Polypeptide modulators of caspase recruitment domain (CARD)-CARD-mediated protein-protein interactions

Abstract

The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display.

FIGURE 1.

Recombinant CARDs adopt α-helical conformation. Circular dichroism spectra for the Apaf-1 CARD (♢), PC9 CARD (−), and NLRP-1 CARD (○) domains are shown. CD spectra were recorded at 4 μm in 50 mm phosphate, pH 7.0, and the average of 20 scans is represented. The percentages of α-helical structure (inset) were evaluated by the K2D software available in Dichroweb.

FIGURE 2.

CARD-mediated activation of procaspase-9. Apaf-1 CARD, PC9 CARD, and NLRP-1 CARD were incubated at 1, 2, and 5 μm with recombinant non-active PC-9 in S buffer. Caspase-9 activity was measured in the presence of the fluorogenic Ac-LEHD-afc substrate. Caspase-9 activity is represented as increase in fluorescence arbitrary units/s (AU/s). Control (C) refers to the basal activity of PC-9. Data correspond to the mean ± S.D. (error bars) of three independent experiments.

FIGURE 3.

Multiple-protein alignment of CARDs. A, structural overlapping of CARDs. The coordinates were obtained from the Protein Data Bank (accession number 3YGS for Apaf-1 and PC9 CARDs, in black and yellow respectively; 2NZ7 for NOD1 CARD in green; 3KAT for NLRP-1 CARD in blue; and 2KN6 for ASC CARD in gray). Structural modeling was performed with MISTRAL (the Multiple Protein Structure Alignment Server), and PyMOL (Schrodinger LLC) was used to obtain the graphical representation. B, multiple-sequence alignment (ClustalW2). Underlined fragments represent the sequences considered for the synthetic peptides. Conserved amino acids are indicated in red.

FIGURE 4.

Synergistic inhibitory effect of Apaf-1-CARD-derived peptides in cellular extracts. A, peptides A2.Y24G33 and A3.F34V43 were incubated at different ratios in the micromolar range in the reconstituted apoptosome assay in cell-free extracts. Caspase-3 activity was determined in the presence of the fluorogenic Ac-DEVD-afc substrate. B, similar procedures were used for the PC9 CARD-derived peptides C1.L8E17 and C4.R52I60. Data represent the inhibition percentages taking as a control a sample incubated in the absence of peptide. Data correspond to the mean ± S.D. (error bars) of three independent experiments.

FIGURE 5.

Apaf-1, PC9, and ASC CARD-derived peptides do not inhibit caspase-9 enzymatic activity. Caspase-9 preactivated in SC buffer was incubated with CARD-derived peptides (70 μm). Caspase-9 activity was determined in the presence of the Ac-LEHD-afc substrate. Data represent the inhibition percentages with reference to a control sample incubated in the absence of peptides. Data correspond to the mean ± S.D. (error bars) of three independent experiments.

FIGURE 6.

Inhibition of cisplatin-dependent apoptosis in HeLa cells by CARD-derived peptides. HeLa cells were treated with a Lipofectamine/peptide (100 μm) mixture for 4 h, followed by administration of 20 μm CDDP. Cells were harvested after 24 h, and cytosolic extracts were supplemented with caspase assay buffer containing 20 μm Ac-DEVD-afc substrate for caspase-3 activity evaluation. Data correspond to the mean ± S.D. (error bars) of three independent experiments and represent the inhibition percentages obtained with reference to a control sample incubated in the absence of peptides.

FIGURE 7.

Inhibition of CARDs and CARD-derived peptides over procaspase-1-activating inflammasome complexes. CARD domains (5 μm) and CARD-derived peptides (100 μm) were incubated with THP-1 cell extracts. Caspase-1 activity was determined in the presence of the Ac-WEDH-afc substrate. Data represent the inhibition percentages with reference to a control sample incubated in the absence of peptides. Data correspond to the mean ± S.D. (error bars) of three independent experiments.