intracellular bacterial components and enable caspase-1 activation to trigger release of cytokines. NLRC4 is a direct transcriptional target of p53 and contributes to p53-mediated cell death (29,30). Recent evidence shows that NLRC4 also contributes to cell death and brain injury in mice (31). Here, we show that NLRC4-H443P engages SUG1 and FADD to induce caspase-8-dependent cell death independent of caspase-1 activation. Interestingly, our results show that two other disease-associated mutations, T337S and V341A, known to induce caspase-1 activation, do not activate caspase-8, nor do they induce cell death. NLRC4 is an ATPase and is known to remain bound to an ADP molecule in its inactive state. The ADP molecule is stabilized via extensive hydrogen bonding with His 443 and residues Pro 119 , Pro 128 , Thr 135 , Gly 172 , Gly 174 , Lys 175 , Ser 176 , and Thr 177 of NBD (amino acids 95-298), which is also the SUG1-binding region (amino acids 91-253) of NLRC4. In case of the mutation, H443P, it is likely that the ADP binding interactions are disrupted, triggering intramolecular  6). ***, p Ͻ 0.0005. Expression of proteins was checked by SDS-PAGE using HA and GFP antibodies. GAPDH was used as loading control. I, immunoprecipitation assay showing increased ubiquitination of cellular proteins coprecipitated with NLRC4-S533D compared with WT-NLRC4. Indicated plasmids were overexpressed in HEK293T cells, and lysates were subjected to immunoprecipitation using GFP antibody. Immunoprecipitates were analyzed by Western blotting for abundance of ubiquitinated proteins and endogenous SUG1. Con, control; Exp, expressing; IP, immunoprecipitation; UT, untransfected; WB, Western blot; WCL, whole cell lysate. reorganization and resulting in enhanced affinity of NBD for binding partners like SUG1, thus enabling NLRC4-H443P to induce SUG1 mediated caspase-8 activation and cell death (Fig. 8).
Unlike His 443 , the residues Thr 337 and Val 341 are not known to be involved in intramolecular interactions involving SUG1binding region, and therefore, mutations of these residues may not alter the affinity of NLRC4 toward SUG1, and these mutants may not show phenotypes similar to H443P with respect to caspase-8 activation. However, the mutations T337S and V341A must be causing some alteration that results in autoactivation of NLRC4 to induce caspase-1-mediated inflammatory response.
Although the mechanism of caspase-1 activation by NLRC4 is evident from the fact that NLRC4 and casapse-1 interact through heterodimerization of their CARDs or via another CARD containing protein ASC (32,33), the molecular details involved in caspase-8 activation are not known. FADD has been shown to enable caspase-8 recruitment to death-inducing silencing complex and initiate its activation (34,35). We observed that NLRC4-H443P or ⌬LRR-NLRC4 or NLRC4 coexpressed with SUG1 induce caspase-8 activation and cell death dependent on FADD, suggesting that FADD may play a role in forming an intracellular caspase-8 activation platform. Our results show that NLRC4 as well as ⌬LRR-NLRC4 can complex with dnFADD. Whether the interaction between NLRC4 and FADD is direct or is aided by SUG1 is presently not known. Interaction of FADD with NLRC4 is independent of the LRR domain of NLRC4 and death effector domain of FADD. Our observation that mCaspase-8 interacts more strongly with ubiquitinated NLRC4 compared with WT-NLRC4 suggests that caspase-8 is brought into the complex through interaction with ubiquitinated NLRC4. It is known that in addition to death-inducing signaling complex recruitment, other molecular events like polyubiquitination, which are not fully understood, are required for full caspase-8 activation (36). Because the H443P mutant of NLRC4 is also dependent on SUG1 for caspase-8 activation just like ⌬LRR-NLRC4, it is possible that SUG1 may be an additional player required for caspase-8 acti-vation. In this context it may be mentioned that we have earlier shown that TNF-␣ and cycloheximide-induced cell death is dependent on SUG1 (15).
Modifications like phosphorylation and ubiquitination enable transient changes in the properties of molecules. Our experiments demonstrated that H443P mutant but not V341A mutant shows increased association with ubiquitinated cellular proteins. Cell death induced by the H443P mutant was inhibited strongly by K48R mutant of ubiquitin and partially by K63R-ubiquitin, suggesting that specific ubiquitin linkages are engaged to trigger cell death signal. The constitutively active S533D mutant also shows enhanced ubiquitination of associated cellular proteins, although it does not interact better with SUG1 compared with WT-NLRC4. Phosphorylation of Ser 533 by PKC-␦ is required for caspase-1 activation (10). Phosphorylation at Ser 533 of NLRC4 keeps it in a primed but inactive state (10,11). We show that ⌬LRR-NLRC4 or co-expression of NLRC4 with SUG1 activates caspase-8 dependent on Ser 533 phosphorylation. NLRC4-H443P mutant can activate caspase-8 even when Ser 533 is mutated to a non-phosphorylable form. Because His 443 interacts with the phosphate of ADP, mutation in this residue may result in constitutive activation and may not require structural reorganization achieved by phosphorylation at Ser 533 to activate caspase-8. Salient findings showing the mechanism of caspase-8 activation and cell death induction by NLRC4-H443P are shown as a model in Fig. 8.
The fact that ⌬LRR-NLRC4 is also dependent on Ser 533 phosphorylation for activation of caspase-8 indicates that phosphorylation at Ser 533 is essential even though NLRC4 is in an open configuration. Unlike NLRC4-H443P, the phosphomimetic mutant S533D does not show enhanced affinity for SUG1 and can activate caspase-8 independent of SUG1. However, enhanced ubiquitination and requirement of FADD remain important steps in cell death signaling mediated by NLRC4-S533D. Because addition of ubiquitin molecules to NLRC4 releases dependence on Ser 533 phosphorylation, it may be inferred that mimicking Ser 533 phosphorylation in NLRC4-S533D or SUG1 interaction results in ubiquitination, which eventually causes caspase-8 activation.
Recent studies have discovered functions of NLRC4 in intestinal epithelial lining and in brain cells (12,13,31). Our study prompts further investigation into how caspase-8 dependent function of NLRC4 contributes to immune response or lung pathology like breakdown of epithelial barrier function caused by cell death. Caspase-8 is known to mediate caspase-1 activation and inflammatory response (26,27). We have presented evidence for a caspase-1-independent cell death function of caspase-8 downstream of NLRC4. More detailed studies are required to understand what determines caspase-8 activation versus caspase-1 activation by NLRC4. In conclusion, our results show that an autoinflammatory syndrome causing mutant H443P of NLRC4 constitutively activates caspase-8 dependent on adapter protein FADD. This mutant shows enhanced interaction with SUG1 and ubiquitinated proteins; in addition it shows increased ubiquitination. Ubiquitination plays an important role in caspase-8 activation and cell death induced by this mutant. NLRC4 requires Ser 533 phosphorylation for optimal caspase-8 activation, and H443P mutation overcomes requirement of this phosphorylation. Overall our results provide insight into mechanism of caspase-8 activation by NLRC4 and its H443P mutant. Therefore, individuals with the H443P mutation are likely to show other defects in addition to autoinflammatory disease.

Experimental Procedures
Cell Culture and Transfections-Lung epithelial adenocarcinoma A549 cells and HEK293T cells were maintained in DMEM supplemented with 10% FBS at 37°C in a CO 2 incubator. THP1 cells were maintained in RPMI supplemented with 10% heat-inactivated FBS at 37°C in a CO 2 incubator. For transient expression of proteins, plasmids (purified using Qiagen plasmid mini kit) were transfected using Lipofectamine2000 or Lipofectamine3000 (Invitrogen) as per the manufacturer's protocol. In general, ϳ20% transfection efficiency was achieved in  F, lysates of HEK293 cells transiently expressing GFP-NLRC4 or GFP-NLRC4-Ub2 along with mCaspase-8 were subjected to immunoprecipitation using caspase-8 antibody and Western blotting. The presence of GFP-tagged NLRC4 or NLRC4-Ub2 was detected using GFP antibody and is indicated by arrows. CHX, cycloheximide; Con, control; Exp, expressing; IP, immunoprecipitation; UT, untransfected; WB, Western blot; WCL, whole cell lysate. and control agarose beads (bab-20) for immunoprecipitation were purchased from ChromoTek. HRP-conjugated rabbit and mouse secondary antibodies were obtained from GE Healthcare. FemtoLUCENT TM PLUS HRP kit (786 -10) was procured from GBiosciences. Cy3 (Indocarbocyanine) and Alexa 633conjugated mouse or rabbit secondary antibodies were purchased from Millipore and Thermo Fisher, respectively.
Indirect Immunofluorescence and Microscopy-Immunostaining of cells fixed with formaldehyde was carried out as described previously (37,38). Immunostained cells were observed, and images were taken with an automated Axio-Imager Z.1 (Zeiss) fluorescence microscope using Axiovision software under ϫ40/0.75 NA dry objective.
Quantitation of Cell Death-The cells were grown on coverslips for 24 h before transfection with the desired plasmids. Transfected cells expressing indicated plasmids were fixed 20 h post-transfection (or later as indicated) with 4% formaldehyde and stained with required antibodies as described previously (37,38). The cells were mounted in PBS with 90% glycerol, 1 mg/ml anti-fade (para-phenylenediamine), and 0.5 mg/ml DAPI. Expressing and non-expressing cells were monitored for apoptotic features like cytoplasmic shrinkage, nuclear constriction, loss of refractility, membrane blebbing, and formation of apoptotic bodies under fluorescence microscope (-40/0.75NA dry objective; Nikon). Cell death was quantitated as the percentage of cells showing apoptotic morphology in expressing cells and non-expressing cells from the same coverslips. The data were obtained from three independent experiments carried out on duplicate coverslips (or indicated otherwise) upon observation of at least 200 expressing cells and 800 non-expressing cells from each coverslip and represented as means Ϯ S.D.
Cells transiently expressing desired proteins were assayed for apoptosis using GFP certified apoptosis assay kit, (Enzo Life Sciences, catalog no. ENZ-51002-100) as per manufacturer's protocol. The cells were washed with PBS and incubated with annexin V and 7-aminoactinomycin D (7-AAD) for 15 min at room temperature, washed with PBS, and fixed in 2% formaldehyde, mounted in DAPI-containing medium, and observed under a fluorescence microscope. Apoptotic cells show annexin V binding to externalized phosphatidylserine and/or nuclear staining of 7-AAD. Cells positive for either or both of annexin V and 7-AAD were scored as apoptotic, and the data were gathered as means Ϯ S.D. of the percentage of annexin V/7-AAD positivity after monitoring at least 200 expressing as well as non-expressing cells from at least two independent experiments performed on duplicate coverslips.
Co-immunoprecipitation and Western Blotting-A549 or HEK293T cells were transfected with the desired plasmids for 16 h (or 24 h as indicated) followed by lysis at 4°C for 10 min in buffer containing 20 mM Tris-Cl (pH 7.4), 1% TritonX-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 0.1% BSA, 10 mM N-ethylmaleimide (a deubiquitinase inhibitor; Calbiochem) and protease inhibitor mixture (GBiosciences). Lysate was centrifuged at 10,000 ϫ g for 10 min at 4°C to remove cellular debris. Supernatant was incubated with 2 g of HA antibody or control IgG on a Roto-torque for 8 h at 4°C followed by 2 h of incubation with protein A/G plus agarose beads (Santa Cruz) to immunoprecipitate HA-tagged proteins. The bound fraction was washed, and immunoprecipitates were lysed in SDS containing Laemmli sample buffer. The samples were then subjected to Western blotting analysis as described earlier (37). For immunoprecipitation of GFP-tagged proteins, GFPTrap antibody (ChromoTek) was used as per the manufacturer's protocol. The cells expressing the desired plasmids were lysed in buffer containing 10 mM Tris-Cl (pH 7.5), 0.5% Nonidet P-40 detergent, 1 mM PMSF, 150 mM NaCl, 5 mM EDTA, 10 mM N-ethylmaleimide, and protease inhibitor mixture at 4°C for 30 min. The cell lysates were further incubated with agarose beadconjugated GFPTrap antibody or control antibody for 2 h on a Roto-torque at 4°C. The blots were quantitated using ImageJ software.
Statistical Analysis-Bar diagrams represent means Ϯ S.D. values. Two-tailed Student's t test was used to calculate significance of differences between two means, and one-tailed t test was used to determine significance of relative difference of a test sample compared with control as 1.
Author Contributions-A. K. R., A. S., P. P., G. G., and Y. K. performed the experiments. G. S., V. R., and A. K. R. planned the experiments, analyzed the data, and wrote the paper. G. S. and V. R. conceived and obtained funding for this study. All authors approved final version of the manuscript.