TIBS 24 – FEBRUARY 1999 TALKING POINT

domains implicated in programmed death. The PSI-BLAST method signifi- cantly increases the database-search sen- The domains of death: evolution of sitivity by incorporating information em- bedded in a multiple sequence alignment the machinery into a position-dependent weight matrix (profile), which is employed as the query for iterating the search. This allows the detection even of very subtle sequence L. Aravind, Vishva M. Dixit and Eugene V. Koonin similarities at a statistically significant level5,6; all previously undetected relation- Recent progress in research into programmed cell death has resulted in ships between domains reported the identification of the principal protein domains involved in this process. here are statistically supported (having The evolution of many of these domains can be traced back in evolution random expectation values of 0.01 or to unicellular eukaryotes or even bacteria, where the domains appear to lower). Such an exhaustive search was be involved in other regulatory functions. Cell-death systems in animals particularly important for the detection of and plants share several conserved domains, in particular the family of possible progenitors of some of the apop- apoptotic ATPases; this allows us to suggest a plausible, even if still tosis domains in phylogenetically distant incomplete, scenario for the evolution of apoptosis. species, such as yeast or even bacteria. The functional classification of the ‘do- mains of death’ and their phylogenetic PROGRAMMED CELL DEATH, also distribution are shown in Table 1. known as apoptosis, is a major process in animal and plant development1,2. There is Table 1. Phylogenetic distribution of the principal domains involved in programmed cell death a basic similarity between the cell-death events that occur in development and Domain Vertebrates Dm Ce Plants Yeast Bacteria those that occur in response to Ligands, intracellular domains and membrane pathogens and other stress signals; tumor TNF family + ? –? ? – – suppression is a form of this process2,3. TNFR, extracellular The extraordinary research activity of domain + ? –? + – – LRR + + + + –/+a –/+a the past few years has resulted in the BCL-2/BAX + ? + ? – – characterization of the principal molec- Adaptor (protein–protein interaction) domains ular components of the apoptotic ma- DD + ? + ? – – 4 chinery . Like proteins that participate in DED + ? –? ? – – other signaling networks, many of the CARD + + + ? – – proteins involved in apoptosis contain TIRb +++c +– +d BIRb +++?+– multiple domains, and complex patterns MATHb +++++– of interactions between these domains CART + + + ? – – define specific pathways. Here, we pre- Effector and regulatory enzymes sent an inventory of the most important Caspaseb +++?–+e apoptosis domains and report a number Ap-ATPase + ? + + – +f of previously undetected structural and DAP kinase + ? + ? – – IRAK/Pelle kinase + + + + – – evolutionary connections. Using the RIP kinase + ? –? ? – – rapidly accumulating genome sequences, Cell-cycle control, transcription factors and other nuclear functions we trace the phylogenetic distribution of CDK/cyclin + + + + + – each domain and propose a scenario for CKI (KIP/WAF family) + + + + – – the evolution of the apoptosis machinery. RB + + + + – – P53 + ? –? ? – – MYC + + –? –? – – The domains of death and their phylogenetic NF-κB + + (+)b,g ? (+)b,g – distribution E2Fs/Dp1s + + + ? – – Using the gapped BLAST program as CES-2 + + + ? – – well as the iterative program PSI-BLAST5, CAD/ICAD + + –? ? – – we performed a detailed analysis of the aOnly intracellular forms detected. bSignificantly expanded in this study; multiple alignments are sequences of the principal proteins and available as Supplementary Information. cA diverged form in a single protein. dDiverged forms found in Streptomyces, Bacillus subtilis, Synechocystis sp. and Rhizobium. eA single, diverged form detected in Streptomyces. fDetected only in the Actinomycetes and B. subtilis. gThese proteins (C. elegans olf-1 and L. Aravind is at the NCBI, National Library of yeast SPT23) share a conserved immunoglobulin-like DNA-binding domain with NF-κB but cannot be Medicine, NIH, Bethesda, MD 20894, USA, considered its true orthologs. A plus indicates that the domain is present in the phylogenetic lineage; a and at theDept of Biology, Texas A&M minus indicates those lineages whose complete genome sequences are available. A question mark University, College Station, TX 77843, USA; indicates that as yet the domain is not detectable in the lineage but that there are no complete genome V. M. Dixit is at the Dept of Molecular sequences. For the nearly completed C. elegans genome, –? is indicated for the domains missing in the Oncology, Genentech Inc., 1 DNA Way, South current sequence set. Abbreviations used: Ap-ATPase, apoptotic ATPase; Ce, Caenorhabditis elegans; San Francisco, CA 94080-4918, USA; and CDK, cyclin-dependent kinase; CKI, CDK inhibitor; DAP kinase, death-associated protein kinase; DD, E. V. Koonin is at the NCBI, National Library death domain; DED, death-effector domain; Dm, Drosophila melanogaster; IAP, inhibitor of apoptosis; LRR, leucine-rich repeat; TIR, Toll–interleukin- domain; TNF, tumor necrosis factor; TNFR, TNF of Medicine, NIH, Bethesda, MD 20894, USA. receptor. Email: [email protected] Published by Elsevier Science Ltd. PII: S0968-0004(98)01341-3 47 TALKING POINT TIBS 24 – FEBRUARY 1999

Ligands and receptors. The first stage of through the caspases9; the second contains a DD, IRAK/PELLE kinases and apoptosis signaling occurs through the includes a protein kinase cascade (see MyD88 (Ref. 11; see below). interaction between extracellular li- below) and some proteins of the TNFR- The DD–DED–CARD network and other adaptor gands, such as tumor necrosis factor associated factor (TRAF) family that domains. Important components of all (TNF) and their cognate receptors, such activate NF-␬B (Ref. 10). cell-death pathways are small protein– as members of the TNF receptor (TNFR) Recently, decoy versions of members protein-interaction domains that are family7. Orthologous relationships be- of the TNFR family of receptors, which present in apoptotic proteins, including tween ligands and receptors are de- have missing or truncated intracellular receptors, adaptors and effectors, in a tectable only among vertebrates. domains, have been discovered; this has variety of combinations (Fig. 1a). These However, characteristic cysteine-rich revealed a new level of control to the domains glue the components of the repeats that are conserved in the extra- apoptotic signaling pathways7. These death machinery together, primarily cellular portions of the receptors are proteins bind the intracellular ligands but through homophilic interactions, and also present in the plant receptor ki- do not transmit the signal and, accord- transmit signals from receptors to nases of the Crinkly family8. These ingly, interrupt the cell-death pathways. downstream effectors, such as the cas- receptors trigger at least two distinct The interleukin 1 (IL-1) signaling pases. The best known of these adap- signaling pathways through their pathway, although not involved in apop- tors is the DD12,13. Two other adaptor intracellular domains. The first pathway tosis, includes components that are domains, namely the death-effector do- involves the death domain (DD) cas- structurally similar to apoptotic pro- main (DED)14 and the caspase activation cade (see below) that communicates teins, namely the IL-1 receptor that and recruitment domain (CARD)15,

TLR3 (a) TNFR FAS IL-3R ATRK1 IL-1R

Ig Receptors DR2 Ig

Ig D D T T D

YddK_Bs SC7H1.23_Scoe TRAF1 T T Cr Cr Cr M

Adaptors TRADD RAIDD FADD MYD88 DG17 SSI D C D De D D T Cr M SH2

IRAK RIP RICK FEN1 DAP kinase Kin Kin Kin Kin ATP D Kinases D D C Kin JAK JAK1 JAK2 Caspase-8 CED-3Y48E1B.13_Ce F22D3.6_Ce Caspases De De Casp C Casp Casp Ig Casp

IAP-1 IAP-CHV SPBIRDP NAIP ARC Caspase inhibitors BBBC BB BBB ATP C ␬ NF- B, p100 NFAT SPT23 Ces-2 N-5 D Nuclear factors Igl NfIg D Igl NfIg NfIg Dbd SH2 NILF-3 STAT Domain key Ankyrin LRR WD40 Fibronectin III DD CARD DED Predicted ATPases TIR Kinase

D C De ATP ATP T Kin

DNA-binding Immunoglobulin Math Caspase Bir domain fold Ring DNA-binding Ig bZIP SH2 CART TNFR M Casp Dbd Ig NfIg SH2 Cr

48 TIBS 24 – FEBRUARY 1999 TALKING POINT

The DD–DED–CARD triad of (b) Animal domains is found only in ani- CED-4 C Ap-ATPase mals; furthermore, DED seems to be limited to vertebrates APAF-1 C Ap-ATPase and arthropods (Table 1). By contrast, as emphasized by our analysis here, TIR and Plant N protein MATH are ancient conserved tobacco T Ap-ATPase domains. Multiple copies of TIR are present in animal Arabidopsis apoptotic receptors and adap- N-protein T Ap-ATPase homolog tors, as well as in plant proteins involved in disease 17 Prokaryotic resistance (Fig. 1a,b) . Using iterative PSI-BLAST searches, Sc6A9.38 T Ap-ATPase we detected previously unno- ticed, divergent versions of AfsR H Ap-ATPase TIR in a Caenorhabditis elegans protein and in proteins from four taxonomically diverse TB-ATPase AC Ap-ATPase H bacteria (see Table 1, Fig. 1a and Box 1). The MATH domain was identified originally in the H Ap-ATPase PHAR009 adaptor proteins of the TRAF family and in membrane pro- 18 GutR H Ap-ATPase teases of the meprin family . We detected multiple copies of MATH in a variety of animal and plant proteins; interest- Domain key ingly, a MATH domain is pres- Adenylyl ␣-Helical ent in a distinct set of ubiqui- cyclase TIR CARD HTH RCC1 Modified TPR WD40 LRR domain tin hydrolases, and this domain architecture is vertically con- AC T C H served in eukaryotes from yeast to vertebrates (see Fig. 1a and Box 1). TRAF, an apop- Figure 1 tosis adaptor that consists of (a) Distinct domain architectures of receptors, adaptors and effectors involved in programmed cell RING, CART and MATH domains death and their homologs (see facing page). For the caspases, four different architectures are shown. is present not only in animals A highly conserved paralog of CED-3 in Caenorhabditis elegans (Ce) contains a coiled-coil domain in- but also in Dictyostelium stead of the caspase activation and recruitment (CARD) domain, and the novel predicted caspase-like discoideum, where it is rep- protease contains an immunoglobulin domain. (b) Domain architectures of Ap-ATPases. Bs, Bacillus resented by three paralogs subtilis; CARD, caspase activation and recruitment domain; CHV, Chilo iridiscent virus; DAP kinase, that share the same domain death-associated protein kinase; DD, death domain; DED, death-effector domain; HTH, helix-turn-helix DNA-binding domain; IAP, inhibitor of apoptosis; IL, interleukin; LRR, leucine-rich repeat; TB-ATPase, a architecture (Fig. 1a). family of predicted ATPases from Mycobacterium tuberculosis; TIR, Toll–interleukin-receptor domain; Even in animals, the func- TNFR, tumor necrosis factor receptor; TPR, tetratricopeptide repeat; TRAF, TNFR-associated factor; tions of the adaptor domains Sc6A9.38, predicted transcription regulator from Streptomyces coelicolor (Scoe); PHAR009, an un- might not be limited to pro- characterized protein from the archaeon Pyrococcus horikoshii. grammed cell death. For ex- ample, in addition to known resemble DD in terms of size and pre- Other interaction domains, such as programmed-cell–death adaptors, DD is dicted ␣-helical structure. Iterative the cysteine-rich motif associated with also present in ankyrins (e.g. C. elegans database searches using PSI-BLAST de- RING and TRAF (CART) domains, the UNC-44), in which its function remains tected some sequence similarity be- Toll–interleukin-receptor domain (TIR), unknown. Similarly, meprins have not tween DED and DD, although we could and meprin and the TRAF-homology do- been implicated in apoptosis, and the not demonstrate that this similarity is main (MATH) appear to be structurally role of the MATH domain in these pro- statistically significant (Ref. 15, and L. unrelated to each other or to the teases remains to be determined. Aravind and E. V. Koonin, unpublished). DD–DED–CARD class. The principle of Caspases and their inhibitors. Caspases, Nevertheless, the recently determined adaptor design is the combination of the major effectors of programmed cell three-dimensional structures of DD, two different interaction domains – for death, are cysteine proteases that pos- DED and CARD reveal that these do- example, DD–DED in FADD, DD–CARD in sess a distinct fold and cleave specific mains indeed have similar ␣-helical RAIDD, and DD–TIR in MyD88 (Fig. 1a). aspartate-containing sites in many pro- folds, which is compatible with the This allows the adaptors to connect teins involved in apoptosis, such as cas- hypothesis that they evolved from a receptors and effectors largely through pases themselves (to initiate a proteo- common ancestor. homophilic interactions. lytic cascade), BCL-2, BAX, I␬B, IL-1, 49 TALKING POINT TIBS 24 – FEBRUARY 1999

MEKK and RB (Ref. 9). Caspases typically typically also contain a RING finger that that appears to involve both an interac- contain one of the adaptor domains, might mediate as-yet-uncharacterized, tion between the respective CARD which links them to the rest of the specific protein–protein interactions; domains and an additional interaction signaling machinery (Fig. 1a). Caspases some IAPs also contain a CARD domain, between the ATPase domain and the are present in vertebrates, arthropods which, given the preponderance of homo- protease domain33. In an in vitro system, and nematodes, which suggests that philic interactions in apoptosis, is likely caspase activation by APAF-1 depends they exist in all animals. Three members to interact with the CARDs in the on cytochrome c, and prevention of cyto- of the caspase family have been found caspases (Fig. 1a). chrome c release from mitochondria by in C. elegans, whereas a major expan- Fusion of the BIR domain with two en- BCL-2 prevents programmed cell death; sion is seen in vertebrates. zymes suggests that this domain has however, a direct a role for cytochrome Until now, the caspases appeared to versatile functions. The neuronal apop- c in caspase activation remains to be a tight, highly conserved family that tosis inhibitor NAIP contains a BIR do- be demonstrated32. An alternatively lacked detectable sequence or struc- main and a predicted ATPase domain. spliced form of CED-4 that contains an tural similarity to any other proteins. The latter, together with the ATPase do- insertion within the ATPase domain is a However, our iterative database searches main of the MHC-class-II transcriptional dominant negative inhibitor of caspase revealed a C. elegans protein (F22D3.6) transactivator, belongs to a previously activation33. that is distantly, but statistically signifi- undetected family (Fig. 1a and Box 1). The phylogenetic distribution of the cantly, related to the caspases. This pro- The giant mammalian protein BRUCE AP-ATPases is most unusual. Conserved tein retains the catalytic residues as well contains a BIR domain fused to a ubiqui- AP-ATPase domains are present in as the critical structural elements of the tin-conjugating enzyme; this protein plants, where they combine with TIR do- caspases, which indicates that the three- might mediate a distinct link between mains, leucine-rich repeats (LRR) and dimensional fold is conserved; a highly apoptosis and the ubiquitin system22. regulator of chromatin condensation conserved homolog of this novel cas- We and others23 have detected BIR do- (RCC) repeats in numerous large pro- pase-like protein was detected in human mains in uncharacterized yeast pro- teins that are involved in resistance to ESTs. Furthermore, a PSI-BLAST search teins, which shows that this is an an- pathogens and stress response30 (Fig. initiated by a profile produced from a cient eukaryotic domain whose original 1b). Our searches showed that multiple multiple alignment of the caspases with function might be unrelated to pro- copies of this domain are encoded in their newly identified distant homologs grammed cell death (see Fig. 1a, Table 1 the genomes of a single group of bac- retrieved putative caspase-like pro- and Box 1). Another variation on the teria, the actinomycetes, whereas an- teases from Dictyostelium discoideum, caspase-inhibitor theme is the recently other Gram-positive bacterium, Bacillus Streptomyces coelicolor and a Rhizobium discovered ARC that contains only the subtilis, and the archaeon Pyrococcus sp. plasmid (Box 1). Notably, the bacte- CARD domain24. horikoshii each encode one member of rial caspase-related protein is combined The key regulators: apoptotic ATPases and the family. Interestingly, APAF-1 shows a with a protein kinase domain; the pro- the BCL-2 family. Two, apparently inde- greater sequence similarity to the plant, tein might therefore have a regulatory pendent, pathways of caspase activation and even to the bacterial, members of function. result in apoptosis25. The first involves the Ap-ATPase superfamily than it does These findings show that caspases recruitment of procaspases (e.g. mam- to CED-4. Thus, in a database search ini- belong to an ancient and diverse pro- malian caspase-2 and caspase-8) by tiated with the sequence of the ATPase tease superfamily. It will be of interest adaptors, such as FADD and RAIDD, that domain of APAF-1 (the 350 N-terminal to find out whether the newly predicted are bound to activated receptors (e.g. residues), statistically highly significant distinct members of this superfamily FAS and TNFR1, respectively); this trig- similarity was observed for a variety of are involved in apoptosis, particularly gers procaspase cleavage and activation. plant resistance proteins (scores of given the recent studies on pro- The second pathway appears to be 50–58 bits, which currently corresponds grammed cell death in Dictyostelium19 central to, if not the only one involved to E values of 10Ϫ5–10Ϫ7) and to a puta- (see below). The recent demonstration in, developmental programmed cell tive transcriptional regulator from of a caspase-like proteolytic activity death26,27. It is mediated by members of Streptomyces coelicolor (score of 45 bits; associated with programmed cell death the BCL-2 family of proteins28, which E value ϭ 6 ϫ 10Ϫ4); by contrast, in the in plants20 further reinforces the notion work in conjunction with a distinct same search, CED-4 scored only 43 bits that this protease family is evolution- ATPase (Ap-ATPase) that is represented (E value ϭ 0.003). Given the obvious arily conserved and suggests that plant by C. elegans CED-4 and human APAF- functional similarities between APAF-1 members are likely to be identified as 129–31. The members of the BCL-2 family and CED-430,31, this suggests that animal, genome sequencing progresses. are either anti-apoptotic (e.g. BCL-2) or plant and bacterial members of the Caspases are regulated by at least pro-apoptotic (e.g. BAX). All these pro- Ap-ATPase superfamily share similar three distinct types of inhibitor. Inactive teins are membrane associated; in par- mechanisms of action. caspase homologs, whose catalytic ticular, several of them have been The Ap-ATPase domain is uniquely de- sites are disrupted (FLIPs), have been shown to associate with the outer mito- fined by five motifs discovered in mammals; these proteins chondrial membrane28. Apoptosis is ac- (Fig. 2). Ap-ATPases and their bacterial function as dominant negative modula- companied by cytochrome c efflux from and archaeal homologs typically are tors of the caspase cascade9. Caspase the mitochondrion into the cytosol, and multidomain proteins that, in addition to activity can be inhibited by inhibitor of this process appears to be regulated by the ATPase domain, contain signaling and apoptosis proteins (IAPs) that contain BCL-2-family members32. interaction domains (Fig. 1b). Interest- 1–3 distinct Zn fingers, termed BIR The AP-ATPases activate caspases, ingly, one of the bacterial members of domains, which seem to participate such as the nematode CED-3 and mam- this family from Streptomyces coelicolor directly in the inhibition21. The IAPs malian caspase-9, in a complex fashion contains the Ap-ATPase domain and a 50 TIBS 24 – FEBRUARY 1999 TALKING POINT

Figure 2 Multiple alignment of Ap-ATPases. The first two sequences are those of Caenorhabditis elegans (Ce) and human (Hs) Ap-ATPases; the second block of sequences includes plant resistance-gene products, and the bottom block includes regulatory proteins from actinomycetes and an un- characterized protein from the archaeon Pyrococcus horikoshii (Ph). The sequences were extracted from the GenBank database by iterative searches that used the PSI-BLAST program and the ATPase domain (the 350 N-terminal amino acids) of APAF-1 as the query [each protein’s gene identification number is shown after the species abbreviation, except for two predicted ATPases from Mycobacterium tuberculosis (Mtu), which have not been annotated in the genome sequence]. The alignment was constructed by using the MACAW program51. Five conserved blocks, whose boundaries have been adjusted to maximize the statistical significance of the alignment, are shown; the distances between the blocks and the distances from the protein termini are indicated. The consensus includes positions conserved in 90% of the aligned sequences and, in addition to individual conserved residues, shows aromatic (a; yellow shading), hydrophobic (h; yellow shading), aliphatic (l; yellow shad- ing), small (s; cyan shading), tiny (u; green shading), hydroxy (o; shown in blue) and polar (p; shown in brown) residues; the conserved charged residues are shown in magenta. Motifs I and III are conserved in a vast class of ATPases and GTPases, and include the phosphate-binding site (P-loop) and the Mg2ϩ-binding site, respectively. At, ; Bs, Bacillus subtilis; Lu, Linum usitatissimum; Ng, Nicotiana glutinosa; Sc, Streptomyces coelicolor; Sn, Streptomyces nogalater.

TIR domain – a domain architecture that kinase, which interacts with the apop- (CPP32) might provide one of the links resembles that of plant Ap-ATPases totic effector RAIDD, functions down- between cell-death signaling and cell- (Fig. 2). Typically, however, instead of stream of TRADD and activates a distinct cycle control42. the protein–protein-interaction domains apoptotic pathway36. A novel domain ar- Several other transcription factors found in animal and plant Ap-ATPases, chitecture – a kinase domain combined (e.g. MYC, CES-2/NILF-3-like bZIP pro- the bacterial homologs contain helix- with a CARD domain – is seen in the RICK teins and NF-␬B) connect cell-cycle- turn-helix domains that are predicted to protein that is involved in the regulation related transcriptional regulation and bind DNA, an uncharacterized ␣-helical of CD95/Fas- and TNFR1-dependent cell- cell death. NF-␬B is linked to the TNFR domain and, in some cases, adenylyl death pathways and is recruited to re- apoptosis signaling pathway through the cyclase or TPR repeats (Fig. 1b). Thus ceptor signaling complexes through its adaptors TRADD and TRAF, and a Ap-ATPase superfamily proteins, includ- interaction with TRAF proteins37. Ser/Thr kinase cascade that includes NIK ing those from bacteria, probably In plants, FEN kinase, a homolog of the and IKK1/2; the latter phosphorylates the participate in complex signaling path- animal IRAK/Pelle kinases, is involved in NF-␬B inhibitor I␬B and thereby causes ways through multiple protein–protein the induction of cell death38. The IRAK NF-␬B activation43. NF-␬B-family proteins (and, in bacteria, also protein–DNA) kinases, although not directly implicated contain an immunoglobulin (Ig)-like do- interactions. Studies on S. coelicolor in apoptosis, contain an N-terminal DD main, which is involved in DNA bind- support such a conclusion because the and are recruited to the IL-1 receptor by ing44,45; the largest forms of NF-␬B (p100 ATPase activity of this protein, which means of the DD/TIR-domain- containing and p105) also contain a DD (Ref. 12; see acts downstream of a Ser/Thr kinase, adaptor MyD88 (Ref. 11). Fig. 1a). Unlike the basic cell-cycle ma- is necessary for activation of the Cell-cycle control and apoptosis. Apoptosis chinery, which consists of cyclins and gene for biosynthesis of the antibiotic is connected to cell-cycle regulation be- cyclin-dependent kinases (CDKs) and is actinorhodin34. cause of the opposing options faced by conserved in all eukaryotes, the tran- Protein kinases in programmed cell death. a cell in development: proliferation ver- scription factors that link cell-cycle con- Several Ser/Thr protein kinases are sus death. In animals, this decision ap- trol to apoptosis thus far have been de- specifically involved in apoptosis. The pears to be mediated by a unique ma- tected only in animals. However, interative calmodulin-dependent death-associated chinery that comprises the RB protein database searches showed that proteins protein (DAP) kinase, which is con- and the DNA-binding proteins E2F-1, that contain Ig-like domains homologous served in vertebrates and C. elegans, DP-1 and p5339,40. The RB protein is anti- to the DNA-binding domain of NF-␬B (e.g. contains a DD and is a positive regulator apoptotic and acts by sequestering an yeast SPT23) are ubiquitous in eukary- of interferon-␥-induced apoptosis. DAP E2F1–DP1 dimer. E2F-1 and DP-1 func- otes (Table 1 and Box 1). Furthermore, kinase appears to link apoptosis and tion as transcription factors in normal the global transcription regulator RB and tumor growth, and might play a crucial cell-cycle progression but they also acti- the KIP1/WAF-type CDK inhibitor, which role in regulation of the balance be- vate p53, which under the right physio- are involved in both apoptosis and cell- tween proliferation and apoptosis pro- logical conditions, triggers apoptosis41. cycle control, are conserved in plants gression35. The DD-containing RIP Direct cleavage of RB by caspase-3 and animals46,47. 51 TALKING POINT TIBS 24 – FEBRUARY 1999

New conserved domains in search of a function. have not been ‘invented’ for this function nevertheless, it is interesting to note The increasing interest in apoptosis has but, rather, have been recruited from that it occurs in Streptomyces – a bac- resulted in the identification of many proteins that, in unicellular organisms, terium that also encodes regulatory proteins that seem to be important for perform other regulatory functions. proteins that contain the AP-ATPase different forms of cell death but whose Ancient domains probably recruited domain and a putative protease of the specific roles are unknown. For exam- at the onset of the evolution of the orig- caspase superfamily. ple, a small protein called DAP1 has inal apoptosis machinery include the Subsequent to the emergence of the been implicated in ␥-interferon-induced AP-ATPases, the adaptor domains TIR, hypothetical ancestral apoptosis sys- apoptosis48. We identified a family of CART and MATH, and possibly caspase- tem, the evolution of programmed cell small DAP1-like proteins that are highly superfamily proteases. The PELLE-type death involved extensive diversifi- conserved in Drosophila and C. elegans protein kinases could have regulated cation, which was brought about by (Table 1 and Box 1), and might be in- cell death in subsets of cells in response further recruitment of pre-existing volved in an uncharacterized but impor- to both developmental and environmen- domains, multiple duplications followed tant aspect of apoptosis common to all tal cues that confronted the ancestral by divergence, and accretion of unre- Metazoa. multicellular organism. A more specific lated domains in receptors, adaptors Several proteins have been impli- reconstruction of the hypothetical an- and effectors (Fig. 1). The amount of cated in the post-mortem DNA fragmen- cestral apoptosis system depends on genome-sequence data is not yet suffi- tation typical of programmed cell death, additional genome-sequence data, par- cient for us to link the major steps in the namely the positive effectors CAD and ticularly complete sequences of plant evolution of apoptosis to particular CIDE-A/B, and the negative effectors genomes, and experimental data on cell- nodes in the eukaryotic phylogenetic ICAD, DFF45 and DREP-149,50. Iterative death mechanisms. We expect that, tree, although, given the apparent ab- database searches allowed us to define taken together, these data will answer sence of a number of apoptosis proteins a conserved domain, which we term the the remaining questions – for example, in C. elegans, it seems obvious that a CAD domain, in all these proteins, in- that regarding the emergence of typical dramatic increase in complexity accom- cluding DREP-1 and its two uncharacter- caspases. Such information will allow us panied the emergence of coelomates*. ized paralogs from Drosophila (see Box to define the animal and plant compo- Nevertheless, such observations as the 1). Again, although the CAD domain is nents of the programmed-cell-death ma- existence of homologs of TRAF and probably conserved in a wide range of chinery that have been inherited from STAT with conserved domain architec- animals, its exact function remains to be the hypothetical ancestral system, and ture, as well as the presence of a cas- identified. those that have evolved independently pase homolog in Dictyostelium, suggest in each lineage. that considerable complexity could Origin and evolution of programmed cell death Ap-ATPases are the components of have been reached even prior to the Although some components and the programmed-cell-death system that emergence of Metazoa. Notably, pro- many functional interactions remain un- have the most interesting phylogenetic grammed cell death has been described clear, we are now in a position to start distribution and evolutionary history. in Dictyostelium, although the proteins reconstructing the evolution of the Several lines of evidence suggest that involved have not been characterized19. apoptosis machinery. The available the evolution of this family included an Important examples of domain recruit- data is not sufficient for us to piece ancient (prior to the animal–plant diver- ment include BIR and MATH domains, together all the components of the cell- gence) horizontal gene transfer from both of which are present in yeast (Table death system in plants. Nevertheless, Gram-positive bacteria into the eukary- 1), where their exact functions remain the conservation of proteins and do- otic lineage: (1) Ap-ATPases are present to be elucidated. In all likelihood, these mains that, in animals, are involved in in multicellular but not in unicellular functions have nothing to do with pro- programmed cell death (Table 1) is eukaryotes; (2) many members of this grammed cell death, given that this phe- prominent enough for us to propose family are present in actinomycetes, nomenon does not seem to exist in that the common ancestor of plants and and they show considerable diversity in yeast (at least not in its typical form). animals possessed a simple apoptosis terms of both sequence and domain ar- The helical adaptor domain should machinery. The driving force behind the chitecture; (3) the family is not found in have emerged early in animal evolution, evolution of such a system might have bacteria that are not of the Gram-posi- although, in this case, the source of the been a form of kin selection, which fa- tive lineage and is present in only one domain remains unknown. The diversifi- vored the death of subsets of cells as a archaeal species. The opposite direc- cation of this domain must have oc- defense against pathogens, as well as a tion of horizontal transfer – from eu- curred prior to the divergence of nema- primitive developmental mechanism karyotes to Gram-positive bacteria – todes and coelomates – given that C. that contributed to effective production cannot be ruled out, but seems less elegans and vertebrates share orthologs and dispersal of progeny. We must em- plausible, given the general dominant among CARD proteins (the caspases) phasize that the apoptosis machinery is trend for gene transfer from bacteria and among DD proteins (IRAK and DAP based on several ancient domains that to eukaryotes51. The existence of an kinases). The emergence of the DED do- Ap-ATPase in the archaeon Pyrococcus main and the major expansion of these Box 1. Supplementary material horikoshii further emphasizes the propensity of this family for horizontal *Coelomates, such as chordates, arthropods, Supplementary material for this article, in- gene transfer. mollusks and echinoderms, are animals that cluding multiple alignments of those apop- TIR is another apoptosis domain that possess a true, mesoderm-lined body cavity. totic domains for which we report new find- has bacterial homologs. Its distribution We adhere to the traditional view, which holds that coelomates are monophyletic52, although ings, is available at ftp://ncbi.nlm.nih.gov/ among bacteria is too episodic for us pub/koonin/PCD an alternative evolutionary scenario has been to propose an evolutionary scenario; published53. 52 TIBS 24 – FEBRUARY 1999 TALKING POINT domains are, however, linked to the crown-group eukaryotes already pos- 17708–17712 coelomate lineage. Another adaptor do- sessed a primitive prototype of the cell- 34 Matsumoto, A. et al. 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