SUMMARY 20 Pattern recognition receptors monitor for signs of infection or cellular dysfunction and 21 respond to these events by initiating an immune response. NLRP1B is a receptor that 22 upon activation recruits multiple copies of pro-caspase-1, which promotes cytokine 23 processing and a pro-inflammatory form of cell death termed pyroptosis. NLRP1B 24 detects anthrax lethal toxin when the toxin cleaves an amino-terminal fragment from the 25 protein. In addition, NLRP1B is activated when cells are deprived of glucose or treated 26 with metabolic inhibitors, but the mechanism by which the resulting reduction in cytosolic 27 ATP is sensed by NLRP1B is unknown. Here, we have addressed whether these two 28 activating signals of NLRP1B converge on a common sensing system. We show that an 29 NLRP1B mutant lacking the amino-terminal region exhibits some spontaneous activity 30 and fails to be further activated by lethal toxin. This mutant was still activated in cells 31 depleted of ATP, however, indicating that the amino-terminal region is not the sole 32 sensing domain of NLRP1B. Mutagenesis of the leucine rich repeat domain of NLRP1B 33 provided evidence that this domain is involved in auto-inhibition of the receptor, but none 34 of the mutants tested was specifically defective at sensing activating signals. 35 Comparison of two alleles of NLRP1B that differed in their response to metabolic 36 inhibitors, but not to lethal toxin, led to the finding that a repeated sequence in the FIIND 37 domain that arose from exon duplication facilitated detection of ATP depletion. These 38 results suggest that distinct regions of NLRP1B detect activating signals. 39 40
INTRODUCTION 41 Inflammasomes are multi-protein complexes that activate pro-caspase-1 in 42
response to pathogen-associated molecular patterns (PAMPS) and danger-associated 43 molecular patterns (DAMPS) (1). Activated caspase-1 processes pro-inflammatory 44 cytokines IL-1β and IL-18 and causes cells to undergo a form of death known as 45 pyroptosis. Pyroptosis is important for the elimination of compromised cells and for the 46 recruitment of immune cells to the site of infection or injury (2). Secretion of IL-1β and IL-47 18 promotes recruitment and activation of inflammatory cells and leads to further 48 production of cytokines important for stimulation of the immune response. 49
NLRP1B is a pattern recognition receptor that triggers caspase-1 processing after 50 it detects an activating signal. NLRP1B contains a NACHT domain (domain present in 51 NAIP, CIITA, HET-E, TP-1), a central leucine rich repeat domain (LRR), a ‘function to 52 find’ domain (FIIND), and a carboxy-terminal caspase recruitment domain (CARD) (3, 4). 53 The NACHT domain is a nucleotide-binding domain that is involved in self-association of 54 NLRP1B (5). The LRR domain has been predicted to be involved in ligand recognition 55 and in intra-molecular interactions that mediate auto-inhibition (1, 4, 6). The FIIND 56 domain undergoes an auto-proteolytic event, which facilitates inflammasome assembly 57 (7-9) and the CARD domain of NLRP1B interacts directly with the CARD domain of pro-58 caspase-1 (6). 59
NLRP1B was originally shown to be activated by anthrax lethal toxin (LeTx) (3). 60 LeTx, a binary toxin composed of protective antigen (PA) and lethal factor (LF), is 61 secreted by the bacterium Bacillius anthracis during infection (10). PA binds to host 62 cellular receptors and translocates the protease LF into the cytosol. Recent studies 63
demonstrated that LF cleaves the amino-terminus of NLRP1B and that this cleavage is 64 sufficient for activation (11-13). 65
Reduction in cytosolic ATP is a second activator of NLRP1B (14). This signal 66 might allow NLRP1B to sense when the cell has entered damaged tissue that is low in 67 glucose and oxygen. Additionally, monitoring cytosolic ATP might enable cells to detect 68 intracellular pathogens that diminish nucleotide pools. Recently, the intracellular 69 parasite Toxoplasma gondii was found to induce NLRP1B inflammasome assembly (15-70 17), although the activation signal elicited by Toxoplasma was not determined and may 71 be distinct from ATP depletion. 72
It is unclear how NLRP1B detects the reduction of cellular ATP or if this signal is 73 direct or indirect. It has been suggested that the amino-terminal region of NLRP1B may 74 serve as a sensor domain, not only for LF proteolytic activity, but for a variety of 75 pathogen proteases (11). We wanted to address whether the amino-terminal region of 76 NLRP1B also serves as a sensor of reduced ATP levels or if a distinct region of NLRP1B 77 is involved. By comparing the abilities of LeTx and ATP depletion to activate a series of 78 NLRP1B mutants, we confirmed that the amino-terminal region is required for sensing 79 LeTx and found that this region is dispensable for the detection of ATP reduction. We 80 isolated LRR mutants that were spontaneously active, but none that was non-responsive 81 to activating signals. We did, however, engineer deletion mutants within the FIIND that 82 were not fully activated by ATP depletion, suggesting that this domain contributes to 83 signal sensing. 84 85
MATERIALS AND METHODS 86 87 88 Cell culture and reagents. 89
HT1080 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium 90 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Protective 91 antigen (PA) and lethal factor (LF) were purified as described previously, and applied to 92 cells at a final concentration of 10-8 M (18). Sodium azide (NaN3) and 2-deoxyglucose 93 (2DG) were purchased from Sigma-Aldrich and used at the indicated concentrations. 94 95 Plasmid construction and site-directed mutagenesis. 96
Plasmids encoding NTAP-NLRP1B allele 1, procaspase-1-T7, and pro-IL-1β-97 hemagglutinin (HA) have been described previously (6). 98
QuikChange site-directed mutagenesis (Stratagene) was performed according to 99 the manufacturer’s instructions to generate pNTAP-NLRP1B43-QAQ-45, pNTAP-NLRP1B 100 LRR mutants and pNTAP-NLRP1B allele 5 (for primer sequences see Table S1). 101
pcDNA3-NLRP1B-3XFlag was generated by amplifying NLRP1B from NTAP-102 NLRP1B allele 1 using the forward primer NLRP1BFor and the reverse primer 103 NLRP1BRev. The PCR product was digested with BamHI and XhoI and then ligated into 104 pcDNA3XFlag. 105
NLRP1B N-terminal truncation plasmids were constructed by amplifying 106 fragments from pcDNA3-NLRP1B-3XFlag allele 1. The reverse primer FlagRev was 107 used with the forward primers NLRP1B40F, NLRP1B45F, NLRP1B50F, NLRP1B60F, 108 NLRP1B70F and NLRP1B80F to amplify the designated fragments, NLRP1B40-1233, 109 NLRP1B45-1233, NLRP1B50-1233, NLRP1B60-1233, NLRP1B70-1233, and NLRP1B80-1233. The 110
PCR products were digested with BamHI and NheI and then ligated into pcDNA3. 111 To construct pNTAP-NLRP1BΔ810-870, pNTAP-Nlrp1Δ749-809 and pNTAP-Nlrp1Δ749-112
870, standard molecular biology techniques were used. To construct pNTAP-Nlrp1Δ749-809 113 and pNTAP-Nlrp1Δ749-870, an AscI site was introduced into pNTAP-NLRP1B allele 1 by 114 using the NLRP1BAscI primer and its complement. The plasmid was digested with AscI 115 and used as template for PCR. The reverse primer NLRP1BFIINDdelR was used with 116 the forward primers NLRP1BFIINDdel1F and NLRP1BFIINDdel2F to amplify the 117 designated fragments pNTAP-Nlrp1Δ749-809 and pNTAP-Nlrp1Δ749-870. The PCR products 118 with the designated deletions were ligated together. 119
120 IL-1β release assay. 121
One million HT1080 cells were seeded in a 10-cm dish the day before 122 transfection. On the day of transfection, 1 μg each of pNTAP-NLRP1B or indicated 123 mutant, pcDNA3–procaspase-1-T7, and pcDNA3–pro-IL-1β-HA were transfected using 9 124 μl of 1 mg/ml polyethyleneimine, pH 7.2. Approximately 24 h after transfection, cells 125 were washed with PBS (unless stated otherwise) and then treated with LF (10-8 M) and 126 PA (10-8 M) or 50 mM 2DG and 10 mM NaN3 for 3 h. The cell supernatant was mixed 127 with 50 μl of α-HA antibody for 2 h, followed by the addition of 75 μl of protein A 128 Sepharose (RepliGen) and overnight incubation. Proteins were eluted from the protein A 129 Sepharose beads with SDS loading dye and subjected to immunoblotting using a 130 polyclonal α-HA antibody (Santa Cruz sc805). 131 RESULTS 132 133 The amino-terminus of NLRP1B is not essential for inflammasome activation in 134
response to metabolic inhibitors 135 We demonstrated previously that metabolic inhibitors activate the NLRP1B 136
inflammasome by using a reconstituted system (14). HT1080 human fibroblasts were 137 transfected with plasmids encoding NLRP1B allele 1, pro-caspase-1 and pro-IL-1β and 138 were treated with a glycolysis inhibitor, 2DG, and a mitochondrial electron transport 139 chain inhibitor, NaN3. Treatment with these inhibitors resulted in reduction in cytosolic 140 ATP levels and secretion of processed IL-1β. The mechanism of activation of the 141 NLRP1B inflammasome by these metabolic inhibitors, however, is unknown. Although 142 the amino-terminus of NLRP1B is cleaved by LF and this cleavage is sufficient for the 143 activation of the inflammasome (11-13), we were previously unable to detect cleavage of 144 NLRP1B in response to treatment with 2DG/NaN3 (14). Because it is conceivable that 145 reduced ATP levels led to a small amount of NLRP1B cleavage that was below 146 detection limits, we sought to determine whether an intact LF cleavage site is essential 147 for 2DG/NaN3-induced activation of NLRP1B. Two LF cleavage sites have been 148 identified in murine NLRP1B, cleavage site-1 after lysine 38 and cleavage site-2 after 149 lysine 44 (11, 12). Cleavage site-2 was determined to be the predominant LF cleavage 150 site since mutation of this site abolished NLRP1B activation in response to LeTx. We 151 mutated cleavage site-2 by substituting amino acids 43LKL45 for QAQ as described 152 previously (11) and tested the mutant for inflammasome activation. In agreement with 153 previous reports, the mutation of LF cleavage site-2 abolished processing and release of 154 IL-1β in response to LeTx (Fig. 1A). This mutant was still able to induce IL-1β secretion 155 in response to 2DG/NaN3 (Fig. 1A), however, suggesting that the cleavage site was not 156 important for NLRP1B activation by these inhibitors. 157
We next sought to determine whether the amino-terminus of NLRP1B is essential 158 for this process. We first constructed an amino-terminal deletion mutant, NLRP1B45-1233, 159 that mimics LF-cleaved NLRP1B. A low level of IL-1β secretion was observed from cells 160 expressing NLRP1B45-1233 in the absence of signal, indicating a degree of spontaneous 161 activity (Fig. 1B). LeTx treatment did not induce additional IL-1β secretion (Fig. 1B). In 162 contrast, 2DG/NaN3 induced IL-1β secretion from cells expressing NLRP1B45-1233 at a 163 comparable level as cells expressing wild-type NLRP1B (Fig. 1B). These results indicate 164 that the “pre-cleaved” NLRP1B45-1233 mutant has some spontaneous activity but is still 165 responsive to 2DG/NaN3. 166
We next constructed a series of deletion mutants of NLRP1B to assess the role of 167 the amino-terminus in signal detection. Deletion of 40 residues from the amino-terminus 168 had little effect on IL-1β release in response to LeTx or 2DG/NaN3 as compared to full-169 length NLRP1B (Fig. 1C). Deletion of between 50 and 80 amino acids, however, 170 resulted in low spontaneous activity, non-responsiveness to LeTx, and a higher 171 responsiveness to 2DG/NaN3 (Fig. 1C). Altogether these results suggest that the amino-172 terminus of NLRP1B is essential for inflammasome activation by LeTx, but is 173 dispensable for activation in response to metabolic inhibitors. 174 175 The LRR domain is involved in auto-inhibition of NLRP1B 176
It has been speculated that the leucine rich repeat (LRR) domain of NLRP1B is 177 involved in auto-inhibition and ligand recognition (1, 4, 6): the inactive form of NLRP1B 178 might be stabilized by an interaction between the LRR and the NACHT domains, which 179 is relieved upon recognition of a ligand by the LRR domain. To study the LRR domain of 180
NLRP1B, the Phyre2 protein structure prediction server (19) was used to generate a 181 model (residues 626-751) using structures of known LRR containing proteins (Fig. 2A). 182 The predicted structure was based on the crystal structure of the tropomodulin LRR 183 domain (1IO0); 99% of the sequence was modeled with 99.8% confidence. The LRR 184 domain model exhibits a typical arch shape comprising four 28-residue repeats (Fig. 2A). 185 Five α-helices form the convex surface and four β-strands compose the concave surface 186 of the LRR. Typically, the β-strands that make up the concave surface of the LRR are 187 involved in protein-protein interactions, but several LRR containing proteins have been 188 shown to engage in ligand binding at the convex surface (20). For this reason, we 189 mutated amino acids on both the convex and concave surfaces. 190
Since we suspected that the LRR domain is involved in auto-inhibition, we first 191 analyzed all the LRR mutants for spontaneous activity in the absence of signal by 192 assaying overnight supernatants of transfected cells for processed IL-1β (Fig. 2B and C, 193 O/N Supernatants). We then assessed the activity of the LRR mutants in response to 194 either LeTx or 2DG/NaN3 by replacing overnight supernatants prior to treatment and 195 then assaying for processed IL-1β (Fig 2B and C, Treatment Supernatants). 196
We found that most of the alanine substitutions at the convex surface of the LRR 197 domain (Q644A, N648A, R651A, R670A, S673A, D698A/R701A/M702A, E705A, 198 Q727A, T734A/K738A) had no effect on activity; only one mutant (NLRP1B-E731A) 199 exhibited spontaneous activity and was still responsive to either LeTx or 2DG/NaN3 (Fig. 200 2B). Of the eleven concave surface LRR domain mutants tested, two mutants (W661A, 201 Y689A) behaved similarly to wild-type NLRP1B whereas the rest of the mutants 202 demonstrated spontaneous activity and were either responsive to LeTx or 2DG/NaN3 203
(E629A, V663A/K664A, T686A/E687A, Q691A) or non-responsive to both signals 204 (D632A/S634A, K658A/T659A, D720A, I744A/S746A) (Fig. 2C). All mutants were 205 expressed at similar or slightly higher levels compared to wild-type NLRP1B in the 206 absence of transfected pro-caspase-1 and pro-IL-1β, suggesting that the mutations did 207 not destabilize the protein (Fig. S1). Co-expression of pro-caspase-1 and pro-IL-1β with 208 the non-responsive NLRP1B mutants consistently led to lower levels of these mutant 209 proteins compared to the wild-type protein, presumably because these spontaneously 210 active mutants were more prone to assemble into inflammasomes that were secreted or 211 leaked from pyroptotic cells prior to the treatments (Fig. S2). Thus, the observed non-212 responsiveness of some mutants to the treatments was likely a result of low levels of 213 inflammasome components after the overnight transfection period. Altogether our 214 results suggest that it is the concave surface of the LRR domain that is predominantly 215 involved in the auto-inhibition of NLRP1B. 216
217 Deletions in the FIIND of NLRP1B allele 1 impair inflammasome activation in 218 response to metabolic inhibitors 219
The NLRP1B gene is polymorphic and there are 5 alleles (3). Murine 220 macrophages that express either allele 1 or 5 are susceptible to LeTx-induced 221 pyroptosis, while those that express alleles 2, 3, or 4 are resistant. We sought to 222 determine whether NLRP1B allele 5 responds to 2DG/NaN3 treatment. We established 223 that in the reconstituted system, LeTx induced the release of IL-1β from cells expressing 224 NLRP1B allele 5; the amount released was approximately 80% of that from cells 225 expressing NLRP1B allele 1 (Fig. 3A). Treatment of cells expressing NLRP1B allele 5 226
with 2DG/NaN3 resulted in ~40% of the amount of IL-1β compared to the amount 227 released by cells expressing allele 1 (Fig. 3A). These results suggest that NLRP1B 228 allele 5 has a diminished capacity to be activated by metabolic inhibitors. 229
Alignment of NLRP1B alleles 1 and 5 reveal 13 amino acid differences and a 61 230 amino acid insertion in allele 1 (Fig. S3). This insertion is due to duplication of exons in 231 a region of the gene that encodes the FIIND (3). The first repeat (encoding 61 amino 232 acids) is missing in allele 2 and the second repeat (encoding 61 amino acids) is missing 233 in alleles 2, 3 and 5 (Fig. 3B). To determine whether the duplication increased the 234 responsiveness of allele 1 to the metabolic inhibitors, we created deletions in the FIIND 235 of NLRP1B allele 1 that correspond to the deletion found in allele 2 (NLRP1BΔ749-809) and 236 the deletion found in alleles 3 to 5 (NLRP1BΔ810-870). We found that LeTx was able to 237 induce IL-1β secretion from cells expressing either NLRP1BΔ749-809 or NLRP1BΔ810-870 at 238 levels similar to those from cells expressing wild-type NLRP1B allele 1 (~95% and ~90% 239 respectively) (Fig. 3C). In contrast, NLRP1BΔ749-809 and NLRP1BΔ810-870 were attenuated 240 in their abilities to respond to treatment with 2DG/NaN3: approximately 20-25% of the 241 amount of IL-1β was released compared to wild-type NLRP1B allele 1 expressing cells. 242 This suggested the deletion of either one of the repeats affects the responsiveness of 243 NLRP1B to the metabolic inhibitors. 244
To assess whether deletion of both repeats further decreased responsiveness to 245 the metabolic inhibitors, we constructed NLRP1BΔ749-870. NLRP1BΔ749-870 exhibited no 246 activity in response to either LeTx or 2DG/NaN3 (Fig. 3C). Immunoblots of the deletion 247 mutants showed that NLRP1BΔ749-870 ran as a single band on a gel instead of a double 248 band that was observed for wild-type NLRP1B allele 1, NLRP1BΔ749-809 and NLRP1BΔ810-249
870 (Fig. S4), suggesting that the deletion prevented auto-proteolysis of NLRP1B, which 250 is important for the assembly of the inflammasome (7-9). 251 252 DISCUSSION 253
Sensing of LeTx by NLRP1B occurs when LF cleaves the NLRP1B amino-254 terminal region to relieve auto-inhibition (11, 12). It was suggested that this region of 255 NLRP1B might serve as a detector not only for LF, but for other pathogen proteases 256 (11). Since we previously demonstrated that reduction in cytosolic ATP also induced 257 NLRP1B inflammasome activation (14), we wished to address whether metabolic stress 258 was also sensed through this region, possibly as a consequence of being cleaved by 259 mislocalized host proteases. We did not, however, detect cleavage of NLRP1B in cells 260 depleted of ATP (14) and we found that an NLRP1B mutant lacking the entire amino-261 terminal region was still responsive to metabolic inhibitors (Fig. 1C). That metabolic 262 inhibitors activate this mutant suggests that cleavage of the amino-terminus, although 263 sufficient to induce inflammasome assembly, does not efficiently activate the protein. 264 Consistent with this notion, we found that cleavage of wild-type NLRP1B by LF leads to 265 secretion of more IL-1β than spontaneously active NLRP1B45-1233, which mimics LF-266 cleaved NLRP1B (Fig. 1B). It is possible, therefore, that binding of LF to a region 267 outside the amino-terminal domain contributes to NLRP1B activation. We note that LF 268 did not further activate NLRP1B45-1233, but it could be that LF binds only very weakly to a 269 mutant that lacks the substrate binding site. It is also conceivable that binding and 270 cleavage must occur simultaneously for NLRP1B to be efficiently activated. Overall, this 271
work indicates that distinct mechanisms lead to inflammasome assembly in response to 272 LF and to depletion of ATP. 273
NLRP1B may directly sense cytosolic ATP levels to regulate its activity. This idea 274 was suggested by the finding that mutation of the Walker A motif of the NACHT domain 275 results in spontaneous activity (14). A decrease in cytosolic ATP levels might lead to 276 nucleotide-free NLRP1B that self-assembles into an inflammasome. Although this 277 model is attractive in its simplicity, it needs to be tested experimentally using a 278 combination of biochemical and physiological approaches. We thought, however, that it 279 would also be informative to examine other regions of NLRP1B that might be involved in 280 regulation or in detecting metabolic stress. We initially focused our attention on the 281 LRR domain because LRR domains found in pattern recognition receptors play roles in 282 ligand recognition and auto-inhibition (20, 21) and we previously found that deletion of 283 the NLRP1B LRR domain results in spontaneous activity suggesting that it is involved in 284 negative regulation (6). 285
Mechanistic insight into how LRR domains facilitate auto-inhibition was provided 286 by a study in which the structure of auto-inhibited NLRC4 was solved (22). This study 287 showed that the LRR domain makes contact with the nucleotide-binding and 288 oligomerization domain to keep NLRC4 in a monomeric state; mutations that disrupted 289 this interaction resulted in constitutive activation. We mapped mutations in the NLRP1B 290 LRR domain that caused spontaneous activity to the concave surface of the domain. 291 Although our data suggested that the LRR domain is involved in auto-inhibition, we can 292 not rule out the possibility that it is also involved in ligand detection: mutation of amino 293
acids involved in both auto-inhibition and signal detection could yield a spontaneously 294 active phenotype. 295
We then made use of allelic differences in NLRP1B to design two deletions in the 296 FIIND domain that decreased responsiveness of NLRP1B to metabolic stress. There 297 are five alleles of Nlrp1b and only two of these (alleles 1 and 5) encode proteins that are 298 activated by LeTx (3). We found that NLRP1B encoded by allele 5 had a diminished 299 capacity to be activated by 2DG/NaN3 compared to the allele 1 protein and mapped the 300 difference in sensitivity to a repeated sequence within the FIIND domain. Allele 1 301 contains both repeats, while allele 5 is missing the second repeat (Fig. 3B). This 302 repeated region in the allele 1 protein might detect a signal derived from metabolic 303 stress or indirectly affect signal sensing by another domain. Allele 2 encodes a protein 304 that is missing the first repeat (and has numerous amino acid differences compared to 305 the allele 1 protein) – it is not activated by LeTx and has not been tested for 306 responsiveness to metabolic stress. The allele 3 protein is defective at auto-processing 307 of the FIIND domain, which impairs inflammasome assembly, rendering it non-308 responsive to LeTx and ATP depletion (9). The allele 4 protein lacks the CARD domain 309 that recruits pro-caspase-1 so it is unlikely to form a functional inflammasome. 310
Toxoplasma gondii infection has been shown to induce inflammasome activation 311 that is dependent on NLRP1B in mice and on NLRP1A in rats (15-17, 23). There are two 312 NLRP1 paralogs in rats (Nlrp1a and Nlrp1b) (24): the Nlrp1a paralog is associated with 313 rat macrophage susceptibility to LeTx (25), but little is know about the Nlrp1b paralog. 314 Amino-terminal cleavage of murine NLRP1B or rat NLRP1A in response to Toxoplasma 315 gondii has not been demonstrated and it might be that the mechanism of activation is 316
distinct from that of LeTx. One possibility is that a common activation signal derived from 317 Toxoplasma gondii infection and 2DG/NaN3 treatment is detected by these NLRP1 318 homologs: cellular invasion by Toxoplasma gondii could induce metabolic stress within 319 the cell. Many protozoan parasites, including Toxoplasma gondii and Plasmodium 320 falciparum, cannot synthesize purines and must take up host purines using nucleoside 321 transporters (26, 27). Parasite ecto-nucleoside triphosphate diphosphohydrolases may 322 facilitate purine salvage and deplete host ATP so it will be interesting to learn whether 323 this contributes to inflammasome activation. 324
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415 FIGURE LEGENDS 416 417 Figure 1. The N-terminus of NLRP1B is not essential for inflammasome activation in 418 response to metabolic inhibitors. (A) HT1080 cells expressing pro-caspase-1-T7, pro-IL-419 1β-HA and either wild-type NLRP1B, or LF cleavage mutant NLRP1B43-QAQ-45, were 420 treated with LeTx or 50 mM 2DG/10 mM NaN3 for 3 h. Cell supernatants were 421 immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β by 422 immunoblotting. (B) HT1080 cells expressing pro-caspase-1-T7, pro-IL-1β-HA and either 423 full length NLRP1B1-1233 or NLRP1B45-1233, were treated with LeTx or 50 mM 2DG/10 mM 424 NaN3 for 3 h. Cell supernatants were immunoprecipated with anti-HA antibodies and 425 then probed for HA-tagged IL-1β by immunoblotting. (C) HT1080 cells expressing pro-426 caspase-1-T7, pro-IL-1β-HA and with either full length NLRP1B1-1233 or with the indicated 427 N-terminal deletion mutants of NLRP1B, were treated with LeTx or 50 mM 2DG/10 mM 428 NaN3 for 3 h. Cell supernatants were immunoprecipated with anti-HA antibodies and 429 then probed for HA-tagged IL-1β by immunoblotting. Blots are representative of three 430 independent experiments. 431 432 Figure 2. The LRR domain is involved in auto-inhibition of NLRP1B. (A) Predicted model 433 of the LRR domain of NLRP1B generated by Phyre2 server (19) using PDB 1IO0 as a 434 template. Amino acids whose side chains were predicted to be accessible for protein-435 protein interactions at the convex (left panel) and the concave (right panel) surfaces are 436 shown in red. (B) HT1080 cells were transfected with plasmids encoding pro-caspase-1-437 T7, pro-IL-1β-HA and either wild-type NLRP1B or indicated convex surface LRR 438
mutants. Approximately 24 h after transfection, cell supernatants were either 439 immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β by 440 immunoblotting (left, O/N Supernatants) or were replaced with media containing LeTx or 441 50 mM 2DG/10 mM NaN3 and then were immunoprecipitated with anti-HA antibodies 442 and then probed for HA-tagged IL-1β by immunoblotting (right, Treatment 443 Supernatants). (C) HT1080 cells were transfected with plasmids encoding pro-caspase-444 1-T7, pro-IL-1β-HA and either wild-type NLRP1B or indicated concave surface LRR 445 mutants. Approximately 24 h after transfection, cell supernatants either 446 immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β by 447 immunoblotting (left, O/N Supernatants) or were replaced with media containing LeTx or 448 50 mM 2DG/10 mM NaN3 and then immunoprecipitated with anti-HA antibodies and then 449 probed for HA-tagged IL-1β by immunoblotting (right, Treatment Supernatants). 450 451
452 Figure 3. Deletions in the FIIND domain of NLRP1B allele 1 impair inflammasome 453 activation in response to metabolic inhibitors. (A) HT1080 cells expressing pro-caspase-454 1-T7, pro-IL-1β-HA and either NLRP1B allele 1 or NLRP1B allele 5, were treated with 455 LeTx or 50 mM 2DG/10 mM NaN3. Cell supernatants were immunoprecipated with anti-456 HA antibodies and then probed for HA-tagged IL-1β by immunoblotting. IL-1β was 457 quantified (top) using the immunoblot analysis (bottom) from 6 different experiments. 458 Representative experiment is shown (data represents mean and s.d. **** p<0.0001, 459 Student’s t-test). (B) Sequence alignments of NLRP1B FIIND domains of alleles 1-5. 460 Residues in light gray and dark gray represent conserved and non-conserved missense 461 mutations relative to allele 1. (C) HT1080 cells expressing pro-caspase-1-T7, pro-IL-1β-462 HA and either wild-type NLRP1B allele 1 or NLRP1B allele 1 with the indicated deletion 463 in the FIIND domain, were treated with LeTx or 50 mM 2DG/10 mM NaN3. Cell 464 supernatants were immunoprecipated with anti-HA antibodies and then probed for HA-465 tagged IL-1β by immunoblotting. IL-1β was quantified (top) using the immunoblot 466 analysis (bottom) from 7 different experiments. Representative experiment is shown 467 (data represents mean and s.d., *** p<0.001, **** p<0.0001, ANOVA analysis with 468 Dunnett’s post-test). 469 470
