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. 2012 Dec;19(12):2040-8.
doi: 10.1038/cdd.2012.99. Epub 2012 Aug 24.

Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis

E D Crawford  1 , J E Seaman, A E Barber 2nd, D C David, P C Babbitt, A L Burlingame, J A Wells
Affiliations

Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis

E D Crawford et al. Cell Death Differ. 2012 Dec.

Abstract

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.

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Figures

Figure 1
Figure 1
(a) For human, mouse, and Drosophila experiments, cell lines were grown under standard culture conditions, and induced to apoptose using various toxic agents. The apoptotic cells were lysed, and the subtiligase labeling method was used to enrich for N-termini generated during apoptosis. (b) For C. elegans, whole worms were homogenized, cuticles and other debris were spun out, and the resulting lysate was treated with exogenously expressed CED-3 for 2 h at room temperature. The subtiligase labeling method was again used to enrich for unblocked N-termini, in this case many of them derived from the added protease. (c) Abundance levels of all human proteins in the PaxDB database were plotted on a log scale in blue, and levels of the human proteins identified as caspase substrates in our human reference data set are plotted in red. Inset shows a histogram derived from the same data
Figure 2
Figure 2
(a) IceLogo diagrams depicting primary structure preferences for the P4–P4′ residues for cleavages following aspartic acid in each of the four species. Letters above the axis indicate residues enriched over background, and letters below the axis indicate residues depleted with respect to background. (b) Secondary structure predictions for the P4–P4′ residues for cleavages following aspartic acid in each of the four species. L=loop, H=alpha helix, and E=beta sheet. The height of the letter indicates the fraction of sites with the corresponding secondary structure prediction, based on predictions using the NetSurfP server. Top row represents caspase cleavage sites; bottom row represents all 8-mers with D at the P1 position in the same proteins. Asterisks indicate statistically significant enrichment of loops in caspase sites compared with background. *P<0.05. **P<0.01. ***P<0.001
Figure 3
Figure 3
Data analysis pipeline. For each caspase cleavage observed in mouse, Drosophila, or C. elegans, we searched the EggNOG database for human orthologs. If the human ortholog found was also present in our human caspase substrate database, an alignment was created to determine whether the orthologs were cleaved at the same site or different sites. If the human ortholog found was not known to be a caspase substrate, we searched IPA's list of ‘Canonical Pathways' to determine whether it functioned in any pathway(s) known to be enriched for caspase substrates. Each mouse, Drosophila, or C. elegans protein is thus assigned to one of five categories: (1) no human ortholog (species-specific protein), (2) human ortholog is not known to be a substrate (species-specific caspase cleavage), (3) pathway-level conservation, (4) protein-level conservation, or (5) motif-level conservation. Supplementary Table 1 shows all mouse, Drosophila, and C. elegans data, organized into these categories
Figure 4
Figure 4
(a) Venn diagram indicating the overlap in the meNOGs associated with caspase substrates in human, mouse, Drosophila, and C. elegans. (b) When Mouse, Drosophila, or C. elegans caspase cleavage sites aligned with known cleavage sites in human orthologs, the aligned sites shared an average of between 5 and 7 identical residues (considering the P4, P3, P2, P2′, P3′, and P4′ positions, as the P1 position is fixed to Asp). In contrast, when a mouse, Drosophila, or C. elegans protein has a human ortholog with a cleavage site at a different location, the two observed cleavage sites share only an average of between zero and two identical residues (considering the same seven positions). Error bars represent S.D. (c) The protein %ID was calculated for pairwise alignments of each mouse, Drosophila, or C. elegans protein with its closest human ortholog. Pairwise alignments were calculated in Jalview. Triangles represent individual pairwise alignments, while large circles represent the average %ID for each category, with error bars representing the S.D. In some cases, datapoints in the ‘not substrate' category may be false negatives (resulting from our human data set being less than fully complete). (d) In cases where the human orthologs were either not known substrates, or were cut at different sites, the Asp residue was conserved between 15 and 65% of the time
Figure 5
Figure 5
Results of this study show that caspases tend to recognize and cleave particular motifs only across short evolutionary distances (represented by the human–mouse comparison, spanning <100 million years of evolution), but that the same proteins will remain targets across longer distances, and the same pathways over even longer distances (represented by the human-Drosophila and human-C. elegans comparisons, both represented by roughly 600 million years of evolution). Ultimately, the phenotype of apoptosis is conserved across the whole metazoan lineage. A small number of false negatives resulting from our human data set being incomplete would likely only serve to shift all trends depicted in this figure towards higher overall conservation
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References

    1. Zmasek CM, Zhang Q, Ye Y, Godzik A. Surprising complexity of the ancestral apoptosis network. Genome Biol. 2007;8:R226. - PMC - PubMed
    1. Van Damme P, Martens L, Van Damme J, Hugelier K, Staes A, Vandekerckhove J, et al. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods. 2005;2:771–777. - PubMed
    1. Dix MM, Simon GM, Cravatt BF. Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell. 2008;134:679–691. - PMC - PubMed
    1. Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell. 2008;134:866–876. - PMC - PubMed
    1. Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database. Nucleic Acids Res. 2010;38:D227–D233. - PMC - PubMed

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