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. 2007 May 8;104(19):8041-6.
doi: 10.1073/pnas.0611496104. Epub 2007 May 2.

Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling

Joseph A Duncan  1 , Daniel T Bergstralh, Yanhong Wang, Stephen B Willingham, Zhengmao Ye, Albert G Zimmermann, Jenny Pan-Yun Ting
Affiliations

Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling

Joseph A Duncan et al. Proc Natl Acad Sci U S A. .

Abstract

The CATERPILLER (CLR/NLR) gene family encodes a family of putative nucleotide-binding proteins important for host defense. Although nucleotide binding is thought to be central to this family, this aspect is largely unstudied. The CATERPILLER protein cryopyrin/NALP3 regulates IL-1beta processing by assembling the multimeric inflammasome complex. Mutations within the exon encoding the nucleotide-binding domain are associated with hereditary periodic fevers characterized by constitutive IL-1beta production. We demonstrate that purified cryopyrin binds ATP, dATP, and ATP-agarose, but not CTP, GTP, or UTP, and exhibits ATPase activity. Mutation of the nucleotide-binding domain reduces ATP binding, caspase-1 activation, IL-1beta production, cell death, macromolecular complex formation, self-association, and association with the inflammasome component ASC. Disruption of nucleotide binding abolishes the constitutive activation of disease-associated mutants, identifying nucleotide binding by cryopyrin as a potential target for antiinflammatory pharmacologic intervention.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Recombinant cryopyrin expressed in insect cell culture can be purified to homogeneity by affinity chromatography. (A) Schematic of the recombinant tags on dual-tagged cryopyrin. N-terminal hexahistidine tag and TEV protease site and C-terminal FLAG tag are indicated. The Pyrin domain, NBD, and LRR domains indicate domains of the native protein. (B) Dual-tagged cryopyrin was expressed in Hi5 Insect cells by infection with recombinant baculovirus (multiplicity of infection = 0.5). After the indicated times, the cells were harvested, flash-frozen, resuspended in lysis buffer, and sonicated. After centrifugation at 15,000 × g for 1 h, the supernatants (S, soluble fraction) were removed and the pellets (P, insoluble fraction) were resuspended in an equal volume of 1× SDS/PAGE loading buffer. The soluble (S, supernatant) and insoluble (P, pellet) fractions (Left) were subjected to immunoblot analysis with anti-FLAG antibody. The soluble lysates from the infected cells fractions are run alone (Right) and demonstrate that immunoreactive material in the second lane in Left is not bleed-over from a neighboring lane. (C) Recombinant cryopyrin was purified by sequential affinity chromatography. Protein eluted from the nickel (N, 200 ng) followed by the anti-FLAG (F, 50 ng) columns were subjected to silver staining (Left) or immunoblot with indicated antibodies (Center and Right).
Fig. 2.
Fig. 2.
Recombinant cryopyrin is an ATP-binding protein. (A) ATPγ S binding activity of the eluted fractions was assayed by filter binding assay as described in SI Materials and Methods using 200 nM ATPγ S. (B) WT cryopyrin eluted from NiNTA agarose was incubated with control or ATP-conjugated agarose. Specific ATP-binding proteins were eluted by incubation with ATP-containing buffer and analyzed by immunoblot with anti-FLAG antibody. (C) PO4 release from [γ-32P]ATP in the presence of highly purified cryopyrin (see Fig. 1C) was measured as described in SI Materials and Methods. Error bars indicate standard deviation of triplicate samples. (D and E) NiNTA and anion-exchange chromatography-enriched cryopyrin was used to assess the specificity of cryopyrin-associated nucleotide-binding activity. Cryopyrin was incubated with 2 nM 35S-ATPγ S (≈10,000 cpm/pmol) and the indicated concentration of unlabeled competitor nucleotides at 4°C for 30 min (at which time bound nucleotide was increasing linearly with increasing time), and bound ATPγ S was assayed. Curves represent nonlinear regression fit to single-site competition model (Y = 100/(1 + 10(X-Log(EC50)))).
Fig. 3.
Fig. 3.
Mutation of the Walker A motif of cryopyrin abrogates its ATP-binding activity. (A) WT and Walker A mutant variants of cryopyrin. Mutated Walker A sequence is shown. Walker B mutation, used in SI Table 1, is noted. Disease-associated mutations, R260W and A439V, used in Fig. 5, are also noted. (B) WT and Walker A mutant (WA) cryopyrin were purified by sequential affinity chromatography as described in Fig. 1A. Silver staining (Left) and immunoblot analysis (Right) of 50 ng of each of the purified proteins is shown. (C) ATPγ S binding activity of the indicated proteins was assayed at 60 min in the presence of 200 nM ATPγ S. Error bars represent standard deviation of ATP-binding measurements determined in triplicate.
Fig. 4.
Fig. 4.
ATP binding by cryopyrin is essential for cryopyrin-induced IL-1β production. THP1 cells were transduced with recombinant retrovirus expressing the following proteins: empty vector (EV), no cryopyrin; WT cryopyrin; and Walker A mutant (WA) cryopyrin. (A) IL-1β secretion was measured 24 h after transduction by ELISA. (B) IL-1β measured in A was corrected for the number of GFP+ cells in the culture. (C) Secreted caspase-1 was assayed by ELISA. Error bars represent standard deviation between transductions performed in triplicate. (D) Retroviral transduction efficiency was determined by monitoring GFP expression with flow cytometry. (A–D) Error bars represent standard deviation between transductions performed in triplicate. (E) Expression levels of recombinant cryopyrin were assessed by immunoblot analysis with anti-FLAG antibody.
Fig. 5.
Fig. 5.
Constitutive activity of disease-associated mutant cryopyrin requires intact ATP-binding activity. (A and B) THP1 cells were transduced with recombinant retrovirus expressing the following proteins: empty vector (EV), no cryopyrin; WT cryopyrin; Walker A mutant (WA) cryopyrin; FCAS mutant cryopyrin (A439V); and FCAS mutant cryopyrin with Walker A mutation (A439V/WA). (A) IL-1β secretion was measured 12 h after transduction by ELISA. (B) IL-1β measured in A was corrected for the number of GFP+ cells in the culture. Error bars represent standard deviation between transductions performed in triplicate. (C) THP1 cells were transfected with plasmids expressing GFP and the following proteins: empty vector (EV), no additional protein; WT cryopyrin; FCAS/Muckle–Wells syndrome mutant cryopyrin (R260W); and R260W with Walker A mutation (R260W/WA). Secreted IL-1β was assayed by ELISA 24 h after transfection. Error bars represent standard error from duplicate measurements.
Fig. 6.
Fig. 6.
Cryopyrin self-association and association with ASC require ATP binding. (A) Extracts from HEK293 cells transfected with plasmids encoding FLAG-tagged WT (Upper) or Walker A mutant (Lower) versions of cryopyrin were subjected to size-exclusion chromatography (as detailed in Materials and Methods). The chromatograms show UV absorbance plotted against volume of elution. Arrows indicate volume of elution for standards of the indicated molecular mass. Cryopyrin content (shown below the chromatographs) is determined by immunoblot analysis with anti-FLAG antibodies. (B) HEK293 cells were transfected with indicated plasmids. Lysates from the cells were subjected to immunoprecipitation with anti-HA antibodies (Left) and anti-FLAG antibodies (Right), and samples were analyzed by immunoblot analysis with the indicated antibodies. (C) THP1 cells were transduced with recombinant retrovirus expressing the following proteins: empty vector, no cryopyrin; FLAG-tagged cryopyrin; or Walker A mutant (WA) FLAG-tagged cryopyrin. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and analyzed by immunoblot with anti-FLAG and anti-ASC. The second and fourth lanes in Left have no sample.
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