Evolution of Apoptotic Signaling Pathways Within Lophotrochozoans

GBE Evolution of Apoptotic Signaling Pathways Within Lophotrochozoans Helen R. Horkan Gabriel Krasovec 1, *, Nikolay Popgeorgiev * 1,4, 2,3 , Michel Vervoort 4,† , Eve Gazave 4 , Centre for Chromosome Biology, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland 2 Centre de Recherche en Cancérologie de Lyon, U1052 INSERM, UMR CNRS 5286, Centre Léon Bérard, Université Claude Bernard Lyon 1, Lyon, France 3 Institut Universitaire de France (IUF), Paris, France 4 Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France † Deceased. *Corresponding authors: E-mails: ; ; . Accepted: September 17, 2024 Abstract Apoptosis is the main form of regulated cell death in metazoans. Apoptotic pathways are well characterized in nematodes, flies, and mammals, leading to a vision of the conservation of apoptotic pathways in metazoans. However, we recently showed that intrinsic apoptosis is in fact divergent among metazoans. In addition, extrinsic apoptosis is poorly studied in nonmammalian animals, making its evolution unclear. Consequently, our understanding of apoptotic signaling pathways evolu tion is a black box which must be illuminated by extending research to new biological systems. Lophotrochozoans are a major clade of metazoans which, despite their considerable biological diversity and key phylogenetic position as sister group of ec dysozoans (i.e. flies and nematodes), are poorly explored, especially regarding apoptosis mechanisms. Traditionally, each apoptotic signaling pathway was considered to rely on a specific initiator caspase, associated with an activator. To shed light on apoptosis evolution in animals, we explored the evolutionary history of initiator caspases, caspase activators, and the BCL-2 family (which control mitochondrial apoptotic pathway) in lophotrochozoans using phylogenetic analysis and protein inter action predictions. We discovered a diversification of initiator caspases in molluscs, annelids, and brachiopods, and the loss of key extrinsic apoptosis components in platyhelminths, along with the emergence of a clade-specific caspase with an ankyrin pro-domain. Taken together, our data show a specific history of apoptotic actors’ evolution in lophotrochozoans, further dem onstrating the appearance of distinct apoptotic signaling pathways during metazoan evolution. Key words: apoptosis, caspases, BCL-2 family, lophotrochozoans, phylogeny, evolution. Significance Apoptosis, a form of programmed cell death, has been long studied in model organisms such as flies and mice and in humans. The restricted focus on these models has led to an overall view that the evolution of genes involved in apoptosis is highly conserved across all animals. The advent of next-generation sequencing has led to a boom in the omics data available across the tree of life. Thanks to this, we explored the evolution of key genes involved in apoptosis in the clade Lophotrochozoa (i.e. molluscs, annelids, flatworms, and brachiopods), one of the three large clades that make up bila terian animals. We found a complex evolutionary history of apoptosis genes, with multiple losses, gains, divergences, and redundancies, highlighting the value of exploring gene evolution and apoptotic mechanisms in lophotrochozoans. © The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact . Genome Biol. Evol. 16(10) https://doi.org/10.1093/gbe/evae204 Advance Access publication 25 September 2024 1 1 GBE Horkan et al. Introduction 2 Genome Biol. Evol. 16(10) https://doi.org/10.1093/gbe/evae204 Advance Access publication 25 September 2024 Apoptosis is a form of regulated cell death that sculpts the animal’s body during embryonic development and allows the removal of obsolete tissues or supernumerary cells (Jacobson et al. 1997). Present at the metazoan scale (Vega Thurber and Epel 2007; Ballarin et al. 2010; Kiss 2010; AnvariFar et al. 2017; Krasovec et al. 2019, 2021; Jeffery and Gorič ki 2021; Krasovec et al. 2022), apoptosis is defined by a conserved set of morphological features that depend on a multigenic family, the caspases (Cohen 1997; Hengartner 2000). While caspases are found in all animals, they have mainly been studied in nematodes, flies, and vertebrates, in which apoptotic signaling pathways are well described (Hengartner 2000; Fan et al. 2005; Steller 2008). Historically, two main apoptotic signaling pathways were described, currently known as extrinsic (formerly the death receptor pathway) and intrinsic (formerly the mito chondrial pathway) apoptosis (Galluzzi et al. 2018). In both cases, caspases play a central role upstream (as initi ator caspases) and downstream (as executioner caspases) of the apoptotic signaling pathways (Hengartner 2000; Fan et al. 2005; Kumar 2007). Importantly, each apoptotic pathway is characterized by a specific initiator caspase, it self activated by a protein complex in which a specific acti vator is involved (Fig. 1). While all caspases are composed of the common P10 and P20 domains, initiator caspases also contain an additional long pro-domain, either a CARD or DED domain, specific for intrinsic and extrinsic apoptoses, respectively. In mammals, intrinsic apoptosis is activated through mitochondrial outer membrane permeabilization (MOMP), a process controlled by BCL-2 family proteins such as BAX or BAK (Fig. 1) (Hengartner 2000). MOMP leads to the re lease of cytotoxic molecules such as cytochrome c into the cytosol. Cytochrome c forms the apoptosome complex with the specific adaptor protein APAF-1 (apoptotic prote ase activating factor 1) and initiator caspase-9, leading to the activation of executioner caspase-3 (Hengartner 2000; Galluzzi et al. 2018; Kalkavan and Green 2018). Key components of the intrinsic pathway are present in flies and nematodes as well (Driscoll 1996; Hengartner 2000; Lettre and Hengartner 2006; Steller 2008; Bender et al. 2012). However, there are several fundamental mechanis tic differences between these models and mammals. For in stance, cytochrome c is crucial to trigger apoptosis in mammals, whereas it is dispensable in flies and nematodes. Indeed, since BCL-2 members BAX and BAK have not been identified in Drosophila melanogaster and Caenorhabditis elegans genomes, MOMP does not occur. In D. melanoga ster, the APAF-1 homologue, DARK, oligomerizes into an eigh (...truncated)