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. 2009 Mar 10;106(10):3935-40.
doi: 10.1073/pnas.0809414106. Epub 2009 Feb 17.

Mechanism of Bcl-2 and Bcl-X(L) inhibition of NLRP1 inflammasome: loop domain-dependent suppression of ATP binding and oligomerization

Benjamin Faustin  1 , Ya Chen, Dayong Zhai, Gaelle Le Negrate, Lydia Lartigue, Arnold Satterthwait, John C Reed
Affiliations

Mechanism of Bcl-2 and Bcl-X(L) inhibition of NLRP1 inflammasome: loop domain-dependent suppression of ATP binding and oligomerization

Benjamin Faustin et al. Proc Natl Acad Sci U S A. .

Abstract

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed "inflammasomes," which are involved in proteolytic processing of interleukin-1beta (IL-1beta) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-X(L) inhibit NLRP1. Bcl-2 and Bcl-X(L) inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with K(i) approximately 10 nM. Bcl-2 and Bcl-X(L) were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-X(L) was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-X(L), which are located between the first and second alpha-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-X(L) inhibit NLRP1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Bcl-2 and Bcl-XL suppress in vitro reconstituted NLRP1 inflammasome. (A) Reaction mixtures contained His-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP (Alexis) and GST-Bcl-2, or GST-Bcl-XL, or His-Bcl-XLΔLoop, or GST, or Bid (17 nM). Caspase-1 activity was measured after 60 min by hydrolysis of Ac-WEHD-7-amino-4-methylcoumarin (AMC) substrate (20 μM) (Calbiochem), expressing data as mean ± SD, n = 3. RFU, relative fluorescence units. Asterisks indicate P < 0.05. (B) His-6-NLRP1 with or without GST-Bcl-2, GST-Bcl-XL, or His-Bcl-XLΔLoop (molar ratio: 1/5) was incubated 15 min in ice, then with pro-caspase-1 in the presence of 1 mM ATP, 1 mM Mg2+, 20 μg/mL MDP for 30 min at 37 °C with or without recombinant GST-pro-IL-1β (Novus Biologicals). Proteins were separated by SDS/PAGE and then immunoblotted using anti-p10 caspase-1 antibodies (Santa Cruz Biotechnology) (Upper), or anti-IL-1β (cell signaling) (Lower). (C and D) Analysis of NLRP1 binding to proteins by GST pull-down assay. His-6-NLRP1 was incubated with GST-Bcl-2, GST-Bcl-XL, or GST-Bfl-1 fusion proteins (C), or GST-NLRP1 was incubated with His-6-Bcl-XL versus His-6-Bcl-XLΔloop (D). Proteins associated with glutathione-Sepharose were analyzed by protein staining using Sypro Ruby. Note that gel mobility of GST-NLRP1 was reproducibly altered in reactions containing Bcl-XL, suggesting perhaps induction of a SDS-resistant conformational change in the protein.
Fig. 2.
Fig. 2.
Bcl-2 and Bcl-XL suppress caspase-1 activation induced by NLRP1 but not caspase-1. (A) Reactions contained His-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP and various concentrations of recombinant GST-Bcl-2 (black symbols) or GST-Bcl-XL (white symbols). Caspase-1 activity was measured after 60 min by hydrolysis of Ac-WEHD-AMC substrate (20 μM), expressing data as mean ± SD, n = 3. (B) Purified active p10/p20 caspase-1 [5 nM (black bars) or 0.625 nM (white bars) (Gift from G. Salvesen/Burnham Institute for Medical Research)] in presence or absence of GST-Bcl-2, or GST-Bcl-XL, or Ac-WEHD-CHO was incubated with 10 mM DTT 10 min at 37 °C. Caspase-1 activity was measured after 60 min by hydrolysis of Ac-WEHD-AMC substrate (20 μM), expressing data as mean ± SD, n = 3. Note that the lower concentration (0.625 nM) of active caspase-1 was used to achieve enzyme activity levels comparable to those obtained in reactions where NLPR1 was used to activate pro-caspase-1, thus achieving similar levels of protease activity.
Fig. 3.
Fig. 3.
Bcl-2 and Bcl-XL inhibit ATP binding to NLRP1. (A) His-6-NLRP1 (0.125 μM) was incubated for 15 min in ice in the presence of GST-Bcl-XL, His-Bcl-XLΔLoop, GST-Bcl-2, GST, or Bid (1 μM). The mixture was then incubated for additional 15 min in ice with 1 μM MDP-LD, FITC-conjugated ATP analog (10 nM) and Mg2+ (0.5 mM). ATP binding was analyzed by FPA (n = 3), measuring milliPolars (mP), and the percentage of inhibition was determined vs. NLRP1 incubated only with MDP-LD (mean ± SD). (B) His-6-NLRP1 (0.1 μM) was incubated for 15 min in ice with various amounts of GST-Bcl-2 (black squares), GST-Bcl-XL (white triangles), or GST-Bcl-XLΔLoop (black triangles) and then incubated 15 min on ice with 1 μM MDP-LD, FITC-conjugated ATP analog (10 nM) and Mg2+ (0.5 mM). ATP binding to NLRP1 was analyzed by FPA (n = 3), measuring milliPolars (mP), and the percentage of inhibition calculated (mean ± SD). Percentage inhibition values were derived by subtracting non-specific FITC-ATP binding, based on experiments where an excess of unlabeled ATP (100 nM) was applied to determine the competitive component of FITC-ATP binding (Fig. S2).
Fig. 4.
Fig. 4.
Bcl-XL inhibits oligomerization of NLRP1. Purified His-6-NLRP1 monomers obtained from gel filtration were incubated 15 min in ice with or without GST-Bcl-XL, His-Bcl-XLΔLoop, or GST (1/10 molar ratio). The mixture was then incubated without [no treatment (NT)] or with Mg2+ (1 mM), ATP (1 mM) and MDP-LD (20 μg/mL) for 30 min at 37 °C. Proteins were separated by a first native-PAGE dimension, then by a second denaturing SDS/PAGE dimension, and stained using Sypro-Ruby (7). Arrows at bottom indicate positions of NLRP1 monomers and oligomers.
Fig. 5.
Fig. 5.
Bcl-2 Loop peptide inhibits NLRP1. (A) Reactions contained His-6-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP, and Bcl-2 Loop peptide 35–83 (50 nM). Asterisk indicates P < 0.05. (B) Purified active p10/p20 caspase-1 (5 nM or 0.625 nM) was incubated with or without Bcl-2 Loop peptide 35–83 or Ac-WEHD-CHO (10 nM). (C) Reactions contained His-6-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP, and various 20-mer Bcl-2 Loop peptides (50 nM), or Ac-WEHD-CHO (10 nM). Asterisks indicate P < 0.05. (D) Reactions contained His-6-NLRP1ΔLRR (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, with (black bars) or without (white bars) 0.1 μg/mL MDP, and with or without Bcl-2 Loop peptide 35–83, 71–90 peptide (50 nM), or Ac-WEHD-CHO (10 nM). (E) Reactions contained His-6-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP, and various amounts of Bcl-2 Loop peptide 35–83, 71–90 Loop peptide, or BH4 peptide 9–30. For all experiments, caspase-1 activity was measured after 60 min by hydrolysis of Ac-WEHD-AMC substrate (20 μM), expressing data as mean ± SD, n = 3.
Fig. 6.
Fig. 6.
Enzymology of NLRP1 inflammasome inhibition by Bcl-2, Bcl-XL, and Bcl-2 Loop peptide 71–80. (A) Caspase-1 activity induced by stimulation of NLRP1 with MDP and ATP was measured in the presence or absence of various peptides. Reactions contained His-6-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP, and various 10-mer or 5-mer Bcl-2 Loop peptides (50 nM). Asterisk indicates P < 0.05. (B) ATP binding by NLRP1 was measured in presence or absence of peptides. Reactions contained His-6-NLRP1 (0.125 μM), GST-Bcl-2 protein or Bcl-2 loop peptides (2 or 20 μM), 1 μM MDP[LD], FITC-ATP analog (10 nM), and Mg2+ (0.5 mM). FITC-ATP binding was analyzed by FPA (n = 3), measuring milliPolars (mP), and values corrected for non-specific FITC-ATP binding as determined by competition with excess unlabeled ATP. The percentage inhibition was determined compared to NLRP1 incubated only with MDP-LD (mean ± SD). (C and D) Enzyme kinetics analysis of NLRP1 inhibition. Reactions contained His-6-NLRP1 (8.5 nM), pro-caspase-1 (8.5 nM), 0.25 mM ATP, 0.5 mM Mg2+, 0.1 μg/mL MDP, with or without 2 nM GST-Bcl-2, or GST-Bcl-XL, or (D) 2 nM Bcl-2 loop peptides 31–50 or 71–80. Caspase-1 activity was measured after 60 min by hydrolysis of various concentrations of Ac-WEHD-AMC substrate, expressing data as mean ± SD, n = 3. Experimental data were analyzed by linear regression to fit the Lineweaver–Burk equation.
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