Summary
Apoptosis is characterised by an analogous set of morphological features1 that depend on a proteolytic multigenic family, the caspases2,3. Each apoptotic signalling pathway involves a specific initiator caspase, upstream of the pathway regulation, which finally converges to common executioner caspases. Intrinsic apoptosis, previously known as the mitochondrial apoptotic pathway, is often considered as ancestral and evolutionary conserved among animals2,4–8. First identified in the nematode Caenorhabditis elegans, intrinsic apoptosis was next characterised in fruit fly Drosophila melanogaster and mammals. Intrinsic apoptosis depends on the key initiator caspase-9 (named Ced-3 and Dronc in Caenorhabditis and Drosophila, respectively), the activator Apaf-1 and the Bcl-2 multigenic family2,6,9. Many functional studies have led to a deep characterisation of intrinsic apoptosis based on those classical models. Nevertheless, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspases activation remain highly heterogeneous and hide profound molecular divergences among apoptotic pathways in animals8,10. Independent of functional approaches, the phylogenetic history of the signal transduction actors, mostly the caspase family, is the Rosetta Stone to shed light on intrinsic apoptosis evolution. Here, after exhaustive research on CARD-caspases, we demonstrate by phylogenetic analysis that the caspase-9, the fundamental key of intrinsic apoptosis, is deuterostomes-specific, while it is the caspase-2 which is ancestral and common to bilaterians. Our analysis of Bcl-2 family and Apaf-1 confirm the high heterogeneity in apoptotic pathways elaboration in animals. Taken together, our results support convergent emergence of distinct intrinsic apoptotic pathways during metazoan evolution.
RESULTS AND DISCUSSION
Apoptosis, a regulated cell death, occurs during metazoan development, tissue homeostasis and regeneration11–13. Pioneering works in Caenorhabditis established a molecular network of apoptosis decision, execution, and engulfment-degradation2,4,14. Next, investigation of apoptotic cascade key components from Drosophila and mammals imposed the paradigm that the intrinsic apoptotic “molecular program” is conserved throughout animal evolution2,4–7. However, recent researches revealed evolutionary divergences between these models, with major functional implications15,16. Based on evolutionarily conserved or divergent features of the cell death machinery among metazoans, we investigated the evolutionary history of several major actors of intrinsic apoptosis. We conducted exhaustive research of Apaf-1 and extensive phylogenetic analyses of initiator CARD-caspases and Bcl-2 multigenic family proteins from major animal phyla and describe their evolutionary patterns. It is apparent that in spite of common functional similarities, each actor of these pathways has a distinct evolutionary history that led us to consider the structural and functional organisation of intrinsic apoptotic components as the result of evolutionary convergence among animals. Thus, the three animal models (nematode, fly, mouse) which were used to elaborate a unified concept of intrinsic apoptosis machinery, not only present major functional divergences, but are markedly distinct in their protein architecture, whose origin and evolutionary history followed very different molecular pathways.
Initiator caspases of the intrinsic apoptosis pathways are not homologous
Initiation and execution of apoptotic signalling pathways are fundamentally linked to the complex diversification of the caspases multigenic family, having a widely extended repertoire encompassing all metazoan phyla17–20.
Caspases are a class of proteases composed of three protein domains; the pro-domain, the small P10, and the large P209,21. Initiator caspases, specific to an apoptotic pathway, have a specific Caspase Recruiting Domain (CARD) pro-domain or two Death Effector Domains (DED) pro-domains. The two distinct intrinsic and extrinsic apoptosis involve specifically the caspase-9 (CARD pro-domain) and the caspases-8-10 (DED pro-domains), respectively. Both pathways converge on common executioner caspases activation, triggering apoptosis execution2,3.
Due to the pivotal role of caspase-9 as initiator in intrinsic apoptosis, our main analyses focused on CARD-caspases. We confirmed their distribution in most animal phyla and reconstructed their phylogenetic relationships (Figure 1, Table S1). Regardless of the phylogenetic methodology used for the analysis, three strongly supported (PP> 0.99) monophyletic groups were identified: caspase-9, caspase-2 and a more heterogeneous group named here [Inflammatory Caspases + Caspase-Y], respectively. Although relationships among these three clades differ depending on the methodology employed, their monophyly remains robust and together they form a clade strictly corresponding to bilaterian animals (PP = 0.87; BS>90), while the sequences of [Cnidaria + Ctenophora + Placozoa] form a divergent paraphyletic group named Caspase-X (Figure 1, Supplementary Figure 1).
Caspase-2 appears to be widely distributed among bilaterians and topology of the gene is congruent with the three major groups of eumetazoan [Deuterostomia + Ecdysozoa + Lophotrochozoa/Spiralia], reflecting an ancestral origin in bilaterians. However caspase-9 is restricted to deuterostome animals. Conversely, the clade [Caspase-Y + Inflammatory Caspase] appears to be restricted to [Vertebrata and Lophotrochozoa]. The robustness of the deutostomes-specific caspase-9 clade was explored (Supplementary Figure 2), and its strict diversification among [Vertebrata + Cephalochordata + Echinodermata + Hemichordata] was confirmed (the relative position of the cephalochordate Branchiostoma belcheri 9A paralogous gene remains unstable). However, consistent with previous studies on the ascidian Ciona intestinalis22, the five representatives of ascidian genomes studied are devoid of any caspase-9, probably confirming the loss of this gene in urochordates, the sister-group of vertebrates23. Within bilaterians, the clade-specific acquisition of caspase-9 is a major event, probably leading to the specific functionalities observed in mammals, such as the mitochondrial outer membrane permeabilisation (MOMP) allowing cytochrome c release.
Unexpectedly, Drosophila Dronc and Caenorhabditis Ced-3 are distinctly identified as orthologous to caspase-2 of vertebrates, and not to caspase-9, as was previously reported and largely accepted2,6. All Dronc/Ced3 proteins of insects, horseshoe crab (Xiphosura), and nematodes form a well-defined, strongly supported (PP = 1), monophyletic clade showing only one orthologous gene per species (except horseshoe crab). Thus, the caspase-2 clade of ecdysozoans is the sister group of caspase-2 of [Lophotrochozoa + Deuterostomia]. The absence of identifiable caspase-2 in echinoderms or hemichordates can probably be interpreted as a specific loss in the latter group (i.e. Ambulacraria).
Taken globally, our analysis shown that the usually considered conserved and ancestral initiator caspases of intrinsic apoptosis are not orthologs but divergent.
The caspase activation regulator apoptosome is highly divergent in metazoans
In intrinsic apoptosis, initiator caspase activation depends on their recruitment by a pivotal and considered shared component, the apoptosome platform2,15. Apoptosome formation relies on critical protein interactions which are Caenorhabditis Ced-4, Drosophila Dark, and human Apaf-1 with their respective initiator caspases, Ced-3, Dronk, and procaspase-915. CARD and other protein domains (NOD, arm) are highly conserved in Apaf-1, Dark and Ced-424–26. However, excepted the majority of nematode species16, Apaf-1 possesses WD40 repeats at its C-terminus to bind to the cytochrome c. This binding is required in mammals for Apaf-1 oligomerisation and apoptosome formation26,27, while Ced-4 and Dark do not require cytochrome c for their assembly into an apoptosome15,28. Taken with major structural and regulating assembly differences between the octameric Dark, tetrameric Ced-4 and heptameric Apaf-1 apoptosomes, it reveal evolutionary divergences between animal apoptosomes formation and procaspases activation mechanisms, probably differentially modulating the cell death execution pathway29.
Here we conducted exhaustive analysis by reciprocal BLAST and phylogenetic analyses of Apaf-1 homologs (data not show) and confirmed its ubiquity in the majority of metazoan phyla (Table S2). Remarkably, Apaf-1 orthologs were absent from all urochordates and molluscs in accordance with previous work22,25,30–33, and from Capitella teleta (Annelida), suggesting independent losses in those animals or, a less parsimonious hypothesis, multiple independent evolution of modular proteins from the common ancestor of metazoans25 (Figure 2). In non-bilaterians, Apaf-1 gene family has not been found in Pleurobrachia pileus genome (Ctenophora), but seems present in other early diverging metazoan phyla [Porifera, Cnidaria and Placozoa] (25,34,35, our data). However, their CARD domains were highly divergent from caspase-9 domains of vertebrates and comparative analysis revealed unrelated complex apoptosis networks.
Importantly, analyses from several genomes (cnidarian, nematode, fly, amphioxus, sea urchin, human) clearly identified three well-defined independent clades of paralogous genes among metazoans, highlighting that Ced-4, Dark and Apaf-1 are not homologous between ecdysozoans and vertebrates25. The divergent evolution history of structural actors of the apoptosome platforms confirm functional analyses carried out so far suggesting that caspase structure and interaction differs among taxa and clades16.
Despite its fundamental role in the apoptotic cell death pathways and also in non-apoptotic functions36–38, diverse metazoan phyla have independently experienced adapter protein Apaf-1 ortholog losses or independent molecular evolution. Consequently, the modality of apoptosome formation and the subsequent caspases activation are convergent among metazoan, and the similar mechanisms observed are hypothetised to be related to relaxation of functional constrains on molecular Apaf-1-like molecules oligomerisation process.
Finally, in-depth analysis of apoptosome structure argues in favour of distinct evolutionary origins, most likely modulated by functional interactions involving distinct initiator caspases.
The regulation of apoptosis by the Bcl-2 family is convergent among metazoans
Intrinsic apoptosis is ultimately regulated by the Bcl-2 proteins, composed of several Bcl-2 homologous (BH) domains39,40. In mammals, the balance between pro-survival (four BH1-BH4 domains) and pro-apoptotic proteins (Bax/Bak/Bok and BH3-only) of the Bcl-2 controls initiation of intrinsic apoptosis. Conformational changes of the three-dimensional structures and interactions between Bcl-2 actors enables the assembly of pore-like structures controlling MOMP41.
Multiple sequence alignments of metazoan Bcl-2 family proteins (Supplementary Figure 3, Table S3) (but with over-represented chordates reflective of greater availability of vertebrate genomes) confirm the distribution of Bcl-2 in Metazoa16,24,40,42. Consistent with functional Bcl-2 classification, proteins clustered into five monophyletic groups (i.e. three ‘pro-apoptotic’ clades: Bok, Bak, Bax and two less supported pro-survival - ‘anti-apoptotic’ groups: Bcl-2/W/XL and a more complex Bcl-B / Mcl-1 / Bfl-1 clade) (Supplementary Figure 3). However, respective relationships among each of these well-supported groups were not clearly resolved, and some divergent or less characterised sequences from molluscs such as Biomphalaria glabrata (Bcl-like2, Bcl-like3), from the cnidarian Hydra vulgaris (Bcl-like1) or from the urochordate Ciona intestinalis (Bcl-like1) were not strictly attributed to a particular class.
Caenorhabditis is deprived of pro-apoptotic Bcl-2 and possesses only the pro-survival Ced-9 (orthologous to vertebrates Bcl-2/w/xl), and two BH-3 only proteins (Egl-1 and Ced-13). Conversely, only two pro-apoptotic Bok-like close paralogs (Debcl and Buffy) were present in Drosophila (Figure 2, Supplementary Figure 3), but their functions remains unclear43. In both animals, there is no MOMP and apoptosome assembly does not require cytochrome c binding44. Presence of both anti- and pro-apoptotic Bcl-2 in molluscs underlines the divergent particularities observed inside Protostomia. Contrarily, similarities of Bcl-2 family composition are observed within some deuterostomes (mammals and echinoderms).
Finally, if multiple Bcl-2 genes were acquired early in metazoan evolution, and despite conservation of almost homologous genes, key differences accumulate, making initiation mechanisms in intrinsic apoptotic signalling pathways convergent among animals.
Apoptotic mitochondrial pathways are convergent among metazoans
Functional evidences emphasise that caspase-2 members play a critical role in various cell deaths, but have independently involved in a range of non-apoptotic functions, including cell cycle regulation, DNA repair and tumour suppression45–48. This implication of caspase-2 in a myriad of signalling pathways and interaction with a panel of adaptor molecules demonstrates its functional versatility45,46,49–52. As previously reported and unlike other initiator caspases studied, our phylogenetic analyses corroborate the wide distribution of caspase-2 in bilaterians and suggest its ancestral multifunctionality.
The major structural and functional similarities that led to an erroneous interpretation of the phylogenetic position of Ced-3 and Dronc underlines a probable common evolutionary origin of caspase-2 and -9 genes. We propose here that caspase-9 originates from deuterostome-specific duplication of a caspase-2-like gene, followed by functional specialisation of paralogs. In the case of vertebrates (and probably in Cephalochordates), the two families of paralogs have been preserved. Caspase-2 retains multifunctional activity, and in mammals can interact with PIDDosome platform containing P53, adapter molecules RAIDD, and signalling complex DISC, activating both extrinsic, intrinsic, and DNA damage pathways51,53–55. Conversely, the caspase-9 gene underwent a functional divergence in connection with its specialisation in allosteric interactions with the apoptosome3.
Due to its pivotal role as a mediator of genomic stability through involvement in cell proliferation, oxidative stress, aging and cell death, the molecular divergence of the caspase-1 gene is highly constrained during evolution, probably because destabilisation of any signalling cascade is sufficient to initiate tumorigenesis. Therefore, purifying selection is likely important during caspase-2 evolution, except during the radiation of deuterostomes, when it could have been relaxed due to the duplication and the modification of the functionalisation of caspase duplicates (cf. Duplication-Degeneration-Complementation model)56–58. This model could explain the loss of caspase-9 concomitantly with a caspase-2 duplication in urochordates (Figure 1, Supplementary Figure 2). Similarly, orthologs of caspase-2 have been lost, or strongly diverged, while caspase-9 orthologs present a de novo relative expansion in Strongylocentrotups purpuratus (Echinodermata) and Saccoglossus kowalevskii (Hemichordata).
Due to generalised gene losses in ecdysozoans and in comparison with other bilaterians, Caenorhabditis and Drosophila apoptotic pathways are generally considered simpler than those of vertebrates9,10. However, what distinguishes ecdysozoans from lophotrochozoans (molluscs, annelids, and their relatives) and deuterostomes is not only a smaller number of genes but also above all the shape of pathways organised around different paralogous genes. The absence of orthologous relationships among genes results in a very different structural organisation of platforms but also generates important functional divergences (i.e. mechanisms of regulating assembly, CARD-CARD interactions with procaspases)15.
Consistent with mammalian caspase-2 functions, Caenorhabditis Ced-3 has both initiator as well as executioner activities but is activated by the Ced-4 platform in a specific manner8,51,59,60. Unlike the organisation of the mammal apoptosome, the Caenorhabditis Ced-4 platform presents neither MOMP nor the release of cytochrome c4,10. Due to its interaction with Dark (Apaf-1 paralog), the only CARD-caspase in Drosophila (Dronc) has been wrongly classified as caspase-96,61. Likewise, involvement of Dronc in various processes such as compensatory cell proliferation, inhibition of cell migration or spermatid differentiation, brings this protein closer to the functionalities of caspase-2 clade62,63.
Like other protostomes, molluscs are deprived of caspase-9 but caspase-2 orthologs have been identified in bivalves and were suspected to function in “a caspase-9-like manner”64. Hence, caspase-2 is responsible for apoptotic process during larval metamorphosis in the oyster Crassostrea gigas, but more surprisingly, despite the absence of caspase-9 and Apaf-1 (and thus, of a mammalian-like apoptosome), this peculiar pathway is amazingly associated with cytochrome c release30,33,65–68. The complexity of intrinsic apoptosis in molluscs seems to be important, but divergent from what was observed in ecdysozoans or vertebrates, and more specifically shows a putative expansion of initiator and executioner caspases that participate both in immunity, stress responses, and apoptosis (Figure 2)33,64,69–72.
Unexpectedly, mammals present almost the unique case (with probably the cephalochordates) in which both caspase-2 and caspase-9 are conserved and involved in apoptosis. This putative functional redundancy (i.e. recruitment, autoactivation or transactivation, homodimerisation and subsequent interchain proteolytic cleavage) undoubtedly led to the functional specialisation observed for caspase-9. This appears to be fundamentally linked to the mammalian mitochondrial pathway and non-apoptotic activity most often indirectly via caspase-3 activation73–76. Finally, echinoderms seem to uniquely have an intrinsic apoptosis similar to mammals, with a caspase-9, Bcl-2, Apaf-1, and a MOMP with cytochrome c release (Figure 2)5,77.
Although we can envisage a weak parsimonious scenario that showcases a common ancestral apoptotic pathway in deuterostomes (but implying independent secondarily losses in hemichordates, cephalocordates and urochordates), the similarities observed between echinoderms and mammals more probably reflect functional convergences based on independent recruitment of apoptotic actors.
CONCLUSION
The apoptotic networks of Caenorhabditis and Drosophila do not exemplify ancestral conditions from which mammalian-grade apoptotic complexity emerged but are on the contrary, and as suggested recently, the result of a derived condition specific to ecdysozoans among animals 24,69,78,79.
The core components of intrinsic apoptotic pathways, especially initiator caspases and the apoptosome platform, are not ancestral in metazoans. Our phylogenetic analyses highlight an unexpected evolutionary history: while the bilaterian caspase-2-mediated apoptotic toolkit emerged ancestrally and remains multifunctional, the caspase-9 mediator of the mammalian apoptosome is specific to deuterostomes.
The major functional divergences in mitochondrial apoptotic pathways observed among animals8,80–82 mainly originated in the recruitment of paralogous actors from the same multigenic families reflecting the adaptive processes specific to each taxon, which lead ultimately to convergent evolutionary histories. Interestingly, this richness of the apoptotic genetic repertoire was suggested to be links to the persistence of stem cells in adults from different phyla83,84.
Finally, mitochondria-mediated apoptosis, like other programmed cell deaths, has likely evolved before and throughout metazoan diversification to shape developmental processes, immune response, or to adapt cellular environment to environmental constraint.
RESOURCE AVAILABILITY
Lead contact
Further information and requests should be address to the Lead Contact, Gabriel Krasovec (gabriel.krasovec{at}nuigalway.ie).
Materials availability
Any request should be address to the Lead Contact.
Data and code availability
No code was generated during this study. Source data are available upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
No experimental model was used during this study.
METHOD DETAILS
Sequences dataset construction
Putative metazoans CARD-caspases were identified by using tBLASTn and BLASTp searches with human caspases (caspase-1 to 10), Ced-3, and Dronc as query on NCBI, ANISEED (ascidians), EchinoBase (Strongylocentrotus purpuratus), and neurobase.rc.ufl.edu (Pleurobrachia bachei) databases, and followed by reciprocal BLAST. After identification of CARD-caspases in target species, sequences were added as query to conduct BLAST searches in close relative (i.e. identified CARD-caspases of Crassostrea gigas were used as query to look for in other molluscs). Sequences with an e-value inferior to 1e-10 was retained. All identified sequences were analysed with ScanProsite (ExPaSy)85 and InterProScan (EMBL-EBI)86 to double check the presence of specific caspases domains. Another sequence was added to verify its identification proposed as a caspase-2 in literature: Crassostrea angulata casp-268. Caspases family are short proteins (containing the large common P20 and the small P10 domains) with a high number of genes per species that rapidly limits the relevance of the phylogenetic analyses. To reduce the artefact branching and unreadable topology, the dataset was built using CARD-caspase gene repertoires of selected species and in order to maximize phylogenetic diversity across Metazoa. A full list of all caspase sequences is provided in Table S1.
Metazoan Bcl-2 were identified by using tBLASTn and BLASTp searches with human Bcl-2 as query on NCBI, are followed by reciprocal BLAST. All identified sequences were analysed with ScanProsite (ExPaSy) and InterProScan (EMBL-EBI) to double check the presence of BH domains. Because of their too short sequences, BH3-only were not taking in account. A full list of all Bcl-2 sequences is provided in Table S3.
Multiple alignments of protein sequences were generated using the MAFFT software version 787 with default parameters and also Clustal Omega88 to verify the congruence of the different alignments. All sequences were then manually checked in BIoEdit 7.2 software89 to verify the presence of the specific domains previously identified. Gblocks version 0.91b90 was used to remove vacancies and blur sites. Final alignments are composed of 230, 235, and 147 amino acids for metazoan CARD-caspases alignment, deuterostomian CARD-caspases alignment, and metazoan Bcl-2 alignment, respectively.
APAF-1 detection
Identification of metazoan Apaf-1 was made by tBLASTn and BLASTp using human APAF-1, nematode Ced-4, and fly Dark as query on NCBI, ANISEED (ascidians), and neurobase.rc.ufl.edu (Pleurobrachia bachei) databases, and followed by reciprocal BLAST. E-value threshold was specified to be 0.1 to increase the chance of finding sequences that match. Potential resulting sequences were analyzed with ScanProsite and also InterProScan. A full list of all Apaf-1 sequences is provided in Table S2.
Phylogenetic analysis
Phylogenetic analyses were carried out from the amino-acid alignment by Maximum-Likelihood (ML) method using PhyML 3.191, combined ML tree search with 1000 bootstrap replicates, and tree were visualized using Seaview92. Best amino-acid evolution model to conduct analysis were determined using MEGA1193 and are WAG and LG model for CARD-caspases alignments and Bcl-2 alignment, respectively.
Bayesian analyses were performed using MrBayes (v3.2.6)94 under mixed model. For each analysis, one fourth of the topologies were discarded as burn-in values, while the remaining ones were used to calculate the posterior probability. The run for metazoan CARD-caspases alignment was carried out for 2 000 000 generations with 15 randomly started simultaneous Markov chains (1 cold chain, 14 heated chains) and sampled every 100 generations. The run for deuterostomian CARD-caspases alignment was carried out for 500 000 generations with 5 randomly started simultaneous Markov chains (1 cold chain, 4 heated chains) and sampled every 100 generations. The run for metazoan Bcl-2 alignment was carried out for 5 000 000 generations with 20 randomly started simultaneous Markov chains (1 cold chain, 19 heated chains) and sampled every 100 generations.
ML boostrap values higher than 50% and Bayesian posterior probabilities are indicated on the Bayesian tree (Figure 1; Supplementary Figures 2, 3) For the metazoans caspase-CARD phylogeny, outgroup used is the only one caspase with a pro-domain Card of the Porifera Amphimedon queenslandica (XP_003383519) (Figure 1). Analyses of caspase-Card were made independently at the deuterostomes scale with four different outgroup to test their effect on the stability of the topology: i) caspase-Card-Y of the annelid Capitella teleta (ELT97848.1), ii) caspase-Card 2 of the mollusk Aplysia californica (XP_005113266), iii) caspase-Card-X2 of cnidarian Hydra vulgaris (NP_001274285.1) iv) caspase-Card Ced-3 of the ecdysozoan Caenohabditis elegans (AAG42045.1) (Supplementary Figure 2). For the metazoans Bcl-2 phylogenies, outgroup used to test their effect on the stability of the topology are: i) Bcl-2 like1 (XP_003383425.1) and Bcl-2 like2 (XP_003387574.1) of Porifera Amphimedon queenslandica (Supplementary Figure 3).
AUTHOR CONTRIBUTIONS
JP and EQ managed the project. GK made BLAST, phylogenetic analysis, and figures. GK and EQ wrote the manuscript.
DECLARATION OF INTERESTS
Authors declare no competing interests.
KEY RESOURCES TABLE
ACKNOWLEDGEMENTS
Authors acknowledge Sébastien Darras (Sorbonne Université, Banyuls-sur-mer), Christine Vesque (Sorbonne Université, Paris), Jérôme Gros (Institut Pasteur, Paris), Sabine Hennequin (Sorbonne Université, Paris), Uri Frank (NUIG, Galway), and Helen Horkan (NUIG, Galway) for helpful comments. GK was supported by a Ph.D. fellowship from the French Ministry of Education, Research and Innovation.
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