Caspases from scleractinian coral show unique regulatory features.

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity.

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

The CaspBase: a curated database for evolutionary biochemical studies of caspase functional divergence and ancestral sequence inference.

Sequence databases are powerful tools for the contemporary scientists' toolkit. However, most functional annotations in public databases are determined computationally and are not verified by a human expert. While hypotheses generated from computational studies are now amenable to experimentation, the quality of the results relies on the quality of input data. We developed the CaspBase to expedite high-quality dataset compilation of annotated caspase sequences, to maximize phylogenetic signal, and to reduce the noise contributed from public databanks. We describe our methods of curation for the CaspBase and how researchers can acquire sequences from CaspBase.org. Our immediate goal for developing the CaspBase was to optimize the ancestral protein reconstruction (APR) of caspases, and we demonstrate the utility of the CaspBase in APR studies. We also developed the Common Position (CP) system for comparing human caspase family paralogs and suggest the CP system as an update to current reporting methods of caspase amino acid positions. We present a standardized multiple sequence alignment (MSA) for the CP system and show the advantage of using large databases such as the CaspBase in defining structural positions in proteins. Although the results described here pertain to caspase evolution and structure-function studies, the methods can be adapted to any gene family.

Life or death: disease-tolerant coral species activate autophagy following immune challenge.

Global climate change has increased the number and severity of stressors affecting species, yet not all species respond equally to these stressors. Organisms may employ cellular mechanisms such as apoptosis and autophagy in responding to stressful events. These two pathways are often mutually exclusive, dictating whether a cell adapts or dies. In order to examine differences in cellular response to stress, we compared the immune response of four coral species with a range of disease susceptibility. Using RNA-seq and novel pathway analysis, we were able to identify differences in response to immune stimulation between these species. Disease-susceptible species Orbicella faveolata activated pathways associated with apoptosis. By contrast, disease-tolerant species Porites porites and Porites astreoides activated autophagic pathways. Moderately susceptible species Pseudodiploria strigosa activated a mixture of these pathways. These findings were corroborated by apoptotic caspase protein assays, which indicated increased caspase activity following immune stimulation in susceptible species. Our results indicate that in response to immune stress, disease-tolerant species activate cellular adaptive mechanisms such as autophagy, while susceptible species turn on cell death pathways. Differences in these cellular maintenance pathways may therefore influence the organismal stress response. Further study of these pathways will increase understanding of differential stress response and species survival in the face of changing environments.