The apoptosome is an Apaf-1 cytochrome c complex that activates procaspase-9. The three-dimensional structure of the apoptosome has been determined at 27 A resolution, to reveal a wheel-like particle with 7-fold symmetry. Molecular modeling was used to identify the caspase recruitment and WD40 domains within the apoptosome and to infer likely positions of the CED4 homology motif and cytochrome c. This analysis suggests a plausible role for cytochrome c in apoptosome assembly. In a subsequent structure, a noncleavable mutant of procaspase-9 was localized to the central region of the apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the cleavage of procaspase-9 is not required to form an active cell death complex.

Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases. The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems.

Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no effect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These findings define a novel function for Hsp27 and provide the first evidence that a heat shock protein represses cas-3 activation.

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of approximately 150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G->>E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted beta-strand/beta-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis.

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Recently, a large subfamily of nucleotide-binding and oligomerization domain-containing proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats has been described. In this study, we identified PYNOD, a novel member of this family that lacks the leucine-rich repeats. We found that human PYNOD mRNA is expressed in various tissues and at high levels in heart, skeletal muscle and brain. It is also expressed in various cell lines, including haematopoietic cell lines. PYNOD oligomerizes and binds to ASC, an adaptor protein that plays a role in apoptotic and inflammatory signal transduction, and to caspase-1 and IL-1beta. PYNOD inhibits apoptosis-associated speck-like protein containing a CARD (ASC)-mediated NF-kappaB activation and apoptosis, and caspase-1-mediated IL-1beta maturation, and it does so in the presence and absence of constitutively active mutants of CARD12 and PYPAF1, which are enhancers of these processes. Thus, PYNOD is a novel regulator of apoptosis and inflammation.

The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.

Inactivation of the tumor suppressor Rb in the mouse induces cell death, which depends entirely (in lens, CNS) and only partly (PNS, skeletal muscles) on Apaf1/Ced4, an apoptosomal factor thought to be required for processing procaspase-9 following mitochondrial permeabilization. Here, we report that in response to cytotoxic drugs, Apaf1(-/-) primary myoblasts but not fibroblasts undergo bona fide apoptosis. Cell demise was associated with disruption of mitochondria but not endoplasmic reticulum. Processing of procaspase-9 occurred in Apaf1(-/-) myoblasts but not fibroblasts, and ablation of Casp9 prevented drug-induced apoptosis in both cell types. Deregulation of the Rb pathway by overexpression of E2F1 also induced caspase-9-dependent, Apaf1-independent apoptosis in myoblasts. Despite its requirement for apoptosis in vitro, mutation in Casp9 abrogated cell death in the nervous system and lens but only partly in skeletal muscles of Rb-deficient embryos. In addition, developmental cell death in fetal liver and PNS was not inhibited in Casp9(-/-) embryos. Therefore, loss of pRb elicits apoptosome-dependent and apoptosome-independent cell death, and the requirement and coupling of caspase-9 to Apaf1 are both context-dependent.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.

OBJECTIVE

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex genetic disorders. CARD15/NOD2, a member of the Ced4 superfamily which includes Apaf-1 and CARD4/NOD1, has recently been associated with genetic predisposition to CD but additional genetic factors remain to be identified. Because CARD4/NOD1 shares many structural and functional similarities with CARD15, we tested its putative role in IBD.

METHODS

The 11 exons of CARD4 were screened for the presence of variants in 63 unrelated IBD patients. The only non-private genetic variation encoding for a substitution in the peptidic chain was genotyped in 381 IBD families (235 CD, 58 UC, 81 mixed, and seven indeterminate colitis families) using a polymerase chain reaction-restriction fragment length polymorphism procedure. Genotyping data were analysed by the transmission disequilibrium test.

RESULTS

Five of nine sequence variations identified in the coding sequence of the gene encoded for non-conservative changes (E266K, D372N, R705Q, T787M, and T787K). Four were present in only one family. The remaining variant (E266K), which exhibited an allele frequency of 0.28, was not associated with CD, UC, or IBD. Furthermore, IBD patients carrying sequence variations in their CARD4 gene had a similar phenotype to those with a normal sequence.

CONCLUSIONS

Our results suggest that CARD4 does not play a major role in genetic susceptibility to IBD.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.

Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N-ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964-10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis.

Apoptosis Protease-Activating Factor 1, APAF1, was originally isolated four years ago and shown to be the mammalian homologue of the C. elegans pro-apoptotic ced4 gene. Since then, the expression of APAF1 has been demonstrated to be involved in several cell death pathways, including the induction of apoptosis by the p53 tumour suppressor protein and neuronal apoptosis. In this review we will focus on the regulation of APAF1 expression, in particular with regard to recent developments in our understanding of the role of APAF1 in both tumourigenesis and mammalian development.

Genome comparison is the main approach to deduce regulatory network from genome sequence. Apoptotic network is one kind of typical regulatory networks. EGL1, CED3, CED4, CED9 and their homologous proteins play essential roles in apoptosis of C.elegans and mammals, and were regarded as the components of the"core apoptotic engine". But in fruit fly, Drosophila melanogaster, this network is incomplete. A series of bioinformatic analyses found the lost chains of "core apoptotic engine" by discovering two homologues of BCL2/CED9 and one of EGL1 in fruit fly genome sequences. These findings proved that the "core apoptotic engine" is indeed widely conserved among multicellular organisms and the evolutionary complexity of this network of Drosophila is between that of C.elegans and mammals.

The mechanisms of plant defense against pathogen attack in plant leaves have been extensively studied. However, our understanding of plant defense mechanisms in plant roots is still limited. In this study, a semi-dominant mutant nrtp1-D (necrotic root tip 1), with a short-root phenotype, was characterized in rice. Map-based cloning revealed that NRTP1 encoded a typical coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) type protein and the mutation caused an amino acid substitution in the Nucleotide-Binding adaptor shared by Apaf1, certain R genes and CED4 (NB-ARC) domain, which may cause constitutive auto-activation of the NRTP1 protein. Gene expression analysis revealed that NRTP1 was preferentially expressed in rice roots. Expression of mutant nrtp1-D in tobacco leaves induced necrotic lesions, which indicated a common mechanism of plant defense response between leaves and roots. Transcriptome analysis revealed that many typical defense-response genes were differentially expressed in homozygous nrtp1-D. In addition, we also found differential expression of genes in pathways which had not previously been described as being associated with pathogen response. Histochemical analysis showed that the level of nitric oxide (NO), but not reactive oxygen species (ROS), was increased in homozygous nrtp1-D mutant roots. These results indicate that, in addition to the mechanism of defense response common to both roots and shoots, a novel pathway may also exist in rice roots, which does not operate in shoots.