Functional degradation: A mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes

Abstract

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

Fig. 1.. The N-terminal domain of NLRP1B does not mediate auto-inhibition.

(A) Schematic of mouse NLRP1B domain architecture. Nt, N-terminus; NBD, nucleotide-binding domain; LRR, leucine-rich repeat; FIIND, function-to-find domain; CARD, caspase activation and recruitment domain. FIIND auto-processing (white triangle) is not complete and thus NLRP1B appears as a doublet [(B to D), upper blot]. Orange triangle, lethal factor (LF) cleavage site. (B, C) The indicated N-terminal amino acids of NLRP1 were mutated to alanine [(B), AAA] or glycine-serine-glycine [(C), GGSGG], and inflammasome activation was induced by co-expression of the tobacco-etch virus (TEV) protease which cleaves a TEV site engineered into the NLRP1B N-terminus. Activation was monitored by immunoblot (IB) for p17, generated upon CASP1 processing of pro-IL-1β. MBP, maltose binding protein tag. (D) The Nt of NLRP1B was replaced with a heterologous sequence from flagellin (FlaNt) and inflammasome activation was assessed as in (B). (E to G) A TEV site was positioned at the indicated positions along the NLRP1B Nt, and inflammasome activation was assessed as in (B). Cleaved NLRP1B was detected by immunoblotting for a C-terminal HA tag [(E), upper blot]. The relative intensity of cleaved IL-1β p17 was plotted relative to the position of the TEV site (F) or the relative level of cleaved NLRP1B protein (G). Gel images are representative of single experiment preformed once (B, C, E) or three times (D).

Fig. 2.. Degradation of NLRP1B is necessary and sufficient for NLRP1B inflammasome activation.

(A) 293T cells transfected with constructs encoding NLRP1B, CASP1 and pro-IL-1β were assayed for inflammasome activation as in Fig. 1 in the presence (+) or absence (−) of proteasome inhibitors (MG132 (10 μM) and Bortezomib (1 μM)) or p97/VCP inhibitor (NMS-873 (0.5 μM)). (B) Immortalized 129 bone-marrow macrophages were treated with lethal factor (LF) to activate the NLRP1B inflammasome ± MG132. Endogenous NLRP1B was detected by immunoblot (IB) with 2A12. Relative band intensities are indicated. (C) The plant auxin-interacting degron (AID) was fused to the N-terminus of indicated GFP-NLRP1B variants. Specific degradation was induced with indole-3-acetic acid (IAA) in TIR1-expressing 293T cells. Inflammasome activation was assessed by immunoblot (IB) for IL-1β p17. Relative band intensities are indicated. Gel images are representative of experiments performed at least three times. S, serine. A, alanine.

Fig. 3.. NLRP1B is activated by “functional degradation”.

(A) A model for NLRP1B activation via “functional degradation”: (i) constitutive auto-processing of the NLRP1B FIIND domain results in two non-covalently associated polypeptides: NBD–LRR–FIIND(ZU5) and FIIND(UPA)–CARD; (ii) Lethal factor (LF) protease cleavage of the NLRP1B Nt exposes a neo-Nt; (iii) N-end rule factor recognition of the neo-Nt results in ubiquitylation of NLRP1B; (iv) NLRP1B is degraded by the proteasome, resulting in (v) release of the FIIND(UPA)–CARD fragment; (vi) The FIIND(UPA)–CARD fragment self-assembles into a high molecular weight oligomer which (vii) serves as a platform for CASP1 maturation and downstream inflammasome signaling. (B) The ability of the indicated C-terminal HA-tagged expression constructs to induce inflammasome activity was tested in 293T cells as in Fig. 1. (C) 293T cells were transfected with the indicated amounts (ng) of plasmid encoding HA-tagged FIIND(UPA)–CARD fragment (left) or full-length (FL) NLRP1B (± co-transfected TEV protease) and inflammasome activation was measured as in Fig. 1. L, Ladder of protein molecular weight standards. (D) 293T cells were transfected with CASP1 and HA-tagged constructs as depicted in (B). Lysates were immunoprecipitated (IP) with anti-CASP1 and immunoblotted (IB) as indicated. (E) 293T cells were transfected with CASP1 and HA-tagged constructs as depicted in (B) and analyzed by native PAGE and immunoblot with anti-NLRP1B antibody (2A12). Proteins were native or denatured with sodium dodecyl sulfate (SDS) as indicated. (F) 293T cells were transfected with an expression construct encoding AID-FIIND(ZU5+UPA)–CARD and treated with IAA or MG132 as indicated. Relative band intensities are shown. Gel images are representative of experiments performed at least three times for (B, D, E, F) or a single (C) experiment is shown. (G) 293T cells were transfected with expression constructs for ASC (blue) and TEV-cleavable NLRP1B encoding a C-terminal FLAG (green) tag and HA (magenta) tag (inserted after the TEV site, as shown). The number of ASC specks per field (+/− SD) was quantified for TEV-treated samples, compared to cells expressing a S984A FIIND auto-processing NLRP1B mutant. Representative images depict cytosolic FLAG and HA signal in untreated samples, with FLAG colocalization with ASC specks (white arrows) and concomitant loss of the HA signal in TEV-expressing cells. The total number of ASC specks, or ASC specks positive for both FLAG and HA (n=7) or only FLAG (n=32) or HA (n=0) in TEV-treated samples is quantified from 12 fields from three independent experiments. Scale bar, 10 microns. Significance was determined by student’s t-test ***, P < 0.001.

Fig. 4.. The secreted Shigella flexneri IpaH7.8 E3 ubiquitin ligase activates NLRP1B.

(A, B) 293T cells were transfected with expression constructs for the 129 (A, B) or B6 (B) alleles of NLRP1B, plus CASP1, pro-IL-1β, and GFP-tagged IpaH1.4, 4.5, 7.8 or 9.8. NLRP1B expression and inflammasome activation was assessed as in Fig. 1. (C) As in (A), but cells were transfected with a serine (S) 984 to alanine (A) mutant of NLRP1B. (D) As in (A) but cells were transfected with expression constructs for mutant IpaH7.8: CA, catalytic mutant; ΔE3, deletion of Ub ligase domain; ΔLRR, deletion of LRR. (E) An in vitro ubiquitylation assay (see Methods) was used to assess the ability of IpaH7.8, IpaH7.8 catalytic mutant (7.8CA) or IpaH9.8 to ubiquitylate the 129 or B6 alleles of NLRP1B-FLAG. Reactions were immunopreciptiated with anti-FLAG before immunoblotting with anti-ubiquitin or with anti-NLRP1B (2A12). Images are representative of at least three independent experiments (C, D, E), except for (A, B), which were performed twice. (F) WT, Casp1^–/–, Nlrp1b^–/– or Nlrc4^–/– RAW264.7 cells were infected (MOI 30) with WT Shigella flexneri strain 2457T (black circle), BS103 (virulence plasmid-cured, white box), Δ7.8 (ipaH7.8 deletion, blue triangle), p7.8 (Δ7.8 strain complemented with pCMD136 ipaH7.8, green inverted triangle), or vec (Δ7.8 strain complemented with pCMD136 empty vector, red diamond). Inflammasome-induced pyroptotic cell death was monitored by assaying for lactate dehydrogenase (LDH) activity in culture supernatants 30 minutes post-infection (+/− SD). (G) Immortalized 129 (i129) bone-marrow-derived macrophages were infected with S. flexneri strains as in (F). Cell lysates were immunoblotted with anti-NLRP1B (2A12) or anti-mouse CASP1, or cell death was measured by LDH as in (F) 2 hours post-infection (+/− SD). Data in (F, G) is representative of at least three independent experiments. Data sets were analyzed using one-way ANOVA. P-values were determined by Dunnet’s multiple comparison post-hoc test. *, P < 0.05; ** P < 0.01; ***P < 0.001.