Surprising complexity of the ancestral apoptosis network

Abstract

Background: Apoptosis, one of the main types of programmed cell death, is regulated and performed by a complex protein network. Studies in model organisms, mostly in the nematode Caenorhabditis elegans, identified a relatively simple apoptotic network consisting of only a few proteins. However, analysis of several recently sequenced invertebrate genomes, ranging from the cnidarian sea anemone Nematostella vectensis, representing one of the morphologically simplest metazoans, to the deuterostomes sea urchin and amphioxus, contradicts the current paradigm of a simple ancestral network that expanded in vertebrates.

Results: Here we show that the apoptosome-forming CED-4/Apaf-1 protein, present in single copy in vertebrate, nematode, and insect genomes, had multiple paralogs in the cnidarian-bilaterian ancestor. Different members of this ancestral Apaf-1 family led to the extant proteins in nematodes/insects and in deuterostomes, explaining significant functional differences between proteins that until now were believed to be orthologous. Similarly, the evolution of the Bcl-2 and caspase protein families appears surprisingly complex and apparently included significant gene loss in nematodes and insects and expansions in deuterostomes.

Conclusion: The emerging picture of the evolution of the apoptosis network is one of a succession of lineage-specific expansions and losses, which combined with the limited number of 'apoptotic' protein families, resulted in apparent similarities between networks in different organisms that mask an underlying complex evolutionary history. Similar results are beginning to surface for other regulatory networks, contradicting the intuitive notion that regulatory networks evolved in a linear way, from simple to complex.

Figure 1

Overview of the initiation of the intrinsic apoptosis pathway. Annotations and domain compositions for N. vectensis (sea anemone), S. purpuratus (sea urchin), and B. floridae (amphioxus) are based on analyses performed in this work, whereas data for C. elegans, D. melanogaster, and Homo sapiens are based on literature [1,2,11]. (Protein and domain lengths are not to scale. In our analysis we noticed a few additional, spurious domains in some CED4/Apaf-1 family members; these are not shown in this diagram.) On the left side, a current view of metazoan phylogeny is shown [13].

Figure 2

Phylogeny and domain organization of CED-4/Apaf-1 homologs. This phylogeny was calculated using a Bayesian approach (MrBayes) based on a MAFFT alignment of the NB-ARC domains. Posterior probability values are shown for each branch (top numbers). Bootstrap support values for branches that are supported by a minimal evolution method (FastME) based on a PROBCONS alignment are also shown (bottom numbers; for detailed information, see Materials and methods). Furthermore, phylogenies based on full-length alignments of the subset of all Apaf-1 homologs exhibiting a CARD-NB-ARC-WD40 domain composition (all vertebrate sequences, 1_BRAFL, 18_NEMVE, and Dark_DROME) as well as 28_DROPS, CED4_CAAEL, and 31_CAEBR showed precisely the same picture: a clade of vertebrate, amphioxus, and Nematostella sequences under exclusion of insect and nematode sequences. For a detailed list of protein sequences see Additional data file 2. For clarity, sequences from S. purpuratus (2), and B. floridae (6), which appear to be redundant and/or results of erroneous assemblies, are not included in this figure; however, their inclusion/exclusion does not change the quality/interpretation of this phylogeny. All sequences are from complete genomes, except the individual sequences from Aedes aegypti, Caenorhabditis briggsae, Drosophila pseudoobscura, and Tribolium castaneum.

Figure 3

Phylogeny of the multi-motif Bcl-2 family. This phylogeny was calculated using a Bayesian approach (MrBayes) based on a MAFFT alignment of Bcl-2 domains. Posterior probability values are shown for each branch (for detailed information, see Materials and methods). Species abbreviations: BRAFL, Branchiostoma floridae (amphioxus); BRARE, Brachydanio rerio (zebrafish); CAEBR, Caenorhabditis briggsae; CAEEL, Caenorhabditis elegans; CANFA, Canis familiaris (dog); CHICK, Gallus gallus (chicken); CIOIN, Ciona intestinalis (sea squirt); DROME, Drosophila melanogaster (fruit fly); FUGRU, Fugu rubripes (Japanese pufferfish); GEOCY, Geodia cydonium (sponge); HYDAT, Hydra attenuata; LUBBA, Lubomirskia baicalensis (freshwater sponge); NEMVE, Nematostella vectensis (starlet sea anemone); STRPU, Strongylocentrotus purpuratus (purple sea urchin); SUBDO, Suberites domuncula (sponge); TETNG, Tetraodon nigroviridis (green pufferfish); and XENTR, Xenopus tropicalis (western clawed frog). For a detailed list of protein sequences see Additional data file 3. All sequences are from complete genomes except the individual sequences from C. briggsae, G. cydonium, H. attenuata, L. baicalensis, and S. domuncula.