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.     

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.     

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.     

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.     

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly.     

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.     

Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1's proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.     

Men are but worms: neuronal cell death in C elegans and vertebrates

Awarding the 2002 Nobel Prize in Physiology or Medicine to Sydney Brenner, H Robert Horvitz, and John E Sulston for 'their discoveries concerning the genetic regulation of organ development and programmed cell death (PCD)' highlights the significant contribution that the study of experimental organisms, such as the nematode Caenorhabditis elegans, has made to our understanding of human physiology and pathophysiology. Their studies of lineage determination in worms established the 'central dogma' of apoptosis: The BH3-only protein EGL-1 is induced in cells destined to die, interacts with the BCL-2-like inhibitor CED-9, displacing the adaptor CED-4, which then promotes activation of the caspase CED-3. The vast majority of cells undergoing PCD during development in C. elegans, as in vertebrates, are neurons. Accordingly, the genetic regulation of apoptosis is strikingly similar in nematode and vertebrate neurons. This review summarizes these similarities - and the important differences - in the molecular mechanisms responsible for neuronal PCD in C. elegans and vertebrates, and examines the implications that our understanding of physiological neuronal apoptosis may have for the diagnosis and treatment of acute and chronic human neurodegenerative disorders.     

Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae

Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.     

Programmed cell death: a missing link is found

Two families of proteins have advanced our understanding of the molecular basis of programmed cell death (PCD) in animal cells - the caspases and Bcl-2-related proteins. While caspases lie at the heart of the death programme, Bcl-2-related proteins act as key intracellular regulators. Although there has been considerable progress in elucidating the biochemical functions of caspases, how Bcl-2-related proteins regulate caspase activation and thereby PCD, has remained a mystery. One key to resolving this mystery seems to lie with a new third family of proteins related to the Caenorhabditis elegans cell-death protein CED-4, which connects Bcl-2-related proteins to caspases. An important step in defining this new family has been made by the identification of a human CED-4 homologue.     

CED-9及其在细胞凋亡中的调控机制

细胞凋亡与生物体发育、疾病等息息相关,已成为近年来生命科学领域的研究热点之一。线虫作为一种模式生物是动物细胞凋亡研究的极好材料。CED-9是Bcl-2抗凋亡蛋白家族中的成员,是调控线虫细胞凋亡的关键蛋白质。与Bcl-2家族中的其他抗凋亡蛋白相比,它具有自身独特的结构特点。通过与促凋亡蛋白CED-4结合,对CED-4在细胞内的定位产生影响,从而改变下游的CED-3的活性,最终调控细胞凋亡。同时,CED-9自身的活性受到另一种促凋亡蛋白EGL-1的调控,EGL—1诱导CED-9的构象发生变化,两者形成稳定的复合物,同时CED-4从CED-9上脱离并释放到细胞质中,进而激活CED-3,使细胞发生凋亡。目前,从线虫中克隆出来的ced-9基因转入其他生物比如动物细胞、高等植物和酵母中进行研究。在论述CED-9的独特结构的基础上,进一步阐述了CED-9对CED-4在细胞内定位的影响,与EGL-1、CED-4相互作用机制,以及近年来它在高等植物和酵母中的研究进展。     

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.     

Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.     

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.     

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 Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast

A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.     

BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons

The Bcl-2 family of proteins are key regulators of programmed cell death. A distinct subfamily of BH3-only molecules has been identified, but their exact mechanism of action remains unclear. Here we show that the BH3-only Bcl-2 family members, Dp5/Hrk and Bim, are induced upstream of the Bax checkpoint in neuronal apoptosis in a manner that shows significant dependence on JNK signaling. We also show that Dp5 and other BH3-only proteins kill cerebellar granule neurons in a Bax-dependent manner. These studies demonstrate that BH3-only members do not act independently in their proapoptotic activities but rather require the action of multidomain proapoptotic Bcl-2 family members to produce cell death.     

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 C. elegans B-cell lymphoma 2 (Bcl-2) homolog cell death abnormal 9 (CED-9) associates with and remodels LIPID membranes

Bcl-2 proteins associate with and remodel mitochondria to regulate apoptosis. While the C. elegans Bcl-2 homolog CED-9 constitutively associates with mitochondria, it is unclear whether or not this association reflects an innate ability of CED-9 to directly remodel mitochondrial membranes. To address this question, we have characterized the effects of recombinantly expressed and purified CED-9 on synthetic lipid vesicles. We found that CED-9 associates with anionic lipid vesicles at neutral pH, and that association can occur independently of the C-terminal transmembrane domain. Membrane association changes the environment of CED-9 tryptophans and results in an apparent increase in α-helical structure. Upon association, CED-9 alters the permeability of membranes resulting in leakage of encapsulated dyes. Furthermore, this membrane remodeling promotes membrane fusion upon protonation of CED-9. Bypass of this protonation trigger can be achieved by mutating two conserved glutamates (E187K/E190K) or removing the N-terminal 67 residues. Together, these in vitro results suggest that CED-9 retains the amphitropic ability of mammalian Bcl-2 proteins to associate with cellular membranes. We therefore discuss the possibility that CED-9 and other Bcl-2 homologs localize at mitochondria to regulate mitochondrial homeostasis by either modulating mitochondrial membrane permeability or fusion.     

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.