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. 2009 Feb 17;106(10):3935–3940. doi: 10.1073/pnas.0809414106

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.