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• W2015449243 abstract "Interleukin (IL)-1β maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called “inflammasomes.” Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens. Interleukin (IL)-1β maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called “inflammasomes.” Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens. Caspase-1 is an intracellular protease that cleaves the precursors of IL-1β and IL-18 to yield active cytokines (Salvesen, 2002Salvesen G.S. Caspases and apoptosis.Essays Biochem. 2002; 38: 9-19Crossref PubMed Scopus (153) Google Scholar). Caspase-1-deficient mice are protected from several acute and chronic inflammatory diseases, including sepsis and colitis (Li et al., 1995Li P. Allen H. Banerjee S. Franklin S. Herzog L. Johnston C. McDowell J. Paskind M. Rodman L. Salfeld J. et al.Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock.Cell. 1995; 80: 401-411Abstract Full Text PDF PubMed Scopus (1248) Google Scholar, Siegmund et al., 2001Siegmund B. Lehr H.A. Fantuzzi G. Dinarello C.A. IL-1 beta-converting enzyme (caspase-1) in intestinal inflammation.Proc. Natl. Acad. Sci. USA. 2001; 98: 13249-13254Crossref PubMed Scopus (332) Google Scholar). Thus, pharmacological strategies for regulating caspase-1 represent attractive approaches for disease intervention. Caspase-1 deficiency, however, also leads to increased susceptibility to bacterial infection (Lara-Tejero et al., 2006Lara-Tejero M. Sutterwala F.S. Ogura Y. Grant E.P. Bertin J. Coyle A.J. Flavell R.A. Galan J.E. Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis.J. Exp. Med. 2006; 203 (Published online May 22, 2006): 1407-1412https://doi.org/10.1084/jem.20060206Crossref PubMed Scopus (270) Google Scholar), demonstrating the importance of caspase-1 activation for host defense. Among other mechanisms, caspase-1 becomes activated in cells through recruitment to macromolecular complexes called “inflammasomes.” The core components of inflammasomes are NLR-family proteins, a large group (n = 22 in humans) of intracellular proteins that contain a putative nucleotide-binding oligomerization domain called NACHT and several leucine-rich repeat (LRR) domains speculated to bind microbial ligands, analogous to TLR-family proteins involved in innate immunity (Inohara et al., 2005Inohara N. Chamaillard M. McDonald C. Nunez G. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease.Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (768) Google Scholar, Martinon and Tschopp, 2005Martinon F. Tschopp J. NLRs join TLRs as innate sensors of pathogens.Trends Immunol. 2005; 26: 447-454Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, Stehlik and Reed, 2004Stehlik C. Reed J.C. The PYRIN connection: novel players in innate immunity and inflammation.J. Exp. Med. 2004; 200: 551-558Crossref PubMed Scopus (105) Google Scholar, Ting et al., 2006Ting J.P. Kastner D.L. Hoffman H.M. CATERPILLERs, pyrin and hereditary immunological disorders.Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (270) Google Scholar). The NACHT and LRRs within NLR-family proteins are often associated with additional domains that allow direct binding to procaspase-1 or that indirectly link them to procaspase-1 via intermediate adaptor proteins. Based on data from cell-based experiments and crude extracts, it has been speculated that microbial ligands induce NACHT-mediated oligomerization of NLR-family proteins, creating a platform for procaspase-1 activation via an induced proximity mechanism analogous to the mechanism of procaspase-9 activation by the “apoptosome” (Pop et al., 2006Pop C. Timmer J. Sperandio S. Salvesen G.S. The apoptosome activates caspase-9 by dimerization.Mol. Cell. 2006; 22: 269-275Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Hereditary differences in some NLR family members are associated with chronic autoimmune and inflammatory diseases (Ting et al., 2006Ting J.P. Kastner D.L. Hoffman H.M. CATERPILLERs, pyrin and hereditary immunological disorders.Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (270) Google Scholar), suggesting that proper regulation of these proteins is important for normal health. NALP1 (also known as NAC, CARD7, DEFCAP, and CLR17.1) was the first NLR family member characterized with respect to inflammasome assembly and caspase-1 activation (Martinon et al., 2002Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3567) Google Scholar). The cellular roles of human NALP1 and its murine ortholog are yet to be defined, but at least one of the isoforms of murine NALP1 is involved in the mechanisms by which anthrax toxin activates caspase-1 (Boyden and Dietrich, 2006Boyden E.D. Dietrich W.F. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin.Nat. Genet. 2006; 38: 240-244Crossref PubMed Scopus (604) Google Scholar). Human NALP1 is a multidomain scaffold protein, containing an N-terminal PYRIN (PYD) domain, followed by a centrally located NACHT domain, five tandem LRR domains, a FIIND domain, and finally a C-terminal CARD (Chu et al., 2001Chu Z.L. Pio F. Xie Z. Welsh K. Krajewska M. Krajewski S. Godzik A. Reed J.C. A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis.J. Biol. Chem. 2001; 276: 9239-9245Crossref PubMed Scopus (144) Google Scholar). Previous data using cell transfections and crude cell extracts suggested a model for the NALP1 inflammasome wherein the PYD domain linked to procaspase-1 via the bipartite adaptor protein ASC, which contains a NALP1-binding PYD domain and a procaspase-1-binding CARD domain, and where procaspase-5 (a close relative of procaspase-1) is bound via the CARD of NALP1, thereby bringing these two proteases into close apposition and promoting their activation (Martinon et al., 2002Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3567) Google Scholar). However, the minimal composition of the inflammasome for achieving caspase-activating competency is unknown, and in the absence of a reconstituted system, a role for additional cellular proteins cannot be excluded. Also poorly defined are the roles of nucleotides as cofactors for inflammasome assembly and the microbial products that activate NALP1. Moreover, it is unknown whether microbial products are sufficient to activate NALP1 versus requiring additional cofactors. For example, in the case of lipopolysaccharide (LPS), binding to TLR4 is mediated by protein cofactors CD14 and MD-2 (Beutler, 2000Beutler B. Tlr4: central component of the sole mammalian LPS sensor.Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (626) Google Scholar). Using baculovirus-expressed recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized its enzymological properties with respect to requirements of nucleotides, microbial cofactors, and adaptor proteins (e.g., ASC) for NALP1 oligomerization and activation of caspase-1. To reconstitute the NALP1 inflammasome (Figure 1A), we expressed NALP1, procaspase-1, and ASC in Sf9 insect cells using recombinant baculoviruses. The NALP1 protein was expressed as a GST-tagged fusion protein purified by a combination of affinity and molecular sieve chromatography to apparent homogeneity (Figure 1B). The inflammasome adaptor protein ASC was similarly expressed and purified. Procaspase-1 was expressed in insect cells as an His6-tagged protein and purified (Figure 1B). Gel sieve chromatography suggested that GST-NALP1 and GST-ASC are monomers, while His6-procaspase-1 is a mixture of monomers and dimers (see Figure S1 in the Supplemental Data available with this article online). Using these three inflammasome components, we investigated the minimal requirements for NALP1-mediated activation of procaspase-1. In this regard, while the CARD of NALP1 was originally reported to bind human inflammatory procaspases 4 and 5 (Martinon et al., 2002Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3567) Google Scholar), we recently observed that human NALP1 is also capable of binding procaspase-1 (Damiano et al., 2004Damiano J.S. Oliveira V. Welsh K. Reed J.C. Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses.Biochem. J. 2004; 381: 213-219Crossref PubMed Scopus (117) Google Scholar). The binding of procaspase-1 to NALP1 is dependent on the CARD domain, as shown by coimmunoprecipitation assays in which epitope-tagged full-length versus ΔCARD NALP1 was expressed in HEK293T cells with various epitope-tagged CARD-containing caspases (Figure 1C). Furthermore, unlike NALP1, the closely related NLR family member NALP3 (Cryopyrin) lacks a CARD and fails to associate with procaspase-1. We first sought to define the microbial ligand requirements of NALP1 for activating procaspase-1 in vitro. Reactions were supplemented with 1 mM ATP, anticipating that NALP1 may require nucleotide triphosphates for oligomerization. At the concentrations employed, procaspase-1 exhibited ∼200 relative fluorescence units (RFU)/min of background hydrolytic activity against the fluorogenic substrate peptide acetyl-tryptophanyl-glutamyl-histidinyl-aspartyl-amino-fluoro-coumarin (Ac-WEHD-AMC) (Figure 2A). Addition of GST-NALP1 at 1:1 molar ratio had little effect on the basal activity of procaspase-1, in the absence of microbial products. Addition to these reactions of synthetic MDP comprised of L-alanyl and D-isoglutamine (mimicking the native structure found in peptidoglycan) induced a 4- to 5-fold increase in caspase-1 activity. In contrast, the DD-enantiomer of MDP had little effect, thus demonstrating the specificity of these results (Figure 2A). Also, γ-tri-diaminopimelic acid (γ-tri-DAP), a peptidoglycan component that constitutes a ligand of NLR-family protein Nod1, did not activate caspase-1 in reactions containing procaspase-1 and NALP1. Addition of the irreversible caspase-1 inhibitor acetyl-tyrosinyl-valinyl-alaninyl-aspartyl-amino-fluoro-coumarin (Ac-YVAD-fmk) to MDP-LD-stimulated reactions negated caspase-1 activity, serving as an additional control. Similar results were obtained using either GST-NALP1 or His6-NALP1 (Figure S2), while GST control protein had no effect (Figure S3). The stimulation of caspase-1 proteolytic activity by MDP-LD correlated with proteolytic processing of procaspase-1 in NALP1-containing reactions, as determined by immunoblot analysis using an antibody specific for the cleaved small subunit of this protease (Figure 2B). In contrast, when ATP was omitted from the reactions, MDP-LD failed to stimulate proteolytic processing of procaspase-1, demonstrating nucleotide dependence. Also, neither the MDP-DD-enantiomer nor γ-tri-DAP stimulated procaspase-1 processing, confirming specificity. The optimal ratio for caspase-1 activation was obtained with 1:1 molar ratio of NALP1/procaspase-1 (data not shown) showing NALP1-dependent increases in caspase-1 activity when only treated with MDP and ATP/Mg2+ (Figure 2C). Addition of NALP1 to procaspase-1 increased protease activity by >20-fold, when ATP and MDP were included (Figure 2C). In contrast to MDP, we observed that LPS induces only a slight increase of caspase-1 activity in NALP1-containing reactions, possibly due to the contamination with small amounts of MDP that may be present in commercial preparations (Figure 2D). The stimulation of caspase-1 activity by MDP-LD was concentration dependent and saturable, and readily fitted by a hyperbolic curve (Figure 2E). The experimentally determined apparent EC50 for MDP was 1.1 ± 0.6 nM, with apparent Vmax of 2.3 ± 0.1 nM·min−1 and kcat of 4.5 ± 0.2 ms−1, under conditions of 1 mM ATP/Mg2+ and with NALP1 and procaspase-1 at 1:1 stoichiometry (Table 1). We conclude therefore that MDP-LD constitutes an effective cofactor for NALP1-mediated activation of caspase-1, and that no additional protein cofactors are required.Table 1NALP1 Kinetic ParametersStoichiometry C1:NALP1 = 1:1EC50 (nM)Vmax (nM·min−1)kcat (ms−1)MDP-LD1.06 ± 0.562.31 ± 0.124.53 ± 0.24ATP0.74 ± 0.283.37 ± 0.186.61 ± 0.35GTP0.92 ± 0.612.59 ± 0.215.09 ± 0.41CTP2.75 ± 1.232.76 ± 0.215.42 ± 0.42TTP11.7 ± 7.523.59 ± 0.727.00 ± 1.42UTP3.32 ± 1.722.79 ± 0.295.47 ± 0.57Mg2+1.11 ± 0.492.88 ± 0.105.65 ± 0.19Parameters are obtained from the data presented in Figure 2, Figure 3 using a nonlinear regression method to fit the Michaelis-Menten equation (mean ± SD; n = 3). Open table in a new tab Parameters are obtained from the data presented in Figure 2, Figure 3 using a nonlinear regression method to fit the Michaelis-Menten equation (mean ± SD; n = 3). We compared various nucleotides with respect to their ability to support caspase-1 activation in NALP1-containing reactions supplemented with MDP. All reactions were supplemented with 0.5 mM MgCl2, as a cofactor. The ribonucleoside triphosphates (NTPs) ATP, GTP, CTP, TTP, and UTP were all capable of supporting NALP1-dependent caspase-1 activation at nanomolar concentrations, with ATP the most efficient of these NTPs (Figure 3A; Table 1). In contrast, deoxy-ATP (dATP) and dideoxy-ATP (ddATP) did not support caspase-1 activation, suggesting that only ribonucleotides are active. ADP also did not stimulate NALP1-dependent caspase-1 activation, suggesting a requirement for the triphosphate form of nucleotides. In addition, the nonhydrolyzable analogs adenosine 5′-(β,γ-imido)triphosphate tetralithium (AMP-PNP), ATP-γ-S, and GTP-γ-S failed to stimulate NALP1-dependent caspase-1 activation (Figure 3A). ATP-induced stimulation of NALP1-dependent caspase-1 activation was concentration dependent and saturable, and readily fitted by a hyperbolic curve (Figure 3B). The experimentally determined apparent EC50 for ATP was 0.7 ± 0.3 nM, with apparent Vmax of 3.4 ± 0.2 nM·min−1 and kcat of 6.6 ± 0.3 ms−1 under conditions of 0.1 μg/ml MDP and 0.5 mM MgCl2, with NALP1 and procaspase-1 at 1:1 stoichiometry (Table 1). The support of NALP1-induced caspase-1 activation by ATP appears to be dependent on Mg2+, as titrating Mg2+ levels in reactions revealed concentration-dependent and saturable increases in caspase-1 activity in reactions containing NALP1, ATP, and MDP (Figure 3C). The experimentally determined apparent EC50 for Mg2+ was 1.1 ± 0.5 nM, with apparent Vmax of 2.9 ± 0.1 nM·min−1 and kcat of 5.6 ± 0.2 ms−1, under conditions of 0.25 mM ATP and 0.1 μg/ml MDP, with NALP1 and procaspase-1 at 1:1 stoichiometry (Table 1). While Mg2+ is competent to support NALP1 reactions, other divalent cations have not been interrogated. The data in Figure 3D were used to determine the kinetic parameters of the inflammasome shown in Table 2, comparing the catalytic activity of procaspase-1 in the presence and absence of NALP1 at 1:1 molar stoichiometry. Compared to procaspase by itself, addition of NALP1 improved Vmax and kcat by 5-fold, with an ∼3-fold increase in catalytic efficiency (kcat/KM). We noted, however, that purified procaspase-1 exhibited significant background protease activity, producing typically 0.08–0.1 nM/min of Ac-WEHD-AMC hydrolase activity per nanomolar of procaspase-1. Because the procaspase-1 preparations were comprised of a mixture of monomers and dimers, we attempted to separate these two species by gel sieve chromatography and compared their activity with and without NALP1. Spontaneous activity of procaspase-1 dimers was at least double that of the monomers (Figure S4). Addition of NALP1 (with ATP/Mg2+ and MDP) had little effect on procaspase-1 dimers but increased the activity of procaspase-1 monomers by ∼5-fold. Thus, it appears that only monomers of procaspase-1 are activable by NALP1. Because monomers represent approximately one-third of the total procaspase-1, the kinetic parameters reported for the NALP1:caspase-1 complex (Table 2) must be interpreted as minimum estimates.Table 2Inflammasome Kinetic ParametersStoichiometry C1:NALP1KM app (nM Ac-WEHD-AMC)Vmax app (nM·min−1)kcat (ms−1)kcat/KM app (M−1·s−1)1:01154 ± 8670.40 ± 0.060.78 ± 0.26682 ± 301:12228 ± 7352.16 ± 0.174.23 ± 0.691900 ± 947C1:NALP1:ASC2:0:02276 ± 8160.87 ± 0.083.43 ± 0.321510 ± 1772:0:11843 ± 2250.94 ± 0.053.72 ± 0.202019 ± 8972:1:02076 ± 7681.43 ± 0.115.63 ± 0.432715 ± 5642:1:12579 ± 3883.04 ± 0.1311.9 ± 0.534623 ± 770Parameters were obtained as above, except ASC was included to reconstitute the complete inflammasome, using data from Figure 4 (mean ± SD; n = 3). Open table in a new tab Parameters were obtained as above, except ASC was included to reconstitute the complete inflammasome, using data from Figure 4 (mean ± SD; n = 3). ASC is a bipartite adaptor protein containing PYD and CARD domains that has been shown to bridge the PYD of NALP1 to the CARD of procaspase-1 (Martinon et al., 2002Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3567) Google Scholar). Though the above experiments demonstrate that ASC is not required for NALP1-mediated caspase-1 activation, we examined the effects of adding ASC to NALP1-containing reactions. First, we titrated ASC protein into reactions containing NALP1 and procaspase-1 at 1:2 molar ratio, reasoning that when ASC is present, two procaspase-1 molecules would be predicted to bind per molecule of NALP1, because ASC links the PYD domain of NALP1 to procaspase-1, while the CARD domain of NALP1 binds procaspase-1 directly. Indeed, when reactions were supplemented with ATP/Mg2+ and MDP, ASC increased caspase-1 activity in a concentration-dependent manner, reaching maximum effect at ∼1:1 molar ratio with NALP1 (Figure 4A). With further increases in ASC concentration, however, decreases in caspase-1 activity were observed, with caspase-1 activity returning to basal levels when ASC was provided in stoichiometric excess. This bimodal behavior of ASC is typical of reactions that involve three or more components, and is consistent with predictions that ASC bridges NALP1 to procaspase-1. Note that ASC did not activate procaspase-1 when NALP1 was excluded from the reactions (Figure 4B). ASC also enhanced proteolytic processing of procaspase-1, when NALP1, ATP, Mg2+, and MDP were present (Figure S5). Second, we compared the catalytic efficiency of caspase-1 with NALP1 in the presence and absence of ASC. For these experiments, procaspase-1 was added to reactions with ASC, NALP1, or the combination of ASC and NALP1, supplementing reactions with ATP/Mg2+ and MDP. The optimal ratio of procaspase-1 was determined to be 2:1 relative to ASC and NALP1 (data not shown). Then, various concentrations of substrate Ac-WEHD-AMC were added to reactions and caspase-1 activity was measured, using the resulting data for Michaelis-Menten analysis (Figure 4C). Without NALP1, ASC had little effect on caspase-1 activity. When combined with NALP1, ASC more than doubled maximal caspase-1 activity attained, and also approximately doubled the apparent Vmax and kcat, without altering KM (Figure 4C and Table 2). Similar results were obtained using either GST-ASC or purified ASC (Figure S6). We correlated the findings regarding NALP1-mediated activation and proteolytic processing of caspase-1 with oligomerization of NALP1, in experiments in which oligomerization of NALP1 was monitored by nondenaturating gel electrophoresis. Purified GST-NALP1 migrated as a monomer in gels, despite the presence of the GST tag, which can sometimes promote dimerization of proteins (Figure 5A). Addition of ATP had no effect on NALP1 in these experiments. In contrast, MDP-LD caused a retardation of NALP1's migration in gels, suggesting that this microbial cofactor either induces a conformational change in NALP1 that makes it less compact (larger Stokes radius) and therefore slows its migration upon gel electrophoresis, or that NALP1 forms dimers when exposed to MDP. Treating NALP1 with both ATP and MDP-LD induced assembly of large oligomers (Figure 5A). These data thus demonstrate that the combination of MDP and ATP induces oligomerization of purified NALP1 protein. Using gel electrophoresis experiments, we compared oligomerization induced by ATP and MDP with some of the reagents that failed to support caspase-1 activation by NALP1 (see Figure 2, Figure 3 above). For example, when AMP-PNP or ADP was substituted for ATP or when the MDP-DD-enantiomer was substituted for MDP-LD, large oligomers of NALP1 were not observed (Figure 5A). Thus, the cofactors that induce oligomerization of purified NALP1 correlated with the cofactors required for stimulating NALP1-dependent caspase-1 activation. Because ATP by itself did not induce a change in electrophoretic mobility of NALP1, while MDP did, we hypothesized a two-step mechanism for NALP1 oligomerization whereby MDP renders the protein competent to oligomerize in response to ATP. To measure the effect of MDP on the ability of NALP1 to bind ATP, we devised a fluorescence polarization assay (FPA) in which binding of fluorophore-conjugated ATP to NALP1 was measured. Several analogs of fluorescein (FL)- or Texas red (TR)-conjugated ATP were compared in which fluorophore was attached at different points, revealing that fluorophore addition to the purine is tolerated but not the ribose or triphosphate moieties (Figure 5B, left; and Figure S7). FL-ATP did not bind NALP1 prior to addition of the active enantiomer of MDP. In contrast, when MDP-LD was present, FL-ATP bound in a concentration-dependent and saturable manner, with apparent KD of 219 ± 38 nM (Figure 5B, middle). The specificity of the results was confirmed by experiments using the inactive enantiomer MDP-DD, which had little effect on FL-ATP binding to NALP1, and by experiments using procaspase-1 as an alternative to NALP1. Thus, MDP-LD is required to render NALP1 competent to bind ATP. The LRRs of NLRs are speculated to autorepress NLR activation until bound by microbial ligands (Stehlik and Reed, 2004Stehlik C. Reed J.C. The PYRIN connection: novel players in innate immunity and inflammation.J. Exp. Med. 2004; 200: 551-558Crossref PubMed Scopus (105) Google Scholar). We therefore hypothesized that deletion of the LRRs would allow NALP1 to bind ATP independently of MDP. To test this idea, we produced and purified NALP1ΔLRR protein in which the LRRs were selectively deleted from the full-length protein (Figure S8). Unlike full-length NALP1, the ΔLRR mutant bound FITC-ATP equally in the presence or absence of MDP (Figure 5B, right). To further characterize the requirements for NALP1 oligomerization, we used nondenaturing gel electrophoresis to compare the isolated NACHT domain of NALP1 with a mutant K340M NACHT domain in which the predicted NTP-binding site was ablated. The wild-type NACHT domain supplied with ATP migrated as an oligomer, while NACHT (K340M) migrated as a monomer in nondenaturing gels (Figure S9). In contrast to the NACHT domain, the PYD and LRR domains of NALP1 migrated as monomers. We conclude therefore that (1) oligomerization of the NACHT domain of NALP1 requires an intact nucleotide-binding site, and (2) the NACHT domain oligomerizes independently of MDP when expressed in isolation without the LRRs. To further explore the effects of ATP and MDP on NALP1 oligomerization, we used electron microscopy (EM) to image NALP1 particles that had been prepared in the presence or absence of MDP and ATP/Mg2+. Electron micrographs of NALP1 before and after incubation with MDP-LD and ATP/Mg2+ indicated a mixture of small particles (most likely monomers, Figure S10) and large particles (oligomers) in both conditions (Figure 6A). The ratio between large and small particles was ∼3× higher after incubation with MDP and ATP (p < 0.001, Figure 6G). Unlike the enzymology experiments above, note that the NALP1 protein preparations used for EM analysis were not size fractionated by gel sieve chromatography to enrich monomers prior to conducting experiments, due to the loss of protein yield. Thus, the baseline representation of NALP1 oligomers prior to addition of MDP and ATP may be higher than reflected in the prior experiments above (Figure 2, Figure 3, Figure 4, Figure 5). Alignment and averaging studies using the EM images of negatively stained specimens revealed that the oligomers have a ring-like organization, with an outer diameter of ∼13 nm and an inner diameter of ∼4 nm (Figures 6B–6D and Figure S10), with density peaks arranged in two concentric rings. Most of the oligomers fall into two classes, one showing five apparent density units (50%, Figure 6C) and the other showing seven density units (32%, Figure 6D) (see the Supplemental Data). More definitive structural studies are required to determine the stoichiometry of oligomerized NALP1 and to determine internal symmetry of the oligomers. The small particles are roughly globular in structure with a diameter of ∼7–9 nm (Figures 6E and 6F and Figure S10), consistent with the mass of GST- NALP1 monomers, assuming a compact spherical shape. We have defined the minimal components of the NALP1 inflammasome required for achieving caspase-1 activation. Our data suggest a two-step mechanism, whereby bacterial cofactor MDP induces a conformational change in NALP1, which then allows the protein to bind NTPs and oligomerize, thus creating a platform for caspase activation. The mechanism by which MDP primes NALP1 for subsequent NTP-dependent oligomerization is a matter for speculation but presumably entails an interaction of MDP with the LRRs. We propose that MDP binding to the LRRs relieves repression on the NACHT domain, allowing NTP-dependent oligomerization of the NACHT domains, based on the observation that deletion of the LRRs from NALP1 renders the protein constitutively active and able to bind ATP without MDP. Several caveats must be considered in interpreting the kinetic parameters of the NALP1 inflammasome measured here. First, our procaspase-1 preparations consisted of mixtures of monomers and dimers, with dimers having higher spontaneous activity but failing to become activated by NALP1, and thus suggesting these procaspase-1 dimers represent at least a partially active state. However, cleaved caspase-1 was only detected when NALP1, ATP, and MDP were added to procaspase-1. In this regard, dimerization has been associated with activation of initiator caspases without requirement for their proteolytic processing (Boatright et al., 2003Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I.M. Ricci J.E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. A unified model for apical caspase activation.Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). In the in vivo context, dimerization of procaspase-1 may be limited by endogenous antagonists that bind the CARD domain of the zymogen, such as Iceberg and COP (Humke et al., 2000Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. ICEBERG: a novel inhibitor of interleukin-1beta generation.Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, Lee et al., 2001Lee S.H. Stehlik C. Reed J.C. Cop, a caspase recruitment domain-containing protein and inhibitor of caspase-1 activation processing.J. Biol. Chem. 2001; 276: 34495-34500Crossref PubMed Scopus (133) Google Scholar), and" @default.
• W2015449243 created "2016-06-24" @default.
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• W2015449243 date "2007-03-01" @default.
• W2015449243 modified "2023-10-17" @default.
• W2015449243 title "Reconstituted NALP1 Inflammasome Reveals Two-Step Mechanism of Caspase-1 Activation" @default.
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