Intrinsic apoptosis is evolutionarily divergent among metazoans

Abstract

Apoptosis is regulated cell death that depends on caspases. A specific initiator caspase is involved upstream of each apoptotic signaling pathway. Characterized in nematode, fly, and mammals, intrinsic apoptosis is considered to be ancestral, conserved among animals, and depends on shared initiators: caspase-9, Apaf-1 and Bcl-2. However, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspase activation remain highly heterogeneous and hide profound molecular divergence among apoptotic pathways in animals. Uncovering the phylogenetic history of apoptotic actors, especially caspases, is crucial to shed light on the evolutionary history of intrinsic apoptosis. Here, we demonstrate with phylogenetic analyses that caspase-9, the fundamental key of intrinsic apoptosis, is deuterostome-specific, while caspase-2 is ancestral to bilaterians. Our analysis of Bcl-2 and Apaf-1 confirms heterogeneity in functional organization of apoptotic pathways in animals. Our results support emergence of distinct intrinsic apoptotic pathways during metazoan evolution.

Keywords: cell death evolution; initiator caspases; intrinsic apoptosis; phylogeny.

Figure 1.

Schematic representations of intrinsic apoptosis in nematode, fruit fly, and mammals illustrate the dependence on three major components: the Bcl-2 family, a CARD-caspase, and Apaf-1, which forms the apoptosome platform (structurally different between species) with the initiator caspase. In mammals, the initiator caspase is caspase-9, while in ecdysozoans, it is caspase-2 (result of this study). In mammals, the release of cytochrome c forms the apoptosome after mitochondrial membrane permeabilization. The initiation of the fruit fly pathway involves the removal of inhibition by Rpr, Hid, and Grim. The function of mitochondria is minor in ecdysozoans and cytochrome c is not required to initiate apoptosis. Nematodes lack executioner caspases.

Figure 2.

Topology of the metazoan caspase phylogeny obtained by Bayesian inference using full-length sequence alignments with a Reticulomyxa filosa sequence as out group. CARD-caspases cluster together, with only three sequences appearing outside of the group. These caspases belong to Hydra vulgaris and Octopus bimaculoides and are likely divergent when compared to other CARD-caspases. The distribution of CARD-caspases suggests a monophyly of this caspase type. The topology of the CARD-caspase cluster shows a similar species distribution as in the analysis made on CARD-caspases only (Figure 3). Non-bilaterian animals branch early, bilaterians are monophyletic and split across two main groups, the deuterostomian caspase-9 and the bilaterian caspase-2.

Figure 3.

Topology of metazoan CARD-caspase phylogeny obtained by Bayesian inference using full-length sequence alignments. Four strongly supported monophyletic groups are identified: caspase-9, caspase-2, inflammatory caspases and caspase-Y. Together they form a clade strictly corresponding to bilaterian animals. Caspase-2 is widely distributed among bilaterians [Deuterostomia + Ecdysozoa + Lophotrochozoa/Spiralia] reflecting an ancestral origin. Conversely, caspase-9 is strictly restricted to deuterostomian animals. Inflammatory caspases are restricted to vertebrates, and caspase-Y are protostomian specific. Sequences of non-bilaterians (Cnidaria + Ctenophora + Placozoa) form divergent paraphyletic groups. The selected out group are the CARD-caspases of Porifera Amphimedon queenslandica.

Figure 4.

Phylogeny of Apaf-1 at the metazoan scale made with the full-length sequence alignment. Several monophyletic groups are identified but with inconsistent clustering. Apaf-1 is characterized by the presence of CARD and NB-ARC domain, while Apaf-1 like exhibit various unusual domains. Cnidarians are monophyletic, with the exception of three divergent Apaf-1 like of Nematostella. Deuterostomes are grouped in a coherent cluster of Apaf-1, but variations in domain composition of Apaf-1 like in two echinoderms, Strongylocentrotus purpuratus and Lytechinus variegatus and in the cephalochordate Branchiostoma floridae are observed. These Apaf-1 like are grouped inside protostomians, likely due to high divergence and long branch attraction. Protostomians are composed of ecdysozoans and platyhelminths. Ctenophores, mollusks, annelids, and urochordates do not have any Apaf-1 genes. Among metazoans, Apaf-1 is characterized by high sequence divergence and fast independent evolution, making homology hypotheses difficult to establish. The selected out group is Apaf-1 of Porifera Amphimedon queenslandica.

Figure 5.

Topology of Bcl-2 family phylogeny from Bayesian inference and maximum likelihood at the metazoan scale using out groups Amphimedon queenslandica Bcl-like 1 (XP_003383425.1) (A) and Bcl-like 2 (XP_003387574.1) (B). Bcl-2 proteins strictly clustered into five monophyletic groups: three “pro-apoptotic” clades (Bok, Bak, Bax) and two less supported “anti-apoptotic” groups: the Bcl-2 clade (Bcl-2/W/XL) and a more complex (Bcl-B/Mcl-1/Bfl-1) clade. Each group includes bilaterian as well as non-bilaterian sequences which suggests a deep origin of this complex multigenic family. Clustering into these five groups is consistent between analyses, while relationships among them is not well resolved.

Figure 6.

Reconstruction of convergent hypothetical intrinsic apoptotic pathways among metazoans according to molecular actors detected and identified in their genomes. Variability of intrinsic apoptotic pathways among animals emerged from convergences and recruitment of distinct actors with independent evolutionary history. Caspase-2 is bilaterian-specific and the initiator of ecdysozoans intrinsic apoptosis. Caspase-9 is restricted to deuterostomes and the specific initiator of mammalian intrinsic apoptosis. Deuterostomes exhibit several losses (i.e., caspase-2 in cephalochordates, caspase-9 in urochordates) or duplication (i.e., caspase-9 in echinoderms), highlighting a putative evolutionary flexibility in apoptotic pathway establishment. Mitochondrial functions diverge among phyla and cytochrome c (circle) release thanks to mitochondrial outer membrane permeabilization (MOMP) is specific only to mammals and possibly echinoderms. Cross indicate absence/loss. The convergent evolutionary histories reflect a probable phylum-specific adaptive process leading to parallel evolution of mitochondrial apoptotic pathways observed among animals. Animal draws come from PhyloPic (https://www.phylopic.org/).