Inflammatory stimuli regulate caspase substrate profiles - PubMed (original) (raw)

Inflammatory stimuli regulate caspase substrate profiles

Nicholas J Agard et al. Mol Cell Proteomics. 2010 May.

Abstract

The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometry-based proteomics to identify newly cleaved proteins. Analysis of THP-1 monocytic cell lysates treated with recombinant purified caspases identified 82 putative caspase-1 substrates, three putative caspase-4 substrates, and no substrates for caspase-5. By contrast, inflammatory caspases activated in THP-1 cells by mimics of gout (monosodium urate), bacterial infection (lipopolysaccharide and ATP), or viral infection (poly(dA.dT)) were found to cleave only 27, 16, and 22 substrates, respectively. Quantitative stable isotope labeling with amino acids in cell culture (SILAC) comparison of these three inflammatory stimuli showed that they induced largely overlapping substrate profiles but different extents of proteolysis. Interestingly, only half of the cleavages found in response to proinflammatory stimuli were contained within our set of 82 in vitro cleavage sites. These data provide the most comprehensive set of caspase-1-cleaved products reported to date and indicate that caspases-4 and -5 have far fewer substrates. Comparisons between the in vitro and in vivo data highlight the importance of localization in regulating inflammatory caspase activity. Finally, our data suggest that inducers of inflammation may subtly alter caspase-1 substrate profiles.

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Figures

Fig. 1.

Fig. 1.

Degradomics approach for identifying caspase-cleaved peptides. A, to identify cleavage sites, lysates are N-terminally biotinylated with subtiligase and TEVest2. The labeled proteins are captured on streptavidin beads, trimmed to a single peptide with trypsin, and released from the beads with TEV protease. The resulting labeled peptides are identified by LC/MS/MS. B, two modes are used for identifying caspase-cleaved peptides. i, in reverse degradomics, purified caspase is added exogenously to cellular lysates, generating caspase cleavage events. ii, in forward degradomics, inflammatory (or apoptotic) stimuli induce caspase activation in the cell or conditioned media. These stimulated cells are then lysed and analyzed. C, the overlap of forward and reverse degradomics data sets. D, the overlap between forward degradomics data sets for three different inflammatory stimuli.

Fig. 2.

Fig. 2.

Caspase-1-cleaved peptides show propensity for hydrophobic residues at P4. A, sequences surrounding the cleavage site of aspartyl-cleaved protein (P1 = Asp) were aligned and analyzed in a sequence logo. Amino acid letter sizes correspond to the frequency; colors correspond to the side chain functionality as follows: acidic (Asp and Glu), red; hydrophobic (Phe, His, Trp, and Tyr), light blue; aliphatic (Leu, Ile, Met, Val, and Pro), dark blue; small (Ala, Gly, Ser, and Thr), green; and other (Cys, Asn, Lys, Gln, and Arg), black. B, SILAC comparisons between caspase-1-treated and untreated lysates were graphed on a double log plot. Aspartyl-cleaved peptides are color-coded according to their P4 residue: putative apoptotic substrates (Asp and Glu), putative inflammatory substrates (Leu, Ile, Met, Val, Pro, Phe, His, Tyr, and Trp), and unknown (Ala, Gly, Ser, and Thr). Note that the putative inflammatory substrates have a high light/heavy ratio, suggesting that they are produced by the addition of caspase-1. Substrates that were selected for further biochemical examination are circled. C, bar graph representation of the caspase-cleaved peptides in B grouped by SILAC ratio. Note that the caspase-derived peptides with the highest light/heavy ratios contained large hydrophobic residues in P4, consistent with caspase-1 specificity.

Fig. 3.

Fig. 3.

In vitro analyses of caspase-1-cleaved substrates show wide variation in rate and selectivity. A, top, individual substrates were expressed by IVT and treated with 50 n

m

caspase-1 (GSDMD) or 200 n

m

caspase-1 (all others) (representative data is shown). At the indicated time points, aliquots were diluted with SDS loading buffer and stored flash frozen prior to analysis by SDS-PAGE. Arrows indicate the masses of cleaved products. Bottom, substrates were treated with 50 n

m

(GSDMD) or 200 n

m

(all others) caspases-1–9 and incubated for 5 min (GSDMD) or 90 min (all others) prior to analysis by SDS-PAGE. B, fluorescence signals from A were integrated and plotted. Lines were fit to standard first order decay, f = _f_0 + A_0_e_−_kt where f is fluorescence intensity, _f_0 is background fluorescence, _A_0 is the fluorescence intensity of the intact protein, k is the rate of the reaction, and t is time. C, apparent _k_cat/Km was calculated for each substrate by assuming [substrate] ≪ Km and applying k = (_k_cat/Km)app × [caspase-1]. D, the caspase screen described in A was applied to SYAP1 D281A. E, caspase specificity was analyzed by integrating the fluorescence intensity of the full-length protein. +++, >60% reduction in intensity compared with no caspase control; ++, >40% reduction; +, >20% reduction or visual evidence of a cleaved product; −, >80% reduction of caspase intensity and no visible products. AU, arbitrary units; Casp, caspase; ZYX, zyxin.

Fig. 4.

Fig. 4.

DNA transfection mediates caspase activation and substrate release. A, THP-1 cells were transfected with or without DNA for the indicated times. Cell extracts and conditioned media were prepared and probed by immunoblot for the presence of caspases-1, -3, and -7 and IL-1β. B, release of lactate dehydrogenase (LDH) was assessed for THP-1 cells under the same conditions. Casp, caspase; AU, arbitrary units.

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