Surprising complexity of the ancestral apoptosis network

Article (PDF Available)inGenome biology 8(10):R226 · February 2007with22 Reads
DOI: 10.1186/gb-2007-8-10-r226 · Source: PubMed
Abstract
Apoptosis, one of the main types of programmed cell death, is regulated and performed by a complex protein network. Studies in model organisms, mostly in the nematode Caenorhabditis elegans, identified a relatively simple apoptotic network consisting of only a few proteins. However, analysis of several recently sequenced invertebrate genomes, ranging from the cnidarian sea anemone Nematostella vectensis, representing one of the morphologically simplest metazoans, to the deuterostomes sea urchin and amphioxus, contradicts the current paradigm of a simple ancestral network that expanded in vertebrates. Here we show that the apoptosome-forming CED-4/Apaf-1 protein, present in single copy in vertebrate, nematode, and insect genomes, had multiple paralogs in the cnidarian-bilaterian ancestor. Different members of this ancestral Apaf-1 family led to the extant proteins in nematodes/insects and in deuterostomes, explaining significant functional differences between proteins that until now were believed to be orthologous. Similarly, the evolution of the Bcl-2 and caspase protein families appears surprisingly complex and apparently included significant gene loss in nematodes and insects and expansions in deuterostomes. The emerging picture of the evolution of the apoptosis network is one of a succession of lineage-specific expansions and losses, which combined with the limited number of 'apoptotic' protein families, resulted in apparent similarities between networks in different organisms that mask an underlying complex evolutionary history. Similar results are beginning to surface for other regulatory networks, contradicting the intuitive notion that regulatory networks evolved in a linear way, from simple to complex.
Genome Biology 2007, 8:R226
Open Access
2007Zmaseket al.Volume 8, Issue 10, Article R226
Research
Surprising complexity of the ancestral apoptosis network
Christian M Zmasek
¤
*
, Qing Zhang
¤
*
, Yuzhen Ye
and Adam Godzik
*‡
Addresses:
*
Burnham Institute for Medical Research, North Torrey Pines Road, La Jolla, CA 92037, USA.
School of Informatics, Indiana
University, E.10th Street, Bloomington, IN 47408, USA.
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San
Diego, Gilman Drive, La Jolla, CA 92093, USA.
¤ These authors contributed equally to this work.
Correspondence: Adam Godzik. Email: adam@burham.org
© 2007 Zmasek et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Evolution of the apoptotic network<p>A comparative genomics approach revealed that the genes for several components of the apoptosis network with single copies in ver-tebrates have multiple paralogs in cnidarian-bilaterian ancestors, suggesting a complex evolutionary history for this network.</p>
Abstract
Background: Apoptosis, one of the main types of programmed cell death, is regulated and
performed by a complex protein network. Studies in model organisms, mostly in the nematode
Caenorhabditis elegans, identified a relatively simple apoptotic network consisting of only a few
proteins. However, analysis of several recently sequenced invertebrate genomes, ranging from the
cnidarian sea anemone Nematostella vectensis, representing one of the morphologically simplest
metazoans, to the deuterostomes sea urchin and amphioxus, contradicts the current paradigm of
a simple ancestral network that expanded in vertebrates.
Results: Here we show that the apoptosome-forming CED-4/Apaf-1 protein, present in single
copy in vertebrate, nematode, and insect genomes, had multiple paralogs in the cnidarian-bilaterian
ancestor. Different members of this ancestral Apaf-1 family led to the extant proteins in
nematodes/insects and in deuterostomes, explaining significant functional differences between
proteins that until now were believed to be orthologous. Similarly, the evolution of the Bcl-2 and
caspase protein families appears surprisingly complex and apparently included significant gene loss
in nematodes and insects and expansions in deuterostomes.
Conclusion: The emerging picture of the evolution of the apoptosis network is one of a
succession of lineage-specific expansions and losses, which combined with the limited number of
'apoptotic' protein families, resulted in apparent similarities between networks in different
organisms that mask an underlying complex evolutionary history. Similar results are beginning to
surface for other regulatory networks, contradicting the intuitive notion that regulatory networks
evolved in a linear way, from simple to complex.
Background
Apoptosis is the best-known type of programmed cell death
and plays important roles in development and homeostasis as
well as in the pathogenesis of many diseases [1,2]. Classical
studies on apoptosis in the nematode Caenorhabditis elegans
identified at first three (CED-3, CED-4, CED-9) and later a
fourth protein (EGL-1) to be directly involved in apoptosis
[3]. Homologs of the first three proteins were found in
Published: 24 October 2007
Genome Biology 2007, 8:R226 (doi:10.1186/gb-2007-8-10-r226)
Received: 20 July 2007
Revised: 24 October 2007
Accepted: 24 October 2007
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2007/8/10/R226
Genome Biology 2007, 8:R226
http://genomebiology.com/2007/8/10/R226 Genome Biology 2007, Volume 8, Issue 10, Article R226 Zmasek et al. R226.2
genomes of all animals and for all systems studied were
shown to be involved in apoptosis (although, the evidence
that CED-9 homologs regulate apoptosis in Drosophila mela-
nogaster is only indirect) [4,5]. Therefore, they logically were
assumed to form the core of the apoptosis network (for an
overview, see Figure 1) [1].
Compared to C. elegans, the vertebrate apoptosis network is
extensive, both in the number and in the size of the protein
families involved. While C. elegans has one homolog of each
(CED-3, CED-4, and CED-9), human has 12 CED-3 (caspase)
homologs and 13 CED-9 homologs (Bcl-2-like proteins con-
taining multiple BH motifs) as well as a number of highly
divergent proteins that play an analogous role to the EGL-1
protein (BH3 motif only) (three additional caspase related
genes, for which confirmation for a role in apoptosis is absent,
have been found in C. elegans) [6-8]. All mammals, as well as
birds, amphibians, and, to a lesser degree, fish, show some-
what similar expansions of these families [9]. The CED-4/
Apaf-1 family is an exception, being the only protein from the
core of the apoptosis network that was not duplicated in any
of the genomes studied until recently. Therefore, it was logical
to expect that the role of this protein is indeed central and
unique and that all homologs studied to date represent one-
to-one orthologs that have evolved by speciation events only.
Such one-to-one orthologs usually tend to display a high level
of functional similarity and could be effectively used as func-
tional models of each other [10]. In this context, it was some-
what puzzling that an increasing body of experimental
evidence suggested fundamental functional differences
between C. elegans CED-4 and Drosophila Dark and their
homologs in other species. In vertebrates, cytochrome c binds
to Apaf-1 to trigger assembly of the apoptosome [6], which in
turn leads to caspase activation. In contrast, no cytochrome c
binding has been recognized for C. elegans CED-4 and
remains controversial for Drosophila Dark [5,11].
With the recent completion of three marine invertebrate
genomes, namely two from Deuterostomia (the sea urchin
Strongylocentrotus purpuratus and the amphioxus Branchi-
ostoma floridae; unpublished; see Materials and methods)
and one from Cnidaria (the sea anemone Nematostella vect-
ensis), we are now able to obtain a more complete picture of
how the complex vertebrate apoptosis network might have
evolved and how representative the simple networks seen in
insects and nematodes are of the systems present in other
invertebrate animals [12-15].
Overview of the initiation of the intrinsic apoptosis pathwayFigure 1
Overview of the initiation of the intrinsic apoptosis pathway. Annotations and domain compositions for N. vectensis (sea anemone), S. purpuratus (sea
urchin), and B. floridae (amphioxus) are based on analyses performed in this work, whereas data for C. elegans, D. melanogaster, and Homo sapiens are based
on literature [1,2,11]. (Protein and domain lengths are not to scale. In our analysis we noticed a few additional, spurious domains in some CED4/Apaf-1
family members; these are not shown in this diagram.) On the left side, a current view of metazoan phylogeny is shown [13].
CARD (caspase recruitment)
DED (death effector domain)
Death
WD40 repeat
EGL-1
CED-9
CED-4 CED-3
Bad
Bim
Bid
Bik
Puma
Bcl-2
Bcl-x
L
Bcl-w
Mcl-1
A1
Bax
Bak
Bok
Apaf-1
cyt c
release
Debcl/Drob-1/dBorg-1/dBok
Buffy/dBorg-2
DroncDark
Caspase-9
Apical (initiator) caspases
Adaptors
C. elegans
D. melanogaster
H. sapiens
S. purpuratus
N. vectensis
B. floridae
NB-ARC
Caspase (P10-, P20-domain)
Bax
Bak
Bok
Bak
Bok
Bak
Bok
Ecdysozoa
Bilateria
Deuterostomia
Chordata
Bcl-2 family
Domains
?
?
Cnidaria
TPR (tetratricopeptide) repeat
TIR (toll/interleukin-1 receptor)
?
?
?
?
?
?
?
?
cyt c
release ?
?
BH3
BH1, BH2
BH4
Bcl-2 Motifs
http://genomebiology.com/2007/8/10/R226 Genome Biology 2007, Volume 8, Issue 10, Article R226 Zmasek et al. R226.3
Genome Biology 2007, 8:R226
Results
The assumption that the major expansion of the apoptotic
networks is specific to vertebrates was challenged by the
results of several studies of individual protein families [16],
such as the presence of multiple Bax- and Bak-like sequences
in the cnidarian Hydra magnipapillata [17], but the assump-
tion was finally laid to rest by the analysis of the recently
sequenced sea urchin genome, which showed that many
groups of proteins related to apoptosis underwent major
expansion in this organism compared not only to C. elegans,
but also to vertebrates (Table 1) [12,18]. Some groups of apop-
tosis-related proteins have ten times more members in sea
urchin than in corresponding families in vertebrates! The
recently sequenced amphioxus genome shows similar expan-
sion. However, the origin of the major expansion of the apop-
tosis network was moved back in time even further by the
analysis of the genome of the morphologically simplest meta-
zoan sequenced to date, the cnidarian N. vectensis. Cnidari-
ans are the sister-group of the bilaterian metazoans, with
both groups splitting about 650-1,000 million years ago [14].
Yet, both the size of most families of apoptosis domains and
proteins as well as the presence of many vertebrate-like sub-
families strongly suggest that the cnidarian-bilaterian ances-
tor had an apoptosis network comparable in its complexity to
that of vertebrates and that the apparent simplicity seen in
insects and nematodes is a result of massive gene loss.
Detailed phylogenetic analysis of the central, nucleotide-
binding domain of the CED-4/Apaf-1 family shows a some-
what unexpected picture (Figure 2). This domain, classified
as NB-ARC (for nucleotide-binding adaptor shared by Apaf-1,
R proteins, and CED-4) is a subfamily member of the very
large family of AAA+ ATPases [19-21]. NB-ARC is distantly
homologous to, but distinctively different from, other nucleo-
tide-binding domains, such as the NACHT domain present in
families of proteins involved in immunity [22]. A well-sup-
ported subtree, containing human Apaf-1 and its vertebrate
one-to-one orthologs, also contains amphioxus, sea urchin,
and Nematostella sequences, but none from nematodes or
insects (subtree A in Figure 2). Evidently, nematode/insect
homologs from this subfamily have been lost, thus leaving
nematodes/insects without orthologs of human Apaf-1. Nem-
atode and insect proteins form their own subtree (B), diverg-
ing from the Apaf-1 branch in a way suggesting that these
proteins belong to a separate subtype that was already
present at the cnidarian-bilaterian split. Interestingly, several
Nematostella and amphioxus homologs form additional sub-
families (C), which were lost in both nematodes/insects and
vertebrates, indicating an evolutionary history for Apaf-1
predecessors rich in gene duplications and gene losses.
The presence of numerous CED-4/Apaf-1 homologs in the
common ancestor of Bilateria and Cnidaria suggests that ini-
tially there might have been several mechanisms to activate
the intrinsic apoptosis pathways and/or several downstream
Table 1
Core apoptosis domains in several completed animal genomes
Classification Species NB-ARC
domain
Bcl-2
(multi-motif)
Caspase CARD
domain
Death
domain (DD)
Death effector
domain (DED)
Vertebrata H. sapiens (human) 1 (1) 17 (12) 11 (11) 23 (22) 31 (29) 8 (8)
M. musculus (mouse) 1 (1) 15 (11) 9 (9) 23 (21) 28 (25) 6 (6)
C. familiaris (dog) 1 (1) 14 (10) 14 (14) 20 (19) 37 (33) 5 (5)
G. gallus (chicken) 1 (1) 13 (7) 13 (13) 13 (12) 30 (24) 6 (6)
X. tropicalis (western clawed frog) 1 (1) 14 (11) 13 (13) 28 (28) 31 (28) 5 (5)
B. rerio (zebrafish) 1 (1) 16 (13) 21 (21) 30 (28) 35 (33) 5 (5)
F. rubripes (Japanese pufferfish) 1 (1) 15 (12) 13 (13) 15 (14) 32 (28) 6 (6)
T. nigroviridis (green pufferfish) 1 (1) 13 (11) 14 (14) 14 (12) 33 (30) 5 (4)
Cephalochordata B. floridae (amphioxus) 16 (16) 7 (7) 53 (53) 84 (84) 139 (136) 57 (57)
Urochordata C. intestinalis (sea squirt) 0* 1 (1) 11 (11) 2 (2) 5 (4) 2 (2)
Echinodermata S. purpuratus (purple sea urchin) 5 (5) 8 (8) 42 (42) 12 (10) 87 (82) 3 (3)
Ecdysozoa D. melanogaster (fruit fly) 1 (1) 2 (2) 7 (7) 1 (0) 5 (5) 0
C. elegans 1 (1) 1 (1) 5 (5) 1 (1) 2 (2) 0
Cnidaria N. vectensis (starlet sea anemone) 4 (4) 11 (11) 10 (10) 8 (8) 5 (5) 9 (9)
The total numbers of full-length protein sequence matches to the corresponding human sequences are shown; the number of hits confirmed by Pfam
and CD-Search under default thresholds displayed in parentheses (see Materials and methods). We have to stress that the number of proteins in all
recently sequenced genomes is approximate because of the diversity of domain sequences and experimental verification of only limited numbers of
gene predictions. Therefore, exact counts of the members of these families strongly depend on significance thresholds for gene predictions and
specific homology-recognition tools used in the analysis. *We were unable to detect an NB-ARC domain in C. intestinalis, probably due to sequence/
assembly problems in this genome.
Genome Biology 2007, 8:R226
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pathways activated by similar signals and that the mechanism
of human Apaf-1 and its vertebrate orthologs presents only
one of several possibilities. This also explains why the bio-
chemical/structural mechanism of C. elegans CED-4 and
Drosophila Dark can be significantly different from human
Apaf-1 [11].
The functional variations among different branches of the
Apaf-1 family are illustrated by their different domain organ-
izations. Human Apaf-1 and its Nematostella, amphioxus,
and sea urchin homologs exhibit the same or similar domain
organization (CARD [two for Nematostella]-NB-ARC-WD40
repeats). Nematode and most, but not all, insect sequences
seem to lack WD40 repeats [23], suggesting that the loss of
the receptor domain of CED-4 is a (relatively) recent event,
specific to nematode/insect Apaf-1 homologs. The expanded
repertoire of CED-4/Apaf-1 homologs in sea urchin, amphi-
oxus, and Nematostella contains proteins with novel domain
combinations. This includes replacement of the single CARD
domain at the amino terminus with pairs of CARD domains
(Nematostella and amphioxus), death domains (amphioxus
and, as previously described in [18], sea urchin), death effec-
tor domains (Nematostella), and TIR domains (amphioxus),
all of which function as protein-protein interaction facilita-
tors [24]. At the carboxyl terminus, the WD40 repeats are
occasionally missing, replaced by TPR repeats [25], or sup-
plemented by double death domain repeats. Therefore, it
seems that functional differences among CED-4/Apaf-1
homologs could include both the sensing mechanism (car-
boxy-terminal receptor domains) and the downstream
recruitment function (amino-terminal protein-protein inter-
action domains). While we can only speculate on how such a
rich set of domain combinations (as seen in amphioxus) came
to be, a correlation between domain versatility and abun-
dance has been observed [26]. Interestingly, the TIR-NB-
ARC domain architecture, present in one of the amphioxus
proteins, resembles plant disease-resistant (R) genes
involved in a process called hypersensitive response [27],
Phylogeny and domain organization of CED-4/Apaf-1 homologsFigure 2
Phylogeny and domain organization of CED-4/Apaf-1 homologs. This phylogeny was calculated using a Bayesian approach (MrBayes) based on a MAFFT
alignment of the NB-ARC domains. Posterior probability values are shown for each branch (top numbers). Bootstrap support values for branches that are
supported by a minimal evolution method (FastME) based on a PROBCONS alignment are also shown (bottom numbers; for detailed information, see
Materials and methods). Furthermore, phylogenies based on full-length alignments of the subset of all Apaf-1 homologs exhibiting a CARD-NB-ARC-
WD40 domain composition (all vertebrate sequences, 1_BRAFL, 18_NEMVE, and Dark_DROME) as well as 28_DROPS, CED4_CAAEL, and 31_CAEBR
showed precisely the same picture: a clade of vertebrate, amphioxus, and Nematostella sequences under exclusion of insect and nematode sequences. For
a detailed list of protein sequences see Additional data file 2. For clarity, sequences from S. purpuratus (2), and B. floridae (6), which appear to be redundant
and/or results of erroneous assemblies, are not included in this figure; however, their inclusion/exclusion does not change the quality/interpretation of this
phylogeny. All sequences are from complete genomes, except the individual sequences from Aedes aegypti, Caenorhabditis briggsae, Drosophila pseudoobscura,
and Tribolium castaneum.
1.0
1.0
1.0
1.0
1.0
1.0
Apaf-1_HUMAN
Apaf-1_MOUSE
12_CANFA
0.65
11_CHICK
16_XENTR
1.0
1.0
14_FUGRU
15_TETNG
17_BRARE
0.96
1_BRAFL
18_NEMVE
23_STRPU
1.0
25_STRPU
26_STRPU
0.76
0.77
1.0
CED4_CAEEL
31_CAEBR
30_TRICA
0.93
1.0
Dark_DROME
28_DROPS
29_AEDAE
1.0
0.84
1.0
0.69
0.94
1.0
34_BRAFL
35_BRAFL
8_BRAFL
1.0
20_NEMVE
21_NEMVE
9_BRAFL
0.99
1.0
36_BRAFL
37_BRAFL
33_BRAFL
1.0
1.0
2_BRAFL
3_BRAFL
19_NEMVE
CARD (caspase recruitment) domain
NB-ARC domain
WD40 repeats
Death domain
DED (death effector) domain
TPR_1 (tetratricopeptide) repeat
TPR_2 (tetratricopeptide) repeat
RVT_1 (reverse transcriptase)
MIF (macrophage migration inhibitory factor)
Collagen triple helix repeat
NB-ARC with similarity to NACHT domain
Similarity to Pfam models (for E-values < 10
-3
):
TIR (toll/interleukin-1 receptor) domain
NB-ARC domain
WD40 repeats
CARD (caspase recruitment) domain
TPR (tetratricopeptide) repeat
Weak similarities (detected by FFAS, InterProScan):
88
100
100
100
100
100
68
89
100
100
89
89
100
100
95
100
61
84
46
100
100
92
95
98
1.0
0.78
CANFA
CHICK
XENTR
FUGRU
TETNG
BRARE
BRAFL
NEMVE
STRPU
CAEEL
CAEBR
TRICA
DROME
DROPS
AEDAE
Canis familiaris (dog)
Gallus gallus (chicken)
Xenopus tropicalis (western clawed frog)
Fugu rubripes (Japanese pufferfish)
Tetraodon nigroviridis (green pufferfish)
Brachydanio rerio (zebrafish)
Branchiostoma floridae (amphioxus)
Nematostella vectensis (sea anemone)
Strongylocentrotus purpuratus (sea urchin)
Caenorhabditis elegans
Caenorhabditis briggsae
Tribolium castaneum (red flour beetle)
Drosophila melanogaster (fruit fly)
Drosophila pseudoobscura
Aedes aegypti (yellow fever mosquito)
Species abbreviations:
A
B
C
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Genome Biology 2007, 8:R226
which bears some similarity to apoptosis in animals [28], sug-
gesting possibly even more distant evolutionary connections.
The evolutionary histories of two other protein families play-
ing central roles in apoptosis, Bcl-2 [2] and caspases [29],
show very similar pictures (Figure 1): members of major sub-
families were most likely present in the early ancestors but
were subsequently lost in nematodes and insects [18,30].
Phylogenetic analysis of multi-motif Bcl-2 family members
shows that the Bax, Bak, and Bok groups of proapoptotic Bcl-
2 homologs appear to be ancient and that each has at least one
well-supported ortholog in Nematostella (Figure 3). The
many other Nematostella Bcl-2 family members are hard to
assign to a specific subtype, although one of them
(140_NEMVE) contains a putative BH4 motif that makes it
similar to the Bcl-2/Bcl-x type. Similarly, Bak and Bok appear
to have representatives in sea urchin and amphioxus, both of
which also contain a multitude of additional Bcl-2 family
genes, which are difficult to consign to a subtype. This is in
sharp contrast to the model organisms D. melanogaster,
which contains only two Bcl-2 family genes belonging to the
Bok group (Debcl and Buffy), and C. elegans, which has one
(CED-9), which is difficult to assign to any vertebrate
subtype.
The final step in apoptosis is proteolysis of a variety of target
proteins in the cell by 'effector' caspases, which are activated
in a proteolytic cascade by several 'apical' ('initiator') caspases
[29]. Both types are clearly present in all animals (Additional
data file 1). Yet, again, Nematostella, amphioxus, and sea
urchin have representatives in more subtypes (defined by
human caspases) than nematodes and insects.
Discussion
It has been proposed that the invention of apoptosis was an
essential requirement for the evolution of multicellular ani-
mals [31], and indeed it has been demonstrated that the apop-
totic pathways involving members of the Bcl-2 family are
present in the most basal metazoan phylum, the sponges
(Porifera) [32,33]. Our results suggest that the bilaterian-cni-
darian ancestor living 650-1,000 million years ago already
had an apoptotic regulatory network composed of Apaf-1, Bcl-
2 and caspase family members. Surprisingly, this ancient
apoptosis network appears to have been more complex than
previously thought and the simple networks seen in present
day insects and nematodes are the result of significant gene
losses. Furthermore, a central protein in the classical apopto-
sis model, the apoptosome forming Apaf-1 [2], which exists as
a single homolog in all genomes studied so far, has multiple
homologs in several morphologically simple invertebrates
and many extant Apaf-1 homologs may not be orthologous.
This suggests that multiple mechanisms triggering apoptosis,
as well as multiple downstream pathways implementing it,
may have existed in early organisms. Many gene copy number
differences are found that can be explained only by lineage-
specific duplications and gene losses. Apparently, different
organisms evolved unique apoptosis networks, which inter-
estingly involved essentially the same gene families, hence
sometimes providing an appearance of similarity between
independently evolved networks. Interestingly, apoptosis
regulators are not the only protein families involved in devel-
opment and disease exhibiting surprising, almost vertebrate-
like complexity in Cnidaria, and thus, presumably, the com-
mon cnidarian-bilaterian ancestor [34,35]. Analyses of
Nematostella Wnt genes revealed unforeseen ancestral diver-
sity: Nematostella and bilaterians share at least eleven of the
twelve known Wnt subfamilies, while five subfamilies appear
to be lost in nematodes/insects [36]. Similarly, proteins with
innate immunity domains have been found to be expanded in
Cnidaria [37]. These results show that biological systems may
not (always) evolve linearly from simple to complex. This
urges caution in interpreting results from studies of C. ele-
gans and D. melanogaster and indeed any model organisms
for understanding apoptosis (or other regulatory pathways)
in human. A more prudent approach might be to carefully
select specific model systems for each protein family studied
in such a way as to minimize the difference between the
model and human. Such a selection process ideally should
include phylogenetic analysis, thus reinforcing the view that
"Nothing in biology makes sense except in the light of evolu-
tion." - Theodosius Dobzhansky (1900-1975).
Conclusion
Phylogenetic inference combined with domain composition
analysis of Apaf-1, Bcl-2, and caspase proteins - central play-
ers in the apoptosis network - reveal a yet unpredicted ances-
tral complexity within each family. In particular, the relative
simplicity of these regulatory networks observed in ecdyso-
zoan species is not the result of a gradual increase in network
complexity correlating with morphological complexity, but
apparently the result of widespread gene losses. Our results
emphasize the importance of explicit phylogenetic analysis
covering a sufficiently large sample of species space, not only
in the detection of orthologous sequences, but also in model
organism selection and in the study of network evolution.
Materials and methods
Sequence database searches
N. vectensis and B. floridae 1.0 genome assemblies and pro-
tein sets were downloaded from the Joint Genome Institute
[38]. The Strongylocentrotus purpuratus assembly
Spur_v2.0 and GLEAN3 gene models were obtained from
Baylor College of Medicine HGSC [39]. The other genome
sequences and corresponding protein sets were downloaded
from Ensembl 38 or SWISS-PROT [40,41]. Several rounds of
PSI-TBLASTN searches were performed against each genome
by using as seeds human NB-ARC, caspase, CARD, death, and
death effector domains as well as Bcl-2 sequences from a vari-
ety of genomes [42]. The hits were then mapped to the corre-
Genome Biology 2007, 8:R226
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Figure 3 (see legend on next page)
0.17
0.9
145_LUBBA
0.25
1.0
136_NEMVE
0.65
138_NEMVE
0.86
127_STRPU
0.64
0.61
113_BRAFL
125_STRPU
0.85
1.0
Debcl_DROME
Buffy_DROME
1.0
0.59
1.0
76_BRARE
77_BRARE
1.0
105_TETNG
87_FUGRU
0.69
106_TETNG
1.0
65_XENTR
0.98
53_CHICK
1.0
BOK_HUMAN
0.95
18_MOUSE
37_CANFA
0.15
0.27
137_NEMVE
140_NEMVE
0.36
0.87
146_SUBDO
128_STRPU
0.2
141_NEMVE
0.49
0.88
142_NEMVE
147_HYDAT
0.44
0.6
1.0
115_BRAFL
0.39
114_BRAFL
117_BRAFL
0.91
121_STRPU
1.0
61_XENTR
1.0
83_BRARE
103_TETNG
0.68
1.0
50_CHICK
1.0
39_CANFA
1.0
B2LA1_HUMAN
30_MOUSE
1.0
0.99
85_BRARE
0.57
80_BRARE
1.0
93_FUGRU
109_TETNG
1.0
0.54
47_CHICK
59_XENTR
1.0
27_MOUSE
0.42
MCL1_HUMAN
33_CANFA
0.17
0.5
122_STRPU
0.76
116_BRAFL
0.99
0.99
0.97
82_BRARE
1.0
88_FUGRU
0.93
104_TETNG
107_TETNG
0.97
67_XENTR
0.93
46_CHICK
1.0
BCL2_HUMAN
0.43
26_MOUSE
45_CANFA
0.76
1.0
66_XENTR
1.0
29_MOUSE
0.59
BCLW_HUMAN
44_CANFA
0.7
0.53
64_XENTR
0.98
54_CHICK
0.92
21_MOUSE
0.89
BCLX_HUMAN
35_CANFA
1.0
1.0
86_FUGRU
108_TETNG
0.8
79_BRARE
1.0
0.95
90_FUGRU
101_TETNG
1.0
100_TETNG
1.0
89_FUGRU
0.98
1.0
94_FUGRU
99_FUGRU
0.47
98_FUGRU
0.92
97_FUGRU
92_FUGRU
0.28
0.66
0.91
0.76
126_STRPU
0.99
118_BRAFL
0.48
143_NEMVE
1.0
55_CHICK
1.0
BAK_HUMAN
0.38
32_CANFA
281_MOUSE
0.95
139_NEMVE
1.0
120_CIOIN
1.0
0.96
110_TETNG
72_BRARE
1.0
68_XENTR
1.0
20_MOUSE
0.75
BAXB_HUMAN
42_CANFA
0.48
1.0
CED9_CAEBR
CED9_CAEEL
0.21
0.46
1.0
73_BRARE
95_FUGRU
0.56
1.0
B2L10_HUMAN
31_MOUSE
1.0
28_MOUSE
0.81
BFK_HUMAN
43_CANFA
0.98
0.87
1.0
123_STRPU
124_STRPU
1.0
1.0
102_TETNG
1.0
74_BRARE
84_BRARE
0.81
62_XENTR
0.98
25_MOUSE
0.99
40_CANFA
B2L13_HUMAN
1.0
1.0
58_XENTR
0.54
1.0
70_BRARE
71_BRARE
1.0
38_CANFA
0.39
B2L12_HUMAN
282_MOUSE
0.87
1.0
81_BRARE
75_BRARE
0.95
60_XENTR
0.36
56_CHICK
1.0
B2L14_HUMAN
0.73
36_CANFA
19_MOUSE
Mcl-1
Bcl-2 like 13
A1
Bfk
Bcl-2 like 12
Bcl-2 like 14
Bok
Bcl-2
Bcl-w
Bcl-x
CED-9
Bcl-2 like 10
Bax
Bak
144_GEOCY
119_BRAFL
http://genomebiology.com/2007/8/10/R226 Genome Biology 2007, Volume 8, Issue 10, Article R226 Zmasek et al. R226.7
Genome Biology 2007, 8:R226
sponding genome protein set to acquire the full-length
protein sequences (for sea urchin and Nematostella, some of
the gene models were in addition predicted by genscan) [43].
All identified genes were checked by reciprocal BLAST analy-
sis, Pfam 21.0 protein searches [44], Conserved Domain
Search (CD-Search), and Reverse PSI-BLAST (RPS-BLAST)
[45].
Multiple sequence alignments and phylogeny
reconstructions
To ensure alignment of homologous domains, sequences
were trimmed to one Pfam 21.0 model (NB-ARC, Bcl-2,
Peptidase_C14 for the caspase domain) [44]. Multiple
sequence alignments were produced by PROBCONS 1.11 [46],
MAFFT 5.861 (localpair, maxiterate 1000) [47], T-COFFEE
4.93 [48], and hmmalign from HMMER 2.3.2 [49,50]. Multi-
ple sequence alignment columns with a gap in more than 50%
of sequences were deleted. MrBayes 3.1.2 was used with
10,000,000 generations, a sample frequency of 1,000, a mix-
ture of amino-acid models with fixed rate matrices and equal
rates, and 25% burn-in [51]. For maximum likelihood
approaches, PhyML 2.4.4 was used with the VT (variable
time) model and four relative rate substitution categories
[52,53]. Pairwise distances (for the Neighbor Joining and
Fitch-Margoliash methods from PHYLIP 3.66 [54-56], and
FastME 1.1 [57]) were calculated by TREE-PUZZLE 5.2 using
the VT model [58]. Tree and domain composition diagrams
were drawn using ATV 4a1 [59]. All conclusions presented in
this work are robust relative to the alignment methods, the
alignment processing, the phylogeny reconstruction meth-
ods, and the parameters used. All sequence, alignment, and
phylogeny files are available upon request.
Domain composition analysis
Domains were analyzed with hmmpfam from HMMER 2.3.2
and Pfam 21.0 [44,49], FFAS03 [60], and InterProScan [61].
Authors' contributions
CMZ performed the phylogenetic, sequence and domain anal-
yses of all the families in this study, as well as prepared the
figures. QZ identified sequences to be analyzed and per-
formed initial analyses. YY contributed to the domain analy-
sis of the proteins involved in this study. AG formulated the
problem and planned the work. All authors contributed to the
interpretation of the results and to writing of the paper.
Additional data files
The following additional data files are available with the
online version of this paper. Additional data file 1 is a figure
illustrating the evolutionary history of caspase protein family
members. Additional data file 2 is a table listing the CED-4/
Apaf-1 protein family members used in this study. Additional
data file 3 is a table listing the multi-motif Bcl-2 protein fam-
ily members used in this study. Additional data file 4 is a table
listing the caspase protein family members used in this study.
Additional data file 1Phylogeny of the caspase familyThis phylogeny was calculated using a Bayesian approach (MrBayes) based on a MAFFT alignment of Peptidase_C14 domains. Posterior probability values are shown for each branch (for detailed information, see Materials and methods). Species abbreviations: BRAFL, Branchiostoma floridae (amphioxus); BRARE, Brachydanio rerio (zebrafish); CAEBR, Caenorhabditis briggsae; CAEEL, Caenorhabditis elegans; CANFA, Canis famil-iaris (dog); CHICK, Gallus gallus (chicken); CIOIN, Ciona intesti-nalis (sea squirt); DROME, Drosophila melanogaster (fruit fly); FUGRU, Fugu rubripes (Japanese pufferfish); NEMVE, Nemato-stella vectensis (starlet sea anemone); STRPU, Strongylocentrotus purpuratus (purple sea urchin); TETNG, Tetraodon nigroviridis (green pufferfish); and XENTR, Xenopus tropicalis (western clawed frog). For a detailed list of protein sequences see Additional data file 4. Para-caspases are excluded from this phylogeny.Click here for fileAdditional data file 2Protein sequences for Figure 2 (phylogeny and domain organiza-tion of CED-4/Apaf-1 homologs)Protein sequences for Figure 2 (phylogeny and domain organiza-tion of CED-4/Apaf-1 homologs).Click here for fileAdditional data file 3Protein sequences for Figure 3 (phylogeny of the multi-motif Bcl-2 family)Protein sequences for Figure 3 (phylogeny of the multi-motif Bcl-2 family).Click here for fileAdditional data file 4Protein sequences for Additional data file 1 (phylogeny of the cas-pase family)Protein sequences for Additional data file 1 (phylogeny of the cas-pase family).Click here for file
Acknowledgements
We thank Drs John C Reed, Guy S Salvesen, and Cheryl Bender for discus-
sions and comments on the manuscript. This research was supported by
NIH grants AI056324 and GM076221. N. vectensis, B. floridae, and Xenopus
tropicalis genome data were produced by the US Department of Energy
Joint Genome Institute [38]. S. purpuratus genome data were produced by
the Sea Urchin Genome Project at Baylor College of Medicine.
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    • "Fifteen caspases have been reported in Hydra, five of which are likely to be initiators based on the presence of N-terminal DED, CARD or Death Domains (DD) [9]. The Zmasek et al. [2] survey identified 10 caspases in Nematostella. Most of the multi-domain pro-apoptotic Bcl-2 family members (Bax, Bak, Bok) known from vertebrates are clearly represented in the coral and sea anemone. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Apoptotic cell death is a defining and ubiquitous characteristic of metazoans, but its evolutionary origins are unclear. Although Caenorhabditis and Drosophila played key roles in establishing the molecular bases of apoptosis, it is now clear that cell death pathways of these animals do not reflect ancestral characteristics. Conversely, recent work suggests that the apoptotic networks of cnidarians may be complex and vertebrate-like, hence characterization of the apoptotic complement of representatives of the basal cnidarian class Anthozoa will help us to understand the evolution of the vertebrate apoptotic network. Results: We describe the Bcl-2 and caspase protein repertoires of the coral Acropora millepora, making use of the comprehensive transcriptomic data available for this species. Molecular phylogenetics indicates that some Acropora proteins are orthologs of specific mammalian pro-apoptotic Bcl-2 family members, but the relationships of other Bcl-2 and caspases are unclear. The pro- or anti-apoptotic activities of coral Bcl-2 proteins were investigated by expression in mammalian cells, and the results imply functional conservation of the effector/anti-apoptotic machinery despite limited sequence conservation in the anti-apoptotic Bcl-2 proteins. A novel caspase type ("Caspase-X"), containing both inactive and active caspase domains, was identified in Acropora and appears to be restricted to corals. When expressed in mammalian cells, full-length caspase-X caused loss of viability, and a truncated version containing only the active domain was more effective in inducing cell death, suggesting that the inactive domain might modulate activity in the full-length protein. Structure prediction suggests that the active and inactive caspase domains in caspase-X are likely to interact, resulting in a structure resembling that of the active domain in procaspase-8 and the inactive caspase domain in the mammalian c-FLIP anti-apoptotic factor. Conclusions: The data presented here confirm that many of the basic mechanisms involved in both the intrinsic and extrinsic apoptotic pathways were in place in the common ancestor of cnidarians and bilaterians. With the identification of most or all of the repertoires of coral Bcl-2 and caspases, our results not only provide new perspectives on the evolution of apoptotic pathways, but also a framework for future experimental studies towards a complete understanding of coral bleaching mechanisms, in which apoptotic cell death might be involved.
    Full-text · Article · Dec 2016
    • "In pro-apoptotic group, the effector proteins (BAX and BOK) contain BH domains 1e3 and a TM domain whereas, BH3-only proteins (BAD and BID) harbor only the BH3 domain and lacked in TM domain [16e18]. BCL-2 family genes have been reported in vertebrates including fish [19], amphibian [20] and mammal [21] and in invertebrates including mollusk [22], sea urchin [23], planarian [24] , sea anem- one [25] and sponge [26] . Recently we have reported bioinformatics characterization and gene expression studies on antiapoptotic gene BCL-2from striped murrel [27] . "
    [Show abstract] [Hide abstract] ABSTRACT: In this study, we have reported a molecular characterization of the first B cell lymphoma-2 (BCL-2) related ovarian killer protein (BOK) from freshwater prawn Macrobrachium rosenbergii (Mr). BOK is a novel pro-apoptotic protein of the BCL-2 family that entails in mediating apoptosis to remove cancer cells. A cDNA sequence of MrBOK was identified from the prawn cDNA library and its full length was obtained by internal sequencing. The coding region of MrBOK yields a polypeptide of 291 amino acids. The analysis revealed that MrBOK contains a transmembrane helix at V(261)-L(283) and a putative BCL-2 family domain at V(144)-W(245). MrBOK also possessed four putative BCL-2 homology domains including BH1, BH2, BH3 and weak BH4. The BH3 contains 21 binding sites and among them five residues are highly conserved with the aligned BOK proteins. The homology analysis showed that MrBOK shared maximum similarity with the Caligus rogercresseyi BOK A. The topology of the phylogenetic tree was classified into nine sister groups which includes BOK, BAK, BAX, BAD, BCL-2, BCL-XL, NR13 and MCL members. The BOK protein group further sub-grouped into vertebrate and invertebrate BOK, wherein MrBOK located within insect monophyletic clad of invertebrate BOK. The secondary structural analysis showed that MrBOK contains 11 α-helices (52.2%) which are connected over random coils (47.7%). The 3D structure of MrBOK showed three central helices (α6, α7 and α8) which formed the core of the protein and are flanked on one side by α1, α2 and α3, and on the other side by α4, α5 and α11. MrBOK mRNA is expressed most abundantly (P < 0.05) in ovary compared to other tissues taken for analysis. Hence ovary was selected to study the possible roles of MrBOK mRNA regulation upon bacterial (Aeromonas hydrophila and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodovirus] infection. During bacterial and viral infection, the highest MrBOK mRNA transcription was varied at different time points. In bacterial infected ovary tissue, the highest mRNA expression was at 24 h post-infection, whereas in viral infection, the expression was highest at 48 h post-infection. Thus we can conclude that MrBOK functions as an apoptotic protein in intracellular programmed cell-death pathway to counteract the anti-apoptotic proteins released by bacterial and viral pathogens at the time of infection. This is the first study that emphasizes the importance of BOK during bacterial and viral infection in crustacean. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · May 2015
    • "Currently, the analysis of genomic information from invertebrates, including cnidarians, echinoderms, mollusks and amphioxus [54,62,96,106,119,127,152], suggests that this apoptotic network already existed in the ancestor of the entire animal kingdom (Metazoa) in a form more complex than expected [34,118,130]. The evolution of the apoptotic pathway does not seem to be a linear process of complexity increase but is inherently dynamic , including lineage-specific gene losses and expansions and the emergence/reemergence of similar proteins [151]. In this context, the apparent simplicity of apoptosis in nematodes and insects might not be the ancestral state but, most likely, a result of more recent simplifications [152]. "
    [Show abstract] [Hide abstract] ABSTRACT: Apoptosis is a type of programmed cell death that produces changes in cell morphology and in biochemical intracellular processes without inflammatory reactions. The components of the apoptotic pathways are conserved throughout evolution. Caspases are key molecules involved in the transduction of the death signal and are responsible for many of the biochemical and morphological changes associated with apoptosis. Nowadays, It is known that caspases are activated through two major apoptotic pathways (the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway), but there are also evidences of at least other alternative pathway (the perforin/granzyme pathway). Apoptosis in mollusks seems to be similar in complexity to apoptosis in vertebrates but also has unique features maybe related to their recurrent exposure to environmental changes, pollutants, pathogens and also related to the sedentary nature of some stages in the life cycle of mollusks bivalves and gastropods. As in other animals, apoptotic process is involved in the maintenance of tissue homeostasis and also constitutes an important immune response that can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults, viruses, and protozoan parasites. The main goal of this work is to present the current knowledge of the molecular mechanisms of apoptosis in mollusks and to highlight those steps that need further study. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Apr 2015
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