Phylogenetic Analysis of the Caspases

Alignment of different proteins of the same family can provide information on their phylogenetic relationship and in some cases indicate which family members execute similar functions. The caspase phylogenetic tree we present in Figure 9 is based on the amino acid sequence containing the p20 and p10 units and, when present, the short interdomain between them. This is referred to as p30 caspase. We have omitted the prodomains because these contain functional domains unrelated to the catalytic properties of caspases and these motifs occur in other proteins as well. A separate phylogenetic analysis of the caspase prodomains is presented in Figure 10. Only the long prodomain caspases have been included in this analysis since short prodomains do not give reliable results.

In Figure 9 a phylogenetic analysis of the enzymatic relevant domains of the known Hydra vulgaris, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Gallusgallus, Mus musculus and Homo sapiens caspases shows divergence in three main clusters. It is immediately apparent that all three clusters contain functionally related members. Cluster I contains vertebrate caspases that are not part of the general apoptotic machinery though some of them might be involved in apoptosis or other forms of programmed cell death in specific tissues15,16,192 or under pathological conditions.82-84 All C. elegans caspases gather in cluster I. All vertebrate and insect apoptosis-related executioner caspases belong to cluster II. These executioner caspases have a short prodomain, with the exception of Drosophila STRICA. The main apoptotic initiator caspases, with a large prodomain, gather in cluster III.

The first cluster contains two branches: Ia comprising solely the three C. elegans caspases and IP consisting of caspase-1, -2, -4, -5, -11, -12 and -14, found only in vertebrates so far. The C. elegans CED-3 was the first gene product identified to be essential for programmed cell death.5,6 CSP-1 and CSP-2 have a long prodomain without obvious functional motifs. In the nematode the apparent lack of short prodomain caspases is bypassed by alternative splicing of CSP-1 and -2. Protease activity has only been reported for CED-3 and CSP-1 and little is known about their possible involvement in proteolytic cascades.193 The genome sequences of the fly and the nematode did not reveal caspase orthologues belonging to branch Ip. Thus this group of caspases, often referred to as inflammatory caspases, may have evolved together with a complex haematopoietic system implicated in inflammatory and immune responses. The prototype of this group is caspase-1, which is mainly implicated in the processing of inflammatory cytokines such as proIL-1P and proIL-18.20,1 7 It is not yet clear if this function exists in Xenopus, as its proIL-1P orthologue seems to lack a clear caspase-sensitive cleavage site at the

Figure 9. Phylogenetic relationship of the caspases based on their p20 and p10 domains. The inflammatory caspases (cluster I), the apoptotic executioner caspases (cluster II) and the apoptotic initiator caspases (cluster III) evolved as separate groups. The sequences were aligned using the CLUSTAL X software (gap weight = 10.00; gap length weight = 0.20) and trees visualized in TreeView.314'315 Hydra vulgaris (hy), Danio rerio (z), Xenopus laevis (x), Gallusgallus (g), Mus musculus (m), Homo sapiens (h).

Figure 9. Phylogenetic relationship of the caspases based on their p20 and p10 domains. The inflammatory caspases (cluster I), the apoptotic executioner caspases (cluster II) and the apoptotic initiator caspases (cluster III) evolved as separate groups. The sequences were aligned using the CLUSTAL X software (gap weight = 10.00; gap length weight = 0.20) and trees visualized in TreeView.314'315 Hydra vulgaris (hy), Danio rerio (z), Xenopus laevis (x), Gallusgallus (g), Mus musculus (m), Homo sapiens (h).

appropriate position.194 The involvement of other group members in inflammation is supported by the inability of LPS to induce endotoxemia in both caspase-1- and caspase-11-defi-cient mice.20,21,127 caspase-11 is most likely an upstream activator of caspase-1. Caspase-1 can also be activated in response to binding of bacterial compounds to Toll-like receptors, suggesting a link between the Toll receptor system and the activation of inflammatory caspases.195 Similar to CED-3 recruitment by CED-4, the CED-4/Apaf-1-like factor Ipaf recruits and activates procaspase-1.196 caspase-12 has been reported to be an ER stress-sensing protease.197 Currently no human orthologue of caspase-12 has been reported. It can be argued that human caspase-4 and -5 are duplicated counterparts of murine caspase-11, especially when considering the full-length proteins including the CARD domain (see also Fig. 9). When comparing

Figure 10. Phylogenetic relationship of the prodomains of caspases.This analysis reveals two separate clusters: CARD and PYRIN containing prodomains (groups A1 and A2) and the DED-containing prodomains (group B). The group A1 CARD prodomains are part of the inflammatory caspases, while group A2 CARD prodomains belong to apoptotic caspases. The sequences were aligned using the CLUSTAL X software (gap weight = 20.00; gap length weight = 0.10) and trees visualized in TreeView.314,315 Hydra vulgaris(hy), Danio rerio (z), Xenopus laevis (x), Gallusgallus (g), Mus musculus (m), Homo sapiens (h).

Figure 10. Phylogenetic relationship of the prodomains of caspases.This analysis reveals two separate clusters: CARD and PYRIN containing prodomains (groups A1 and A2) and the DED-containing prodomains (group B). The group A1 CARD prodomains are part of the inflammatory caspases, while group A2 CARD prodomains belong to apoptotic caspases. The sequences were aligned using the CLUSTAL X software (gap weight = 20.00; gap length weight = 0.10) and trees visualized in TreeView.314,315 Hydra vulgaris(hy), Danio rerio (z), Xenopus laevis (x), Gallusgallus (g), Mus musculus (m), Homo sapiens (h).

human procaspases, procaspase-4 and -5 have an amino acid sequence identity of 77%, the next highest identity score being 55% (between procaspase-1 and -4). Procaspase-4 and -5 amino acid sequences are 59% and 54% identical to procaspase-11, respectively. They are only 48% and 45% identical to procaspase-12, respectively. Furthermore, caspase-4 and -11 mRNA have similar tissue distribution patterns9 and both caspase-5 and -11 are LPS- or IFN-y-induc-ible.198 Most probably caspase-13 is the bovine orthologue of caspase-4,199 although it was first described as a human caspase. Caspase-2 has deviated from the main branch leading to the caspase-1-likes and is implicated in neuronal cell death, with evidence for an apoptotic initiator function for this caspase.200 Like caspase-1, caspase-2 can also mediate apoptosis of macrophages infected with Salmonella.201 Zebrafish caspy and caspy2 are the orthologues of mammalian caspase-1 and -2, respectively, though their prodomains contain a PYRIN motif instead of a CARD.70 The only short prodomain caspase of this cluster is caspase-14. This caspase is mainly expressed in differentiating keratinocytes of the skin and processed in later stages of epidermal differentiation.15,16 It might therefore contribute to the terminal differentiation of skin cells especially since the classical apoptotic cell death cascade is not activated in this form of programmed cell death.16

The caspases gathered in cluster II have in common that they all are activated during the execution fase of apoptosis. In a separate branch (IIa) are the vertebrate caspases-3 and -7. The latter has only been found in frog, mouse and man. In mammalians these two proteases share large substrate specificity but they are not completely redundant and it is still unclear whether both caspases are always activated under the same circumstances. This functional difference is most apparent from the difference in phenotype in the knockout animals (see Chapter 10). Hydra caspase-3B shares more homology with caspase-3 and -7 than with any other mammalian caspase.10 caspase-6, found in all tetrapods represented here, belongs to the (branch of cluster II together with Hydra caspase-3A and the Drosophila caspases DCP-1, DRICE, DECAY, DAMM and STRICA that are considered to be downstream executioner caspases in the fly based on structural and enzymatic properties. DAMM and STRICA are closely related to each other if only their p30 domain is considered. There is nevertheless a striking difference in the prodomain. DAMM has a short prodomain like all members of the cluster, whereas STRICA has a large prodomain (Fig. 1 and 10). Remarkably, Hydra possesses caspases that belong to both branches of cluster II, while C. eleganscaspases gather in cluster Ia.

Cluster III, which contains all upstream initiator caspases separates into two main branches. Branch IIIa contains proteins closely related to caspase-8, in mammalians characterized by their activation in the death receptor complex, and branch IIIp contains caspase-9 that is involved in the initiation of the intrinsic mitochondrial death pathway. Even though prodomains were not included in the alignment, caspases in this cluster are divided over two branches according to the structural domains in their prodomain (Fig. 1). All caspases in branch IIIa have two DED domains while those in branch IIIa have a single CARD. As mentioned, some caspases in cluster I also contain a CARD. Branch III( includes the upstream initiator caspase-8 in M. musculus, caspase-8 and caspase-10 in X. laevis and H. sapiens, and DREDD in D. melanogaster. DREDD seems to play a role in the innate immunity signalling pathway of the Toll receptor by proteolytically processing Relish, an NF-KB-like transcription factor.202 In mammalians, a link between the Toll-like receptor 2 and caspase-8 through the MyD88-de-pendent recruitment of FADD has been proposed.195 Since both caspase-8 and -10 have been identified in Xenopus and man, the apparent absence of caspase-10 in mice is remarkable. Branch III p contains caspase-9 orthologues including DRONC that have conserved their function and activation mechanism during the separation of vertebrates and insects. In mammalians procaspase-9 is bound and activated by Apaf-1 while the Drosophila orthologue that activates DRONC is called DARK/Dapaf-1/HAC-1.203-205 In both cases binding and activation is dependent on the presence of dATP and cytochrome c. This mechanism is different from the apoptosome complex in C. elegans, since Ced-4 does not contain the WD-40 repeats required for cytochrome c binding.206

As mentioned above human caspases and Ced-3 have also been classified in three groups based on screening of a combinatorial tetrapeptide substrate library.38 In general this classification fits the phylogenetic relationships presented here, with the exception of caspase-2, -6, -11 and Ced-3. In other words, evolutionary related caspases usually have related substrate specificities suggesting evolutionary constraints both on the enzymes and their substrates.

The second phylogenetic tree (Fig. 10) we present here illustrates the relationship between the large prodomains found in different caspases. This tree also segregates into three groups, named A1, A2 and B. The first two groups, consisting of CARD-like prodomains, are more related to each other than to the third group, containing the DED-motif prodomains. There is a remarkable resemblance between the phylogenetic analysis of the prodomains and that of the p30 caspases (Fig. 9 and 10). As mentioned before short prodomain caspases, except caspase-14, cluster in one group based on their p30 homology. Both phylogenetic analyses indicate a strong coevolution between the prodomains and the enzymatic part of caspases. Phylogenetic analysis of the prodomains of intermediate length of the two reported Hydra caspases10 did not allow reliable classification in the three clusters.

Group A1 includes the CARD-containing prodomains of the inflammation-related caspases. Furthermore, this group harbours the PYRIN-containing prodomains of zebrafish caspy and caspy2 and the prodomain of STRICA that is related to the prodomain of C. elegans CSP-1. Group A2 holds the CARD-containing prodomains of the apoptosis-related caspases together with the prodomain of CSP-2. The phylogenetic positioning of the zebrafish PYRIN motifs within group A1 suggests a close relationship between PYRIN and CARD motifs both belonging to the DD superfamily. The same may hold for the prodomains of CSP-1, -2 and STRICA. The resemblance of the CARD domains of procaspase-2 an -9 seems to have functional consequences since both specifically interact with the Apaf-1/Ced-4-like proapoptotic caspase adaptor PACAP, a protein that promotes the proteolytic activation of these caspases.207 In addition, the CARD domain of caspase-2 binds the CARD and DD-domain-containing adaptor RAIDD and could be recruited in the TNF-R1 complex.208 However, a precise function for this interaction has not yet been found. It is remarkable that the three Caenorhabditis caspases, which are closely related according to their enzymatic entity (Fig. 9), have highly diverged prodomains. Group B gathers all DED-containing prodomains from caspase-8, -10 and DREDD. DED motifs are used for protein recruitment in the extrinsic cell death pathway initiated with the formation of a DISC complex.67 Interestingly, no DED-containing caspases are found in C. elegans.

The sequence relationship between caspases allows us to speculate about the functional evolution caspases underwent during the separation of the different animal groups. In nema-todes all described functions of the caspases are related to the apoptotic mechanisms in the worm. The two known Hydra caspases are probably also involved in apoptotic programs.10 In more complex organisms caspases acquired more diverse functions. In the fly, like in vertebrates, there is a clear distinction between initiator (cluster III) and executioner caspases (cluster II). However, most caspases still seem to be involved in apoptosis, although DREDD acquired a role in innate immunity.202 In vertebrates many caspases have a function outside the classical apoptotic machinery, such as the maturation of cytokines. These all belong to branch IP, indicating a common origin. Some of these caspases can play a role in specialized forms of programmed cell death, however only in certain tissues16,192,209 or pathological conditions.82-84

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