The Caenorhabditis elegans pvl-5 gene protects hypodermal cells from ced-3-dependent, ced-4-independent cell death

Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. Here we describe pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway.     

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.     

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.     

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.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

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.     

Anti-apoptotic genes of baculoviruses

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.     

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.     

Apoptosis: a process with a (beta)NAC for complexity

Most programmed cell deaths in the nematode C. elegans require ced-3 caspase activity. In a recent paper, reveal a new C. elegans death inhibitor, icd-1, whose loss can promote apoptosis independently of ced-3.     

Role of early and late replication events in induction of apoptosis by baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

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.     

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.     

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 .     

Mutational analysis of the interacting cell death regulators CED-9 and CED-4

The genes ced-3, ced-4 and ced-9 are central components in the cell death pathway of the nematode C. elegans. Ced-9, which functions to inhibit cell death, is homologous to the Bcl-2 family of mammalian anti-apoptotic genes. The ced-3 gene encodes a protein homologous to the caspases, a family of cysteine proteases involved in the execution of programmed cell death. It has recently been demonstrated that CED-4, an inducer of apoptosis for which no mammalian equivalent has been reported, can interact with CED-9 and Bcl-x(L). Here we confirm that CED-9 and CED-4 interact and using a series of deletion mutants, demonstrate that only short N-terminal deletions are tolerated in each molecule without loss-of-interaction. Two loss-of-function point mutations in different regions of CED-4 also lead to a significant loss of interaction suggesting further that the relevant interaction domains are not short linear sequences, but rather, are formed by more complex structural determinants in each molecule. Furthermore, we demonstrate that CED-4 not only interacts with Bcl-x(L) but also with its homologue, Bcl-2, and that the unstructured loop region present in Bcl-x(L) and Bcl-2 can regulate the CED-4 interaction. Lastly, we show that a BH3 peptide that can inhibit Bcl-2 family interactions also inhibits the interaction between Bcl-x(L) and CED-4.     

The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death

Mutations in the genes ced-3 and ced-4 prevent almost all of the programmed cell deaths that occur during Caenorhabditis elegans development. To determine the sites of action of these two genes, we performed genetic mosaic analyses. We generated C. elegans animals that carried a free chromosomal duplication bearing either ced-3(+) or ced-4(+) in an otherwise homozygous ced-3 or ced-4 genetic background. We used other genes on the duplication as markers to identify genetic mosaic animals in which the duplication was present in some but not all cells. The patterns of cell death survivors in these mosaic animals indicated that the products of both ced-3 and ced-4 function within dying cells to cause cell death.     

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.     

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.