Apaf1 and the apoptotic machinery

The molecular characterization of the Caenorhabditis elegans cell death genes has been crucial in revealing some of the biochemical mechanisms underlying apoptosis in all animals. Four C. elegans genes, egl-1, ced-9, ced-4 and ced-3 are required for all somatic programmed cell death to occur. This genetic network is highly conserved during evolution. The pro-death gene egl-1 and the anti-death gene ced-9 have structural and functional similarities to the vertebrate Bcl2 gene family. The killer gene ced-3 encodes a cystein-aspartate protease (caspase), which is the archetype of a family of conserved proteins known as effectors of apoptosis in mammals. Zou and collaborators1 reported the biochemical identification of an apoptotic protease activating factor (Apaf1), a human homolog of C. elegans CED-4, providing important clues to how CED-4 and its potential relatives could work. A number of proteins have been shown to interact with Apaf1 or to be determinant for its activity as an apoptotic adapter. The aim of this review is to provide an overview of the recent progress made in the field of developmental apoptosis by means of the murine Apaf1 targeted mutations. The central role of Apaf1 in the cell death machinery (apoptosome) and its involvement in different apoptotic pathways will also be discussed.     

Mutational analysis of the Caenorhabditis elegans cell-death gene ced-3

Mutations in the gene ced-3, which encodes a protease similar to interleukin-1beta converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.     

Noncanonical cell death programs in the nematode Caenorhabditis elegans

Genetic studies of the nematode Caenorhabditis elegans have uncovered four genes, egl-1 (BH3 only), ced-9 (Bcl-2 related), ced-4 (apoptosis protease activating factor-1), and ced-3 (caspase), which function in a linear pathway to promote developmental cell death in this organism. While this core pathway functions in many cells, recent studies suggest that additional regulators, acting on or in lieu of these core genes, can promote or inhibit the onset of cell death. Here, we discuss the evidence for these noncanonical mechanisms of C. elegans cell death control. We consider novel modes for regulating the core apoptosis genes, and describe a newly identified cell death pathway independent of all known C. elegans cell death genes. The existence of these noncanonical cell death programs suggests that organisms have evolved multiple ways to ensure appropriate cellular demise during development.     

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.     

The ced-8 gene controls the timing of programmed cell deaths in C. elegans

Loss-of-function mutations in the gene ced-8 lead to the late appearance of cell corpses during embryonic development in C. elegans. ced-8 functions downstream of or in parallel to-the regulatory cell death gene ced-9 and may function as a cell death effector downstream of the caspase encoded by the programmed cell death killer gene ced-3. In ced-8 mutants, embryonic programmed cell death probably initiates normally but proceeds slowly. ced-8 encodes a transmembrane protein that appears to be localized to the plasma membrane. The CED-8 protein is similar to human XK, a putative membrane transport protein implicated in McLeod Syndrome, a form of hereditary neuroacanthocytosis.     

C. elegans MAC-1, an essential member of the AAA family of ATPases, can bind CED-4 and prevent cell death

In the nematode Caenorhabditis elegans, CED-4 plays a central role in the regulation of programmed cell death. To identify proteins with essential or pleiotropic activities that might also regulate cell death, we used the yeast two-hybrid system to screen for CED-4-binding proteins. We identified MAC-1, a member of the AAA family of ATPases that is similar to Smallminded of Drosophila. Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1, the mammalian homologue of CED-4. Furthermore, MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9. A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C. elegans, and this protection is enhanced in a ced-9(n1950sd)/+ genetic background. We observe a similar effect in mammalian cells, where expression of MAC-1 can prevent CED-4 and CED-3 from inducing apoptosis. Finally, mac-1 is an essential gene, since inactivation by RNA-mediated interference causes worms to arrest early in larval development. This arrest is similar to that observed in Smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced-4 null mutants. These results suggest that MAC-1 identifies a new class of proteins that are essential for development, and which might regulate cell death in specific circumstances.     

Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced-9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED-9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9.CED-4 complex. These studies suggest that the release of CED-4 from the CED-9.CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.     

C. elegans EIF-3.K promotes programmed cell death through CED-3 caspase

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.     

The phosphoinositide phosphatase MTM-1 regulates apoptotic cell corpse clearance through CED-5-CED-12 in C. elegans

Multicellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P(2) (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.     

No death without life: vital functions of apoptotic effectors

As a result of the genetic experiments performed in Caenorhabditis elegans, it has been tacitly assumed that the core proteins of the 'apoptotic machinery' (CED-3, -4, -9 and EGL-1) would be solely involved in cell death regulation/execution and would not exert any functions outside of the cell death realm. However, multiple studies indicate that the mammalian orthologs of these C. elegans proteins (i.e. caspases, Apaf-1 and multidomain proteins of the Bcl-2 family) participate in cell death-unrelated processes. Similarly, loss-of-function mutations of ced-4 compromise the mitotic arrest of DNA-damaged germline cells from adult nematodes, even in a context in which the apoptotic machinery is inoperative (for instance due to mutations of egl-1 or ced-3). Moreover, EGL-1 is required for the activation of autophagy in starved nematodes. Finally, the depletion of caspase-independent death effectors, such as apoptosis-inducing factor (AIF) and endonuclease G, provokes cell death-independent consequences, both in mammals and in yeast (Saccharomyces cerevisiae). These results corroborate the conjecture that any kind of protein that has previously been specifically implicated in apoptosis might have a phylogenetically conserved apoptosis-unrelated function, most likely as part of an adaptive response to cellular stress.     

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans

We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.     

Cell ablation by ectopic expression of cell death genes, ced-3 and Ice, in Drosophila

We have developed a system for killing specific cells in Drosophila using ectopic expression of cell death genes. CED-3 and ICE (caspase-1) are proteins required for programmed cell death in the nematode Caenorhabditis elegans and in mammals, respectively. Our previous study has shown that both ced-3 and Ice can elicit cell death in Drosophila . By expressing ced-3 or Ice in several kinds of cells using a GAL4-UAS system and examining the resulting morphological defects, we show that these abnormalities are thought to be caused by the action of ced-3 or Ice genes. As cells are killed by apoptosis in our system, we could eliminate the possibility of harmful effects on the neighboring cells. Our system provides an alternative and novel cell ablation method to elucidate mechanisms of cell differentiation and cell-cell interactions during development in Drosophila .     

Cap-independent translation promotes C. elegans germ cell apoptosis through Apaf-1/CED-4 in a caspase-dependent mechanism

Apoptosis is a natural process during animal development for the programmed removal of superfluous cells. During apoptosis general protein synthesis is reduced, but the synthesis of cell death proteins is enhanced. Selective translation has been attributed to modification of the protein synthesis machinery to disrupt cap-dependent mRNA translation and induce a cap-independent mechanism. We have previously shown that disruption of the balance between cap-dependent and cap-independent C. elegans eIF4G isoforms (IFG-1 p170 and p130) by RNA interference promotes apoptosis in developing oocytes. Germ cell apoptosis was accompanied by the appearance of the Apaf-1 homolog, CED-4. Here we show that IFG-1 p170 is a native substrate of the worm executioner caspase, CED-3, just as mammalian eIF4GI is cleaved by caspase-3. Loss of Bcl-2 function (ced-9ts) in worms induced p170 cleavage in vivo, coincident with extensive germ cell apoptosis. Truncation of IFG-1 occurred at a single site that separates the cap-binding and ribosome-associated domains. Site-directed mutagenesis indicated that CED-3 processes IFG-1 at a non-canonical motif, TTTD(456). Coincidentally, the recognition site was located 65 amino acids downstream of the newly mapped IFG-1 p130 start site suggesting that both forms support cap-independent initiation. Genetic evidence confirmed that apoptosis induced by loss of ifg-1 p170 mRNA was caspase (ced-3) and apoptosome (ced-4/Apaf-1) dependent. These findings support a new paradigm in which modal changes in protein synthesis act as a physiological signal to initiate cell death, rather than occur merely as downstream consequences of the apoptotic event.     

Genetic control of programmed cell death in the nematode C. elegans

The wild-type functions of the genes ced-3 and ced-4 are required for the initiation of programmed cell deaths in the nematode Caenorhabditis elegans. The reduction or loss of ced-3 or ced-4 function results in a transformation in the fates of cells that normally die; in ced-3 or ced-4 mutants, such cells instead survive and differentiate, adopting fates that in the wild type and associated with other cells. ced-3 and ced-4 mutants appear grossly normal in morphology and behavior, indicating that programmed cell death is not an essential aspect of nematode development. The genes ced-3 and ced-4 define the first known step of a developmental pathway for programmed cell death, suggesting that these genes may be involved in determining which cells die during C. elegans development.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.