InteractiveFly: GeneBrief

Death caspase-1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Death caspase-1

Synonyms -

Cytological map position -

Function - protease

Keyword(s) - apoptosis - programmed cell death - an effector caspase

Symbol - Dcp-1

FlyBase ID:FBgn0010509

Genetic map position -

Classification - ICE/CED-3 protease

Cellular location - cytoplasmic



NCBI links: | Entrez Gene
Recent literature
Sudmeier, L. J., Howard, S. P. and Ganetzky, B. (2015). A Drosophila model to investigate the neurotoxic side effects of radiation exposure. Dis Model Mech 8: 669-677. PubMed ID: 26092528
Summary:
Children undergoing cranial radiation therapy (CRT) for CNS malignancies are at increased risk for neurological deficits later in life. Using Drosophila as a model, wild-type third-instar larvae were irradiated with single doses of gamma-radiation, and the percentage that survived to adulthood was determined. Motor function of surviving adults was examined with a climbing assay, and longevity was assessed by measuring lifespan. Neuronal cell death was assayed by using immunohistochemistry in adult brains. Irradiating late third-instar larvae at a dose of 20 Gy or higher impaired the motor activity of surviving adults. A dose of 40 Gy or higher resulted in a precipitous reduction in the percentage of larvae that survive to adulthood. A dose-dependent decrease in adult longevity was paralleled by a dose-dependent increase in activated Death caspase-1 (Dcp1) in adult brains. Survival to adulthood and adult lifespan were more severely impaired with decreasing larval age at the time of irradiation. Differences in genotype confered phenotypic differences in radio-sensitivity for developmental survival and motor function. This work demonstrates the usefulness of Drosophila to model the toxic effects of radiation during development.
BIOLOGICAL OVERVIEW

Death caspase-1 (Dcp-1) is the first known Drosophila member of the caspase family of ICE/CED-3 proteases thought to play a role in apoptosis or programmed cell death. ICE was originally described as the cysteine protease required for cleavage of pro-interleukin-1ß in order to generate the active cytokine. CED-3 is a C. elegans cell death gene, with homology to mammalian ICE (Yuan, 1993). The term caspase is based on two catalytic properties of these enzymes. The "c" refers to a cysteine protease mechanism, and "aspase" refers to the group's ability to cleave aspartic acid, the most distinctive catalytic feature of this protease family. Each of these enzymes is synthesized as a proenzyme, proteolytically activated to form a heterodimeric catalytic domain. To date, ten homologs in humans have been discovered (Alnemri, 1996).

Three apoptotic activators (Reaper, Wrinkled/Head involution defective and Grim) have been identified in Drosophila. All three possess death domains, identifying them as proteins that act as mediators between different signaling pathways and the cell death program. The products of these genes appear to activate one or more caspases, because cell killing by Reaper, HID and Grim is blocked by the baculovirus protein p35, a specific inhibitor of caspases (Song, 1997 and references).

Dcp-1 is also capable of inducing cell death. The gene was expressed in several mammalian cell lines. Cells expressing Dcp-1 display the typical apoptotic morphology, such as condensed, rounded cell morphology and severe membrane blebing. A cell-free apoptosis system was used to investigate apoptosis-like nuclear events. In this system, Dcp-1 treatment results in fragmentation of chromosomal DNA that displays the characteristic apoptotic DNA ladder. It appears that Dcp-1 is able to engage at least part of the apoptotic program in mammalian cells (Song, 1997).

No significant abnormalities in the pattern of cell death are seen in dcp-1 mutants. Either there is sufficient Dcp-1 protein encoded maternally, or there are additional caspases performing a redundent function. However, dcp-1 mutation causes lethality during larval stages. Although most of the dcp-1 mutants die before the third instar larval stage, some reach that stage and display several abnormalities. Mutant larvae lack imaginal discs and gonads. In addition, they have fragile trachea. However, the most prominent phenotype found in these larvae is the presence of melanotic tumors located in various parts of the body. Melanotic tumors can result from either the overproliferation of blood cells or from an immune response toward abnormal cells and tissues in the larva. In dcp-1 mutants, there is no evidence for hyperplasia of the lymph glands or overproliferation of blood cells. This suggests an immune reaction toward abnormal tissues or cells, possible resulting from a defect in the ability to carry out cell death (Song, 1997).

The cytoplasmic region of Fas, a mammalian death factor receptor, shares a limited homology with Reaper, an apoptosis-inducing protein in Drosophila. Expression in Drosophila cells of either the Fas cytoplasmic region (FasC) or reaper causes cell death. The death process induced by FasC or reaper is inhibited by crmA or p35, suggesting that in both cases the death process is mediated by caspase-like proteases. Both Ac-YVAD aldehyde and Ac-DEVD aldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases, respectively, inhibited the FasC-induced death of Drosophila cells. However, the cell death induced by Reaper is inhibited by Ac-DEVD aldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activity that preferentially recognizes the YVAD sequence gradually increases in the cytosolic fraction of the FasC-activated cells, whereas the caspase 3-like protease activity recognizing the DEVD sequence is observed in the Reaper-activated cells. Partial purification and biochemical characterization of the proteases indicates that there are at least three distinct caspase-like proteases in Drosophila cells that are differentially activated by FasC and Reaper. The conservation of the Fas-death signaling pathway in Drosophila cells, which is distinct from that for Reaper, may indicate that cell death in Drosophila is controlled not only by the Reaper suicide gene, but also by a Fas-like killer gene (Kondo, 1997a).

The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. This shows that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 leads to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, SesB was found to suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A rescues the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, it was found that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo (DeVorkin, 2014).

The results reveal that starvation-induced autophagic flux occurs in both midstage egg chambers that have not entered the degeneration process as well as in those that are undergoing cell death. Furthermore, it was found that the effector caspase Dcp-1 is required for autophagic flux in degenerating midstage egg chambers in addition to its role in cell death. One mechanism of Dcp-1-induced autophagic flux is mediated through SesB. In humans, there are four mitochondrial ANT isoforms, each with a tissue-specific distribution and different roles in apoptosis. Adenine nucleotide translocase family ANT1 and ANT3 were proposed to be proapoptotic, whereas ANT2 and ANT4 were shown to be antiapoptotic (Brenner, 2011). However, the roles of mammalian ANT proteins in autophagy have yet to be characterized. The data show that reduced Dcp-1 leads to increased levels of SesB protein in fed and starvation conditions during Drosophila oogenesis and in Drosophila cultured cells. No significant change was observed in SesB transcript levels in fed conditions or after 4 h of starvation, but a significant increase was observed in cells after 2 h of starvation. This finding suggests that a transcription-related mechanism may play some role in the observed cellular response but is not sufficient to account for all of the observed changes in protein levels. Although Dcp-1 does not cleave SesB, the proform of Dcp-1 interacts with SesB, and it is predicted that this interaction regulates the stability of SesB. It was also found that SesB is required to suppress autophagic flux during midoogenesis even under nutrient-rich conditions, and reduction of SesB in Dcp-1Prev1 flies rescues the autophagic defect after starvation. This is the first study showing that an ANT functions as a negative regulator of autophagy (DeVorkin, 2014).

The Drosophila genome encodes seven caspases, and to date, only the initiator caspase Dronc and the effector caspase Drice have been shown to localize to the mitochondria (Dorstyn, 2002). In mammalian cells, caspases have been detected at the mitochondria during apoptosis; however, the role of caspases at the mitochondria, especially under nonapoptotic conditions, is poorly understood. The current results demonstrate that Dcp-1 localizes to the mitochondria where it functions to maintain the mitochondrial network morphology. Under nutrient-rich conditions, nondegenerating midstage egg chambers from Dcp-1Prev1 flies contained mitochondria that appeared elongated and overly connected, and ovaries contained increased ATP levels, indicating that Dcp-1 normally functions to negatively regulate mitochondrial dynamics and ATP levels. Consistent with these findings, overexpression of the caspase inhibitor p35 in the amnioserosa suppressed the transition of mitochondria from a tubular to a fragmented state during delamination, further suggesting that inhibition of caspases hinders normal mitochondrial dynamics (DeVorkin, 2014).

Dcp-1 acts to finely tune the apoptotic process, and cell death only occurs when caspase activity reaches a certain apoptotic threshold. Effector caspases involved in nonapoptotic processes may be restricted in time or space to regulate caspase activity. As Dcp-1 functions not only in autophagy and apoptosis but also at the mitochondria to regulate mitochondrial morphology and ATP levels, one question that remains is to how the activity of Dcp-1 is regulated. As Dcp-1 has autocatalytic activity, perhaps Dcp-1 is sequestered in mitochondria to prevent its full activation. Mitochondrial localized mammalian pro-Caspase 3 and 9 are S-nitrosylated in their catalytic active site, leading to the inhibition of their activity. Perhaps mitochondrial Dcp-1 is also S-nitrosylated, serving to limit Dcp-1's activity. In addition, mammalian Hsp60 and Hsp10 were shown to interact with mitochondrial localized pro-Caspase 3 in which they function to accelerate pro-Caspase 3 activation after the induction of apoptosis. Perhaps Dcp-1 associates with Drosophila Hsp60 or Hsp10 in the mitochondria to regulate its mitochondrial related functions. However, further studies are required to identify upstream regulators of Dcp-1 that regulate its mitochondrial, autophagic, and apoptotic functions (DeVorkin, 2014).

Effector caspases are the main executioners of apoptotic cell death; however, it is becoming increasingly evident that caspases have nonapoptotic functions in differentiation, proliferation, cytokine production, and cell survival. For example, Caspase 3 was shown to regulate tumor cell repopulation in vitro and in vivo, and it was also shown to be required for skeletal muscle and macrophage differentiation. In Drosophila, the initiator caspase Dronc maintains neural stem cell homeostasis by binding to Numb in a noncatalytic, nonapoptotic manner to regulate its activity (Ouyang, 2011). In addition, Dcp-1 is required for neuromuscular degeneration in a nonapoptotic manner (Keller, 2011). The current results show that Dcp-1 also has a nonapoptotic role during oogenesis, in which it is required to maintain mitochondrial physiology under basal conditions. Loss of Dcp-1 alters this physiology, leading to increased SesB and ATP levels that in part prevent the induction of autophagic flux after starvation. These data support the notion that caspases play a much more diverse role than previously known and that the underlying mechanisms should be better understood to appreciate the full impact of apoptosis pathway modulation for treatment in human pathologies (DeVorkin, 2004).


REGULATION

Transcriptional Regulation

Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).

The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).

In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).

This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).

When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).

Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).

Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).

It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).

How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).

This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).

Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling; Brahma is regulated by caspase-dependent cleavage

Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. This study shows that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex, and the Hippo (Hpo) signaling pathway directly restricts ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promotes ISC proliferation. These findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation (Jin, 2013).

SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation. Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance. The current study suggests that the chromatin remodeling activity of Brm complex is required for the proliferation and differentiation of Drosophila ISCs. Based on these findings, it is proposed that Brm is critical for maintaining Drosophila intestinal homeostasis. High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels result in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of Drosophila homologs BRG1 and BRM are also detected in adult tissues. BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types. These observations indicate that Brm may act similarly to BRG1 and BRM in controlling proliferation and differentiation (Jin, 2013).

The Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki and inducing activation of pro-apoptotic genes such as caspases directly. This study has identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as diap1 that inhibits Hpo-induced caspase activity cannot be further activated. Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway. The knockdown of Yki in ISCs did not cause any phenotype in the midgut, suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd, which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (Oh, 2013). These results support the model developed in this paper (Jin, 2013).

The current results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating Drosophila caspases to cleave Brm at the D718 site and inhibiting downstream target gene diap1 transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation. One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing BrmD718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of BrmD718A completely rescues the impairment of Hpo-induced ISC proliferation. D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. This study has defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity (Jin, 2013).

It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, attempts were made to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs. The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis, suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between Drosophila and mammals (Jin, 2013).

This study provides evidence that the Brm complex plays an important role in Drosophila ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. The findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma. Thus, this study that has implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets (Jin, 2013).

Protein Interactions

A specific inhibitor of mammalian CPP-32 caspase, Ac-DEVD-CHO, completely inhibits Poly(adenosine diphosphate-ribose) (PARP) cleavage by DCP-1, whereas the ICE-specific inhibitor Ac-YVAD-CHO is ineffective. Therefore, DCP-1 is biochemically more closely related to caspases CED-2 and CPP-32 than to ICE. DCP-1 also cleaves p35 in an identical manner to that of CED-3 (Song, 1997).

Expression of the cell death regulatory protein Reaper (RPR) in the developing Drosophila eye results in a smaller than normal eye, owing to excessive cell death. Mutations in thread (th) are dominant enhancers of RPR-induced cell death. thread encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1/Thread). Overexpression in the eye of DIAP1 or a related protein, DIAP2, suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeat region, a repeat motif found in both viral and cellular proteins associated with death prevention (Hay, 1995).

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, Drosophila and human genes that encode IAP-like proteins (dILP and hILP) have been isolated. Like baculovirus IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis (Ducket, 1996).

ced-9 (Drosophila homolog: death executioner Bcl-2 homologue), a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells induced by reaper, a Drosophila cell-death gene, is partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death (Hisahara, 1998).

Site-specific proteases play critical roles in regulating many cellular processes. To identify novel site-specific proteases, their regulators, and substrates, a general reporter system in Saccharomyces cerevisiae has been designed in which a transcription factor is linked to the intracellular domain of a transmembrane protein by protease cleavage sites. Here, the efficacy of this approach has been explored by using as a model, the caspases, a family of aspartate-specific cysteine proteases. Introduction of an active caspase into cells that express a caspase-cleavable reporter results in the release of the transcription factor from the membrane and subsequent activation of a nuclear reporter. Known caspases activate the reporter; an activator of caspase activity stimulates reporter activation in the presence of an otherwise inactive caspase, and caspase inhibitors suppress caspase-dependent reporter activity. Although low or moderate levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. This observation has been exploited to isolate clones from a Drosophila embryo cDNA library that block DCP-1 caspase-dependent yeast cell death. Among these clones, the known cell death inhibitor DIAP1 has been identified. Using bacterially synthesized proteins, it has been shown that glutathione S-transferase-DIAP1 directly inhibits DCP-1 caspase activity but that it has minimal effect on the activity of a predomainless version of a second Drosophila caspase, drICE (Hawkins, 1999).

Biochemical and genetic interactions between DCP-1 and Grim

In Drosophila, the induction of apoptosis requires three closely linked genes, reaper, head involution defective, and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. DCP-1 has a substrate specificity that is remarkably similar to that of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein is able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter results in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) has little effect. However, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina shows no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 does not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhance the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo (Song, 2000).

The proapoptotic proteins encoded by rpr, hid, and grim all require caspase activity to kill cells. Whether coexpression of caspases and these proapoptotic genes could lead to significantly enhanced cell killing was investigated. For this purpose, flies carrying GMR-rpr, GMR-hid, and GMR-grim were crossed to GMR-fl-dcp-1 and GMR-fl-drICE flies. Two different GMR-fl-dcp-1 transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, with identical results. Likewise, two GMR-fl-drICE transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, again with identical results. Flies carrying one copy of GMR-fl-dcp-1 or GMR-fl-drICE have almost normal eye morphology. Flies transgenic for GMR-hid1M, GMR-grim, or GMR-rpr46 have a mild but easily detectable eye phenotype. The coexpression of hid and full-length drICE produces no obvious enhancement of the eye phenotype, but rather an additive effect of the two transgenes. Also, the expression of hid together with full-length dcp-1 enhanced the eye phenotype only weakly, comparable to what is seen for coexpression of many other proapoptotic gene combinations. In stark contrast, expression of either rpr or grim together with GMR-fl-dcp-1 yields a dramatically enhanced eye phenotype that cannot be simply explained by additive effects. rpr produces a stronger effect than grim. The expression of rpr also enhances the eye phenotype of GMR-fl-drICE flies, whereas grim is not very effective. This finding is consistent with the observation that drICE is activated in rpr-transfected S2 cells. Among the different cell types of the Drosophila retina, the pigment cells appear to be particularly sensitive to DCP-1. Both the truncated and the full-length DCP-1 cause pigment cell death. Judging by the complete loss of eye color, all pigment cells are eliminated in flies that coexpress DCP-1 with either rpr and grim. In order to further investigate the specificity of this interaction, GMR-fl-dcp-1 flies were also crossed to a transgenic line with strong hid expression: GMR-hid 10 flies. Again, the eye phenotype observed for this combination is not significantly enhanced. Overall, rpr and grim were found to interact with dcp-1 much more strongly than hid and interact more effectively with dcp-1 than with drICE. Taken together, these observations suggest that dcp-1 is rate limiting for cell killing by rpr and grim, but not hid. Therefore, it is proposed that rpr and grim function upstream of dcp-1 in vivo (Song, 2000).

These results indicate that Reaper and Grim, but not Hid, can lead to DCP-1 activation. Several other observations also indicate that rpr and grim have cell killing properties that are distinct from those of hid. For example, the Ras/MAPK pathway inhibits hid-induced cell death but has no effect on rpr- or grim-induced death (Bergmann, 1998). In addition, mutations in the diap1 gene of Drosophila have been isolated that enhance rpr- and grim-induced cell killing but suppress hid-induced cell killing (J. Agapite, K. McCall, and H. Steller, unpublished data cited in Song, 2000). The easiest interpretation of all these observations is that rpr and grim kill cells by activating the same (set of) caspases and that hid activates a distinct caspase. Since it has been recently shown that rpr, hid, and grim induce cell death by inhibiting the antiapoptotic activity of diap1, diap1 must control at least two distinct caspase pathways. According to this model, Reaper and Grim and HID would interact selectively with specific DIAP1-(pro)caspase complexes. The binding of Reaper, Grim, or HID to the relevant DIAP1-(pro)caspase complex is thought to result in caspase activation. This model is consistent with a variety of findings from both invertebrate and vertebrate systems. However, the possibility that rpr and grim may also activate DCP-1 through a DIAP1-independent pathway cannot be ruled out. Although several Drosophila caspases have been described, these results indicate that additional caspases, in particular ones activated by HID, remain to be identified (Song, 2000).

Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival

Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. This study used a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man (Creagh, 2009).

At present, three substrates for Drosophila caspases, DIAP1, Lamin DmO, and the Drosophila Apaf-1 homologue, ARK, have been identified. A proteomics-based screen resulted in the identification of 14 proteins that underwent caspase-dependent alterations to their relative mobilities on two-dimensional (2D) SDS-PAGE gels. Two of these substrates were cloned and confirmed that they are efficiently cleaved by the Drosophila caspases DrICE and DCP-1. Bicaudal and its human homologue, βNAC, appear to be essential for cell proliferation and cell survival as RNAi-mediated silencing of the expression of these proteins resulted in a block to cell division, followed by spontaneous apoptosis. This suggests that, as well as targeting substrate proteins that contribute to the ordered destruction of the cell, caspases also inactivate key proteins such as βNAC that are essential for cell survival (Creagh, 2009).

Interestingly, βNAC has also been implicated as a negative regulator of programmed cell death in the nematode C. elegans and ablation of this gene product results in massive and unscheduled apoptosis in developing worm embryos. NAC functions to bind short nascent polypeptides as they emerge from the ribosome. The latter event prevents inappropriate interactions with cellular proteins and non-specific binding by the signal recognition particle and consequent targeting to the ER. NAC also prevents the targeting of non-translating ribosomes to the ER. This fundamental role of NAC is reflected in the catastrophic phenotype of null mutations affecting the βNAC-coding sequence gene in a range of species. Loss of βNAC in developing mice leads to post-implantation lethality and mutation of Drosophila bicaudal promotes developmental arrest, which is associated with duplication of the posterior embryonic regions in the place of the anterior embryonic segments (Markesich, 2000). RNAi-mediated silencing of the C. elegans βNAC homologue, ICD-1, also results in developmental arrest associated with massive cell death (Bloss, 2000). Thus, the disablement of βNAC function through caspase-dependent proteolysis may contribute substantially to cellular demise (Creagh, 2009).


DEVELOPMENTAL BIOLOGY

Embryonic

Preblastoderm embryos, prior to zygotic gene transcription, contain large and uniform amounts of DCP-1 mRNA. Therefore dcp-1 is maternally expressed. At later stages, transcripts continue to be present throughout the embryo. Toward the end of embryogenesis, dcp-1 expression becomes more restricted. Some regions of the embryo, including the head, some cells within the central nervous system, the developing gonads, and portions of the gut, contain higher levels of DCP-1 mRNA. Strong expression can be seen in cells along the midline of the central nervous system (Song, 1997).

Temporal and molecular hierarchies in the link between death, delamination and dorsal closure

One process that occurs during dorsal closure is cell delamination, the seemingly stochastic, rapid apical constriction of cells that culminates in their extrusion from the ectodermal layer. Their number (between 10% and 30%) and position is variable and unpredictable. Extruded cells are engulfed by hemocytes. This behaviour is thought to contribute to up to one-third of the force generated in the amnioserosa for dorsal closure and exhibits a preferential occurrence at the anterior canthus. Its suppression by caspase inhibition has led to the suggestion that ‘apoptosis’ triggers delamination. This study explored whether cell delamination in the amnioserosa, a seemingly stochastic event that results in the extrusion of a small fraction of cells and known to provide a force for dorsal closure, is contingent upon the receipt of an apoptotic signal. Through the analysis of mutant combinations and the profiling of apoptotic signals in situ, spatial, temporal and molecular hierarchies were establish in the link between death and delamination. Although an apoptotic signal is necessary and sufficient to provide cell-autonomous instructions for delamination, its induction during natural delamination occurs downstream of mitochondrial fragmentation. It was further shown that apoptotic regulators can influence both delamination and dorsal closure cell non-autonomously, presumably by influencing tissue mechanics. The spatial heterogeneities in delamination frequency and mitochondrial morphology suggest that mechanical stresses may underlie the activation of the apoptotic cascade through their influence on mitochondrial dynamics. These results document the temporal propagation of an apoptotic signal in the context of cell behaviours that accomplish morphogenesis during development. They highlight the importance of mitochondrial dynamics and tissue mechanics in its regulation. Together, they provide novel insights into how apoptotic signals can be deployed to pattern tissues (Muliyil, 2011).

These results establish the necessity and utility of apoptotic signals in driving cellular delamination in the amnioserosa and in patterning the spatiotemporal dynamics of closure. They invoke the induction of pro-apoptotic genes and thus go beyond earlier observations that inferred the role of an apoptotic cascade through the effects of caspase suppression. The results also provide mechanistic insights into the mode of action of the apoptotic cascade by demonstrating cell-autonomous effects of pro-apoptotic genes and caspase activity (DIAP1 overexpression) on the rates of apical constriction. This suggests that apoptotic regulators must regulate cytoskeletal organisation and cell mechanics. A question that arises is whether both classes of regulators function in the linear hierarchy that was delineated or whether functions independent of the apoptotic cascade contribute to their role in driving delamination. The analysis of the molecular hierarchy shows that caspase activation induced by reaper upregulation is a necessary downstream event. Its late activation in delaminating cells, however, raises the issue of whether it is necessary for apical constriction or just for cell extrusion. Although the complete suppression of delamination by p35 overexpression precludes the analysis of constriction rates, this analysis reveals an absence of rosette patterns that characterise delamination rather than the presence of constricted cells that fail to extrude. This suggests that caspase activation must also be necessary for apical constriction. One explanation is that this marked upregulation of the cascade triggers the almost abrupt transition in cell behaviour, characterised by the rapid fall in cell area in a delaminating cell. This is consistent with the higher rates of decrease in cell area with increases in the amounts of caspases/Reaper. Although the phenotypes associated with DIAP1 overexpression also support a role for caspases in cell constriction, caspase-independent functions of DIAP1 have been reported to influence actin organisation in Drosophila border cells. Thus, apoptotic signals must impinge on a distinct set of regulators of the actin cytoskeleton to facilitate apical constriction and tissue contraction. Caspase activation may also regulate adhesion to facilitate extrusion. Indeed, the adherens junction component armadillo/β-catenin is a caspase substrate during cell death in Drosophila and mammals (Muliyil, 2011).

The results also provide evidence for cell non-autonomous regulation of delamination by components of the apoptotic cascade. Further support for this comes from ongoing observations that caspase inhibition influences actin organisation in the entire amnioserosa. It is speculated that the influence of low undetectable levels of caspase activation not restricted to delaminating cells, regulates tissue mechanics in the amnioserosa and through it also influences cell delamination. The results also show that non-autonomous influences on delamination can originate in the epidermis. Uncovering the molecular players that underlie both autonomous and non-autonomous effects of apoptotic signals on cell behaviour will be interesting avenues to pursue (Muliyil, 2011).

Temporal and epistatic analysis position mitochondrial fragmentation upstream of the induction of pro-apoptotic genes and caspase activation both during delamination and degeneration. This is the first time that the sequence of propagation of an apoptotic signal has been elucidated in the context of cell behaviour in vivo. Mitochondrial fragmentation is thus the earliest indicator of the cellular commitment to delamination. Other studies have placed mitochondrial fragmentation downstream of the pro-apoptotic genes reaper and hid. What, if not the pro-apoptotic genes, then triggers mitochondrial fragmentation in the amnioserosa? Two recent reports have documented the ability of chemical and radiation injuries to trigger changes in mitochondrial morphology and lead to the induction of apoptosis. An attractive candidate for the trigger in the amnioserosa, consistent with the spatial heterogeneities in delamination frequency and mitochondrial morphology observed, is mechanical stress. Two sets of observations support this. First, not all cells that overexpress pro-apoptotic genes delaminate, and the anterior predominance of such events is maintained. This suggests that although an apoptotic signal is necessary, it must cooperate with other permissive signals to accomplish delamination. Second, the studies of native dorsal closure uncover spatial heterogeneities in mitochondrial morphology. Two features characterise this heterogeneity: (1) the early abundance of cells with predominantly fragmented mitochondria in the anterior AS and (2) their delayed transition to tubular/hyperfused morphologies prior to degeneration compared with the posterior. It has been suggested in a different context that low levels of chemical stress can induce hyperfusion as a means of countering stress (through optimisation of mitochondrial ATP production), whereas higher magnitudes of stress lead to fragmentation and apoptosis. A similar reasoning (with the substitution of chemical stresses by mechanical stresses) might underlie the spatial heterogeneities in delamination frequency. Specifically, high magnitudes of stress locally (from head involution) might be responsible for increased mitochondrial fragmentation and subsequent delamination, whereas prolonged lower levels of stresses (from the leading edge) may drive hyperfusion and subsequent degeneration of the amnioserosa. Adhesion anisotropies resulting from differences in the substrate (yolk anteriorly and hindgut posteriorly) could additionally contribute to the force anisotropies between the anterior and posterior amnioserosa (Muliyil, 2011).

Taken together, the results reveal that apoptotic regulators contribute multiple forces to dorsal closure. In the amnioserosa, they act locally to drive delamination but also globally to maintain tissue tension. The latter is attributed to the low levels of caspase activation and pro-apoptotic gene induction. This provides a permissive environment for mitochondrial fragmentation and the subsequent marked upregulation of the cascade in delaminating cells. Additionally, they contribute to forces generated in the epidermis. This is best inferred from anti-apoptotic perturbations. In hid mutants, the rates of dorsal closure are higher despite the absence of delaminations in the amnioserosa. Conversely, delamination in the amnioserosa is 'upregulated' when either caspases or hid is downregulated in the epidermis, but their effects on closure rates are different. These non-autonomous effects must reflect the feedback regulation of forces generated in the epidermis and in the amnioserosa. That multiple forces contribute to dorsal closure and can feedback regulate each other has been long appreciated. These studies identify apoptotic signals as crucial regulators of the balance of forces that drive dorsal closure. Uncovering the basis for feedback regulation and the force hierarchies that lend dorsal closure resilience will be interesting. A recent study reported on a novel, non-apoptotic role for an epidermal caspase, caspase 8: its effect on interleukin signalling resulted in the recapitulation of a wound healing response when deleted in the skin. In light of the above observations, it is interesting that in this analysis of dorsal closure, which recapitulates wound closure, some perturbations that suppress apoptosis also resulted in accelerated closure (Muliyil, 2011).

These explorations demonstrate the primacy of mitochondrial fragmentation in the induction of apoptotic signalling and uncover the complex relationships between death signals, delamination and dorsal closure. Furthermore, they illustrate how an apoptotic signal is deployed multiple times in the same tissue to accomplish heterogeneity in cell behaviour and have helped identify some of the cellular properties they modulate. Understanding the triggers for mitochondrial fragmentation and the precise outcomes and mechanisms of apoptotic signals on cell biological attributes of delaminating cells will be interesting avenues to explore (Muliyil, 2011).

Larval

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

Larval-specific tissues such as larval muscles, the midgut, and the salivary glands undergo programmed cell death during metamorphosis. Genes involved in programmed cell death were identified in these experiments. The apoptosis-activating reaper gene has previously been shown to be ecdysone-inducible, and this is reflected in the data. Expression of the Drosophila caspase-1 gene is also observed during the prepupal ecdysone pulse but not during the late larval pulse. This gene is also an activator of apoptosis, and mutants display melanotic tumors and larval lethality. Induction of a cell death inhibitor gene, thread (also known as Diap1), is observed during the late larval pulse but not the prepupal phase. The DIAP1 protein includes inhibitor-of-apoptosis (IAP) domains and has been identified as a factor that can block reaper activity. Because different tissues begin apoptosis at different stages of development, changes in the expression of inhibitors and activators of apoptosis are expected to be tissue-specific. For example, the expression profiles observed for the caspase-1 activator and the Diap1 inhibitor are those expected in tissues such as the larval salivary glands. Tissue-specific information on the induction of these genes will be important to an understanding of the coordination of apoptosis during metamorphosis (White, 1999).

Apoptosis ensures spacing pattern formation of Drosophila sensory organs

In both vertebrates and invertebrates, developing organs and tissues must be precisely patterned. One patterning mechanism is Notch/Delta-mediated lateral inhibition. Through the process of lateral inhibition, Drosophila sensory organ precursors (SOPs) are selected and sensory bristles form into a regular pattern. SOP cell fate is determined by high Delta expression and following expression of neurogenic genes like neuralized. SOP selection is spatially and temporally regulated; however, the dynamic process of precise pattern formation is not clearly understood. In this study, using live-imaging analysis, it was shown that the appearance of neuralized-positive cells is random in both timing and position. Excess neuralized-positive cells are produced by developmental errors at several steps preceding and accompanying lateral inhibition. About 20% of the neuralized-positive cells show aberrant cell characteristics and high Notch activation, which not only suppress neural differentiation but also induce caspase-dependent cell death. These cells never develop into sensory organs, nor do they disturb bristle patterning. This study reveals the incidence of developmental errors that produce excess neuralized-positive cells during sensory organ development. Notch activation in neuralized-positive cells determines aberrant cell fate and typically induces caspase-dependent cell death, as detected using SCAT3, a fluorescence resonance energy transfer (FRET) indicator for effector caspase activity. Apoptosis is utilized as a mechanism to remove cells that start neural differentiation at aberrant positions and timing and to ensure robust spacing pattern formation (Koto, 2011).

The Drosophila sensory organ is a typical model for the study of Notch/Delta-mediated lateral inhibition. Tracking the process of cell fate determination in each cell lineage is presumed to be effective in revealing the mechanisms behind precise pattern formation (Koto, 2011).

The first finding in this study is that bristle patterning starts in a random fashion: about 20% of neuralized-positive cells are fated to become aberrant SOP-like cells, and Notch signaling is involved in determining the fate of SOP-like cells. However, the mechanisms proposed for the production of SOP-like cells in previous reports do not coincide perfectly with the current observations. Previous studies showed that the conventional model of Notch/Delta-mediated lateral inhibition is not sufficient to produce the precise bristle pattern but that cell-autonomous interaction or filopodia-mediated intermittent Notch/Delta signaling makes lateral inhibition robust enough to suppress the neural differentiation of surrounding cells. In contrast, the current results suggest that lateral inhibition from adjacent SOPs is not the sole source of Notch activation in SOP-like cells, because a portion of SOP-like cells preceded the nearby SOPs. Also, SOP-like cells showed ongoing Notch activity even in the absence of adjacent SOPs. One possible explanation for these SOP-like cells failing to develop into sensory organs may be that they appear too early in the developmental time course and cannot complete the developmental program to become sensory organs in a cell-autonomous manner. In any case, the decrease in SOP-like cells in the N55e11 heterozygous mutant reliably suggests that Notch activation in cells that start neural differentiation contributes to the determination of their cell fate as aberrant SOP-like cells (Koto, 2011).

Dynamic oscillation of the Notch effector gene Hes1 has been observed in neural progenitors of the developing mouse brain with the aid of a short-half-life indicator using ubiquitinated firefly luciferase. In Drosophila sensory organ development, the technical limitations of the GFP reporter make it difficult to confirm this type of oscillation pattern in Notch signaling. However, given that Notch oscillation occurs in cells in the proneural stripe regions at the beginning of SOP selection, it is conceivable that SOP-like cells might be the product of fluctuating Notch signaling at inappropriate times, developmentally speaking (Koto, 2011).

The second finding in this study is that a program of caspase-dependent cell death specifically eliminates SOP-like cells. Ablating an incipient SOP removes Notch/Delta-mediated lateral inhibition and allows a nearby epithelial cell to become the SOP, as has also been observed in the embryonic central nervous system of grasshoppers. However, the adjacent SOP-like cells never develop into sensory organs, suggesting that the fate of SOP-like cells is irreversible. By observing the nuclear morphology along with an indicator for caspase activation, it was noted that in the process of sensory organ development, only SOP-like cells showed the typical features of programmed cell death. These results indicate that programmed cell death ensures robust pattern formation by eliminating aberrantly differentiated cells (Koto, 2011).

The significance of programmed cell death in pattern formation has been well studied, especially in the development of the fly eye. Each ommatidium is composed of eight photoreceptor neurons and six support cells, consisting of four cone cells and two primary pigment cells. Between each ommatidium, remaining cells form the interommatidial lattice. Excess pigment cells are eliminated through programmed cell death. Notch functions within the interommatidial lattice to induce cell death, and the primary pigment cells send a survival signal to adjacent cells. The life-and-death fate of interommatidial cells is decided by their position and the cells to which they are attached. In the case of sensory organ formation, Notch signaling is crucial in determining the aberrant cell fate of SOP-like cells. However, Notch activation alone seems insufficient to induce programmed cell death, because the surrounding epithelial cells do not disappear, even though they exhibit high levels of Notch activation during sensory organ development. Therefore, some factor that marks neural differentiation in SOP-like cells may be required to induce cell suicide. This study found that ectopic neuralized expression did not induce the aberrant cell fate or cell death in epithelial cells, suggesting that neuralized itself is not essential in determining the aberrant cell fate of SOP-like cells. Therefore, to determine how apoptosis is induced in SOP-like cells, the effect of Notch activation in neuralized-positive cells was examined at the one-cell stage using the temporal and regional gene expression targeting (TARGET) system with tub-GAL80ts. As reported previously, activated Notch induced the multiple-sockets phenotype. At the same time, about 50% of neuralized-positive cell lineages died, accompanied by nuclear fragmentation, causing a dramatic bald phenotype that was observed in the adult flies. These findings suggest that the combination of neural differentiation in the SOP lineage and Notch activation switches on cell death signaling. One possible future approach to searching for the killing factor expressed in SOP-like cells would be gene profiling using laser microdissection (Koto, 2011).

When the apoptotic pathway is blocked, the inhibition of cell death results in cell fate transformation. In C. elegans, cell death survivors in ced-3 mutants exhibit an ambiguous cell fate. The most disruptive alternative cell fate occurs when the remaining cells differentiate into tumor-like proliferating cells, as shown in the development of the Drosophila serotonin lineage. Under apoptosis-deficient conditions, other types of cell death occur, such as necrosis or autophagic cell death. These alternate reactions could mask the incidence of programmed cell death; therefore, it is possible that the role of the apoptotic pathway has been missed in the case of sensory organ development. This study has shown that SOP-like cells differentiate into epithelial cells when the cell death pathway is blocked. Time-lapse imaging made it possible to trace the transient fate of dying SOP-like cells, revealing the contribution of programmed cell death in the SOP selection process. Although the function of apoptosis has been emphasized in various developmental processes, the principle message is that several pathways exist to overcome the appearance of excess or aberrant cells and to make the developmental process more robust. This study reveals that programmed cell death plays an important role in overcoming innately induced developmental errors and contributing to robust neural cell selection (Koto, 2011).

Oogenesis

In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages: stage 2a/2b (germarium), stage 8 (midoogenesis), and stages 10-13 (late oogenesis). These cell deaths are subject to distinct regulatory controls, since cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. This report shows that the effector caspase Drice is activated during cell death in both mid- and late-oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice forms localized aggregates during nurse cell death in late oogenesis; however, active Drice is found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity is limited in late oogenesis, an examination was performed to see whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. Premature activation of the effector caspase, Dcp-1, results in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppresses cell death induced by Dcp-1 but has no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte (Peterson, 2003).

The cytoskeletal events induced by expression of activated Dcp-1 (tdcp-1) are significantly different from those normally seen in nurse cells during late oogenesis. Normally during stage 10B of oogenesis, actin bundles form in the cytoplasm of nurse cells, connecting the plasma membrane with the nucleus. After actin bundle formation, actin-myosin-based contraction occurs and drives nurse cell cytoplasm dumping. Expression of tdcp-1 does not lead to actin bundles or nurse cell dumping. Instead, actin forms clumps and disappears as the egg chambers degenerate. Similar cytoskeletal events have been reported for egg chambers that degenerate during stage 8 in response to other stimuli. It is possible that factors required for actin bundle formation are not expressed during midoogenesis. However, egg chambers from midway mutants that degenerate prematurely during stages 8-9 do show actin bundles. Thus, midway, which encodes an acyl coenzyme (A:diacylglycerol acyltransferase) may normally act to control the timing of actin bundle formation and nurse cell death. It has been reported that dcp-1 germline clone mutants are defective in actin bundle formation and nurse cell dumping; however, recent findings suggest that these effects are due to a neighboring gene affected by the P-element alleles and that dcp-1 activity is not required for these cytoskeletal events. Nonetheless, because nurse cell nuclear breakdown has already begun prior to dumping, it has been suggested that the cytoskeletal events are part of the nurse cell death process (Peterson, 2003).

The formation of actin bundles in the cytoplasm of dying late-stage nurse cells differs from the actin rearrangements that have been reported in apoptotic cells and shows similarity to the actin structures in autophagic MCF-7 cells. In cultured mammalian cells undergoing apoptosis, filamentous actin relocalizes to form a dense network in the cytoplasm at the base of the membrane blebs, and later disappears. However, in MCF-7 cells undergoing autophagy, actin forms fibers stretching from the nucleus to the plasma membrane, resembling the actin bundles seen in nurse cells. Autophagic cell death has been observed in groups of cells that die, such as cells of the Drosophila salivary gland during metamorphosis. Caspase activity is required for Drosophila salivary gland autophagy, but it is not known if caspase activity is regulated differently in autophagy and apoptosis. There are several modes of cell death, including deaths that show properties of both autophagy and apoptosis; nurse cell death may fall into this class. Indeed, autophagic vacuoles have been reported in nurse cells, although follicle cells appear to engulf nurse cell material as well (Peterson, 2003).

Egg chambers degenerating during midoogenesis in response to nutrient deprivation or tdcp-1 expression do not show expression of the altered form of cytochrome c, suggesting that this may be a stage-specific event. One explanation is that the factors required to alter cytochrome c may not be expressed in midoogenesis. Alternatively, a mitochondria-independent cell death pathway may be utilized in midoogenesis. The lack of cytochrome c alteration suggests that cytochrome c involvement is not necessary for Drosophila apoptosis when a high level of caspase activity is present. However, it is possible that cytochrome c plays no direct role in apoptotic signaling in Drosophila, and this antibody simply recognizes cytochrome c in dividing or otherwise altered mitochondria (Peterson, 2003).

Interestingly, egg chambers prior to stage 8 were largely resistant to any apoptotic effects of activated Dcp-1. Stage 8 has been shown to be a checkpoint stage for a number of signals, including reduced food availability, ecdysone signaling, treatment with chemicals, ectopic death of follicle cells, or abnormal egg chamber development. Because vitellogenesis begins during stage 8, it has been suggested that the state of the egg chambers is monitored before making the investment of vitellogenesis. However, it is interesting that the egg chambers prior to stage 8 are well-protected from strong death-inducing stimuli, including expression of a truncated caspase. This stage-specific protection may be the result of a high level of caspase inhibitors like IAPs early in oogenesis. Indeed, transcriptional downregulation of Drosophila IAPs during stage 8 has been reported. Overexpression of DIAP1 can indeed block cell death induced by expression of truncated Dcp-1 in midoogenesis (Peterson, 2003).

The limited activation of Drice and the unusual cytoskeletal events that normally occur in late oogenesis suggest that caspase activity is carefully controlled during nurse cell death in late oogenesis. One model to explain the observed cytoskeletal differences is that only a subset of the usual cytoskeletal targets of caspases are cleaved in late oogenesis. This limited cleavage would allow for the formation and persistence of cytoplasmic actin bundles, which are necessary for proper nurse cell cytoplasm transfer. Alternatively, the cytoskeletal events may be controlled by Damm or Decay or may be caspase-independent. However, these models would still require that the activity of Drice and Dcp-1 be curtailed to prevent disassembly of actin cytoskeleton (Peterson, 2003).

Overexpression of the caspase inhibitor DIAP1 does not affect normal nurse cell death. This suggests that nurse cell death may be caspase-independent, or utilize caspases that are not readily inhibitable by DIAP1. Alternatively, mechanisms may exist to compartmentalize caspase activation or to degrade DIAP1, even when it is overexpressed. Support for this idea comes from the observations that full-length DIAP1 does not inhibit naturally occurring cell death in the eye as well as a version of DIAP1 lacking the RING finger. The RING finger has been shown to be critical for rapid turnover of DIAP1 protein (Peterson, 2003).

The controlled caspase activation that occurs in nurse cells during late oogenesis may explain why this form of cell death is not regulated by the cell death activators: Reaper, Hid, and Grim. Reaper, Hid, and Grim induce apoptosis in many cell types by triggering the degradation of DIAP1. Embryos homozygous for the H99 chromosomal deletion, which removes reaper, hid, and grim, are completely lacking in normal programmed cell death (White, 1994). However, flies carrying H99 germline clones undergo normal nurse cell death, indicating that nurse cell death is regulated differently from the vast majority of cell deaths in Drosophila. Perhaps the Reaper, Hid, Grim/DIAP1 mechanism of apoptosis induction would not permit such localized and limited caspase activation. This regulation of caspase activity may be necessary for the systematic destruction of nurse cells while the oocyte is protected from active caspases and other dangerously cleaved proteins (Peterson, 2003).

A mitochondrial-associated link between an effector caspase and autophagic flux

It has become evident that caspases function in nonapoptotic cellular processes in addition to the canonical role for caspases in apoptotic cell death. It has been demonstrated that the Drosophila effector caspase Dcp-1 localizes to the mitochondria and positively regulates starvation-induced autophagic flux during mid-oogenesis. Loss of Dcp-1 leads to elongation of the mitochondrial network, increased levels of the adenine nucleotide translocase sesB, increased ATP levels, and a reduction in autophagy. sesB is a negative regulator of autophagic flux, and Dcp-1 interacts with sesB in a nonproteolytic manner to regulate its stability, uncovering a novel mechanism of mitochondrial associated, caspase-mediated regulation of autophagy in vivo (DeVorkin, 2014).


EFFECTS OF MUTATION

There are no significant abnormalities related to cell death in dcp-1 mutants. Thus zygotic DCP-1 function is not required for most embryonic cell deaths in Drosophila, perhaps because of the existence of additional capspases. However, because DCP-1 has significant maternal expression, it is also possible that sufficient DCP-1 protein is present during embryogenesis for cell death to occur (Song, 1997). dcp-1 mutation causes lethality during larval stages. Although most of the dcp-1 mutants die before the third instar larval stage, some reach that stage and display several abnormalities. Mutant larvae lack imaginal discs and gonads. In addition, they have fragile trachea. However, the most prominent phenotype of these larvae is the presence of melanotic tumors located in various parts of the body (Song, 1997).

During Drosophila oogenesis, nurse cells transfer their cytoplasmic contents to developing oocytes and then die. Loss of function for the dcp-1 gene, which encodes a caspase, causes female sterility by inhibiting this transfer. dcp-1- nurse cells are defective in the cytoskeletal reorganization and nuclear breakdown that normally accompany this process. Breakdown of the nuclear envelope is a central event during apoptosis and is accompanied by caspase-mediated cleavage of nuclear lamins. To address whether nuclear lamins are degraded as nurse cells become permeable, egg chambers were examined for the distribution of Lamin Dm0. Loss of Lamin Dm0 signal in control nurse cells takes place by stage 11, at which time lamin staining appears to be a diffuse cytoplasmic cloud around the nuclei. Mutant nurse cells continue to show distinct nuclear envelope staining as late as stage 14. Thus dcp-1 mutants are defective in the cleavage or dissociation, or both, of nuclear lamins. This failure in lamin breakdown is a likely cause of the defect in nuclear permeability revealed by a beta-Gal marker. Lamin breakdown is likely to be directly due to DCP-1 protease activity. Actin is localized to the plasma membrane during early stages in control and mutant egg chambers. During stage 10B in control egg chambers, actin bundles form throughout the cytoplasm, connecting the nuclei and plasma membrane. In contrast, actin in many dcp-1 mutant egg chambers remains associated with the plasma membrane, even in stage 14 egg chambers. Therefore, dcp-1 activity is required for the proper formation of cytoplasmic actin bundles in nurse cells. The dcp-1- phenotype suggests that the cytoskeletal and nuclear events in the nurse cells make use of the machinery normally associated with apoptosis and that apoptosis of the nurse cells is a necessary event for oocyte development (McCall, 1998).

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).

While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).

Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).

The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than the simple models indicate (Kiessling, 2006).

A genome-wide RNAi screen reveals multiple regulators of caspase activation

A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).

The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).

This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).

diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).

Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).

In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).

To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).

The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).

To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).

Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).

Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage-induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).

In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).

Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).

ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in this screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. A systematic study was conducted of Drosophila cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. It was discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53--as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in Drosophila cultured cells. During Drosophila oogenesis, it was found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in autophagy-related genes Atg1 and Atg7 (Atg signifies an autophagy-related gene) resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. This study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo (Hou, 2008).

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved mechanism for the degradation of long-lived proteins and organelles. During autophagy, cytoplasmic components are sequestered into double membrane structures called autophagosomes, which then fuse with lysosomes to form autolysosomes, where degradation occurs. Currently, there are 31 autophagy-related (Atg) genes in yeast, and 18 Atg proteins are essential for autophagosome formation. Most yeast Atg genes have orthologues in higher eukaryotes and encode proteins required for autophagy induction, autophagosome nucleation, expansion, and completion, and final retrieval of Atg protein complexes from mature autophagosomes (Hou, 2008).

Depending on the physiological and pathological conditions, autophagy has been shown to act as a pro-survival or pro-death mechanism in vertebrates. In the case of growth factor withdrawal, starvation, and neurodegeneration, autophagy has been shown to function in cell survival. In contrast, autophagy has been found to act as a cell death mechanism in derived cell lines where caspases or apoptotic regulators are impaired. The nature and perhaps level of the stress stimulus may also be important in determining whether autophagy promotes cell survival or cell death (Hou, 2008).

Overlaps between components in apoptosis and autophagic pathways have been described. Upstream signal transducers in apoptotic pathways, including TNF-related apoptosis-inducing ligand (TRAIL), TNF, Fas-associated protein with death domain (FADD), and death-associated protein kinase (DAPK), have been shown to play a role in autophagy regulation. In addition, two recent studies demonstrate physical and functional interactions between components of apoptosis and autophagy. First, the antiapoptosis protein, Bcl-2, suppresses autophagy through a direct interaction with Beclin 1, a protein required for autophagy. Second, Atg5, which is cleaved by calpain, associates with Bcl-XL, leading to cytochrome c release and caspase activation. Further examples and discussion of the connections between apoptosis and autophagy can be found in several recent reviews on this topic. The current findings indicate that there is a complex relationship between apoptosis and autophagy, but the regulatory mechanisms underlying the crosstalk between the two processes are still largely unknown (Hou, 2008 and references therein).

Autophagy is observed in several Drosophila tissues during development, and thus Drosophila is useful as a model to study autophagy in the context of a living organism. 14 Drosophila annotated genes share significant sequence identity with the yeast Atg genes, and, overall, eight Drosophila Atg homologues have already been shown to be required for autophagy function. In addition, recent studies demonstrated the role of autophagy in Drosophila physiological cell death. Loss of Atg genes, including Atg1, Atg2, Atg3, Atg6, Atg7, Atg8, Atg12, and Atg18, inhibit proper degradation of salivary glands during development. Overexpression of Atg1 induces premature salivary gland cell death in a caspase-independent manner). In contrast, caspase activity was required for Atg1-mediated apoptotic death in the fat body. Mutation of Atg7 results in an inhibition of DNA fragmentation in the midgut but leads to an increase of DNA fragmentation in the adult Drosophila brain. Together, these results further suggest that the mechanistic role of autophagy in cell death and the interrelations between autophagy and apoptosis may be tissue and/or context dependent (Hou, 2008).

The adult Drosophila ovary contains 15-20 ovarioles comprised of developing egg chambers, which consist of 16 germ line cells (15 nurse cells and 1 oocyte) surrounded by a layer of somatic follicle cells. The germ line cells originate from stem cells that undergo mitosis to form 16-cell cysts in a specialized region called the germarium. In the late stage of oogenesis, the nurse cells support the development of the oocyte by transferring to it their cytoplasmic contents. After this 'dumping' event, the nurse cells undergo cell death, and their remnants are engulfed by the surrounding follicle cells. In addition to this late-stage developmental cell death, egg chambers can be induced to die at two earlier stages, during germarium formation (in region 2) and mid-oogenesis, by factors such as nutrient deprivation, chemical insults, and altered hormonal signaling. In some respects, cell death during Drosophila oogenesis is similar to the death of Drosophila larval salivary glands. Both nurse cells and salivary gland cells are large and polyploid, and the entire tissues undergo cell death simultaneously. Notably, morphological features of autophagy have been described during mid-oogenesis cell death in a related species, Drosophila virilis, which suggests that the cell death process in ovaries and salivary glands share additional similarities (Hou, 2008).

Previous studies have focused on characterizing the role of autophagy genes in cell death and determining the paradoxical functions of autophagy (pro-survival and pro-death) in various cell lines and organisms. However, a systematic approach that investigates the involvement of cell death genes in starvation-induced autophagy has not been conducted. This study presents RNAi analyses to determine whether known cell death-related genes in Drosophila play a role in autophagy regulation in the lethal (2) malignant blood neoplasm (l(2)mbn) cell line. Drosophila genetics was used to investigate a role for the effector caspase death caspase-1 (Dcp-1) and the inhibitor of apoptosis (IAP) family member Bruce in autophagy regulation in vivo during Drosophila oogenesis. Further, the function was studied of autophagy genes Atg7 and Atg1 in starvation-induced germ line cell death in the Drosophila ovary (Hou, 2008).

All Reaper, Hid, Grim (RHG) family members, Rpr, Hid, Grim, and Skl, bind to Drosophila IAP-1 (DIAP1) and inhibit its antiapoptotic activities. To test whether DIAP1 (encoded by th) is a putative downstream mediator of Hid-dependent autophagy in l(2)mbn cells, dsRNA was designed specifically to target th. th-dsRNA-treated cells showed no difference in LysoTracker green (LTG) fluorescence levels compared with Hs-dsRNA (negative control)-treated cells. Interestingly, the data showed that reduced expression of Bruce, another IAP family member protein, further increased the LTG fluorescence levels after starvation treatment (confirmed using nonoverlapping dsRNAs). RNAi of Bruce expression also resulted in an increase in GFP-LC3 puncta (refering to GFP tagged microtubule associated protein 1 light chain 3B) after starvation treatment. These results suggest that Bruce, instead of DIAP1, could be the downstream target of Hid during starvation induced autophagy in l(2)mbn cells (Hou, 2008).

To investigate the requirement of caspases, the final effectors of apoptosis, in starvation-induced autophagy, gene-specific dsRNAs were designed corresponding to seven different Drosophila caspases. RNAi of just one caspase, Dcp-1, but not others resulted in a decrease in the percentage of LTGhigh cells after starvation treatment. A second dsRNA against Dcp-1, nonoverlapping with the first dsRNA, yielded a similar result. Reduction of Dcp-1 expression by RNAi was determined using QRT-PCR. Consistent with the LTG derived data, RNAi-mediated knockdown of Dcp-1 resulted in a decrease in GFP-LC3-positive cells after starvation treatment. These results indicate that Dcp-1 functions as a positive regulator of autophagy in D. melanogaster l(2)mbn cells (Hou, 2008).

Key outstanding questions that need to be addressed are how autophagy and apoptosis pathways interact with each other, and whether common regulatory mechanisms exist between these two processes. This study showed that six known cell death genes and the Ras-Raf-MAPK signaling pathway not only function in apoptosis but also act to regulate autophagy in D. melanogaster l(2)mbn cells. The possibility cannot be ruled out that additional cell death genes that were screened may also function in autophagy but were not detected in the assay that was used because of insufficient knockdown by RNAi, a long half-life of the corresponding proteins, and/or functional redundancy (Hou, 2008).

Consistent with in vitro data, the involvement of Hid in autophagy regulation has been demonstrated in Drosophila. Overexpression of Hid induced autophagy in the fat body, larval epidermis, midgut, salivary gland, Malpighian tubules, and trachea epithelium. Further, expression of the constitutively active Ras form (RasV12), which has been shown to inhibit Hid activity in apoptosis, can also block Hid-induced autophagy. In Drosophila salivary glands, the Ras signaling pathway has also been shown to inhibit the autophagy process. Based on loss-of-function findings and these previous gain-of-function studies, it was speculated that the Ras-Raf-MAPK pathway acts upstream to inhibit Hid activity in autophagy (Hou, 2008).

Poor nutrition has a dramatic effect on egg production in Drosophila. Flies fed on a protein-deprived diet showed an increase in cell death in germaria and midstage egg chambers. These two stages have been proposed to serve as nutrient status checkpoints where defective egg chambers are removed before the investment of energy into them. The molecular mechanisms of germarium cell death are still largely unknown, and Daughterless, a helix-loop-helix transcription factor, was the only known regulator involved in cell death of germaria. Nurse cell death during mid-oogenesis is also different from most developmental cell death in other Drosophila tissues because apoptotic regulators such as rpr, hid, or grim are not required for cell death in these cells. However, the activity of caspases, particularly Dcp-1, was shown to be required for mid-oogenesis cell death. The current findings implicate several additional genes, Dcp-1, Bruce, Atg7, and Atg1, in nutrient deprivation-induced cell death in the germarium, as well as during mid-oogenesis (Hou, 2008).

Other forms of cell death, such as autophagic cell death, have been proposed previously to be involved in the elimination of defective egg chambers during mid-oogenesis. Known signaling pathways, including the insulin and ecdysone pathways, have been shown to be required not only for the survival of nurse cells in mid-oogenesis; they are also known to regulate the autophagy process, supporting the notion that autophagy plays a role in mid-oogenesis cell death. Features of autophagy were observed during D. virilis mid-oogenesis cell death as shown by monodansylcadaverine staining and transmission electron microscopy. The results using GFP-LC3 and LTG demonstrate that autophagy occurs in degenerating midstage egg chambers and also in germaria of nutrient deprived Drosophila. It was found that mutation of Atg7 results in a significant decrease of autophagy in dying mid-stage egg chambers and in germaria of starved flies, further supporting the presence of autophagy during these stages (Hou, 2008).

The role of autophagy in cell survival or cell death is still not well resolved and is likely to be context dependent. The results show that autophagy contributes to the cell death process in the ovary. Loss of Atg7 or Atg1 activity in both dying midstage egg chambers and germaria leads to decreased TUNEL staining, which indicates a reduction in DNA fragmentation. Consistent results were observed previously in the larval midguts of Atg7 mutants, which also showed an inhibition of DNA fragmentation. Interestingly, lack of autophagy function does not appear to affect nuclear DNA condensation in nurse cells. Nurse cells in degenerating stage 8 egg chambers of starved Atg7 mutants or Atg1 GLCs appeared to still have condensed nuclei, as shown by DAPI staining. Thus, based on Atg7 and Atg1 mutant analyses, autophagy contributes to DNA fragmentation but not all aspects of nurse cell death. Future studies are required to determine how autophagy is connected to known pathways leading to DNA fragmentation and chromatin condensation during cell death (Hou, 2008).

The IAP family member Bruce was shown previously to repress cell death in the Drosophila eye (Vernooy, 2002). Bruce was also shown to protect against excessive nuclear condensation and degeneration, perhaps by limiting excessive caspase activity, during sperm differentiation (Arama, 2003). Other IAP family members have been shown to bind caspases via a BIR domain and inhibit apoptosis. The presence of a BIR domain in Bruce suggests that it may also have caspase-binding activity. This study found that lack of Bruce function resulted in an increase in both LTR and TUNEL staining in germaria and degenerating midstage egg chambers. Thus, the Bruce mutant-degenerating phenotype in ovaries suggests that Bruce might function normally to restrain or limit caspase activity in this tissue. Because it was found that Dcp-1 and Bruce are both required for the regulation of autophagy and DNA fragmentation in germaria and dying midstage egg chambers, it is possible that Bruce acts to bind and degrade Dcp-1 in nurse cells under nutrient-rich conditions. Future studies using epistasis and protein interaction analyses will be required to test this prediction. The possibility cannot be ruled out that other IAP proteins, such as DIAP1, and other caspases also play a role during these stages. However, at least in response to starvation signals, Bruce and Dcp-1 play a nonredundant dual role in the regulation of autophagy and cell death in the ovary (Hou, 2008).

Numerous studies have linked caspase function to apoptosis, but recent findings indicate that caspases are also required for nonapoptotic processes including immunity and cell fate determination (for reviews see Kumar, 2004; Kuranaga, 2007). Tnis study has shown that Dcp-1 is also required for starvation-induced autophagy. In the ovary, it appears that both apoptotic and autophagic events occur in the germaria and midstage egg chambers after nutrient deprivation. It is possible that Dcp-1 coordinates autophagy and apoptosis at these two nutrient status checkpoints to ensure elimination of defective egg chambers in the most efficient manner possible. Dcp-1 mutants exhibit intact nuclei in stage 8 defective egg chambers, which indicates a block in both DNA fragmentation and nuclear condensation, and further supports a dual regulatory role for Dcp-1 in mid-oogenesis cell death. Dcp-1 might function to induce autophagosome formation while coordinately acting upon alternate proteolytic targets to complete execution of apoptosis. Future studies to elucidate upstream regulators and downstream substrates of Dcp-1 in cells undergoing autophagy or apoptosis will help to establish the regulatory mechanisms governing the crosstalk between these two cellular processes. Given the multiple cellular effects associated with autophagy, these results also have important therapeutic implications for the use of modulators of caspase or IAP activity in the treatment of cancer and other diseases (Hou, 2008).


EVOLUTIONARY HOMOLOGS

The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease

The baculovirus protein p35 inhibits programmed cell death in such diverse animals as insects, nematodes and mammals. p35 protein has been shown to be a substrate for and inhibitor of the C. elegans cell-death protease CED-3 and a substrate for four CED-3-like vertebrate cysteine protease activities implicated in apoptosis in mammals. A p35 mutation that greatly reduces p35 activity in vitro as a CED-3 substrate and inhibitor abolishes p35 activity in vivo in protecting against cell death in C. elegans. Introduction of the CED-3 cleavage site in p35 into the cowpox virus protein crmA, which inhibits mammalian apoptosis but not programmed cell death in C. elegans, causes crmA to block CED-3-mediated cell death. These observations suggest that p35 may prevent programmed cell death in C. elegans and other species by acting as a competitive inhibitor of cysteine proteases (Xue, 1995).

CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED-9

Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).

Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans

Genetic studies have identified over a dozen genes that function in programmed cell death (apoptosis) in the nematode C. elegans. Although the ultimate effects on cell survival or engulfment of mutations in each cell death gene have been extensively described, much less is known about how these mutations affect the kinetics of death and engulfment, or the interactions between these two processes. Four-dimensional-Nomarski time-lapse video microscopy has been used to follow in detail how cell death genes regulate the extent and kinetics of apoptotic cell death and removal in the early C. elegans embryo. Blocking engulfment enhances cell survival when cells are subjected to weak pro-apoptotic signals. Thus, genes that mediate corpse removal can also function to actively kill cells (Hoeppner, 2001).

In a weak caspase (ced-3) mutant background, cells at early stages (ring and erythrocyte stages) of cell death have three options: progression to full-blown apoptotic cell corpses, direct engulfment without morphological progression to full corpse, or reversion to normality and survival. It is proposed that the relative frequency of the latter two fates might be influenced by the efficiency with which the early corpse is recognized and engulfed. To determine whether preventing engulfment might enhance reversion from 'early death' and hence cell survival, the fate of the early AB cells was followed in ced-6;ced-3 and ced-7;ced-3 double mutant embryos. ced-6 and ced-7 are known to participate in the removal of apoptotic cells in C. elegans (Hoeppner, 2001).

Indeed, the absence of ced-6 or ced-7 function significantly increase the frequency of such reversion events. Furthermore, the fraction of cells that never initiated any overt signs of apoptosis is also greatly increased. How can the engulfment machinery contribute to cell killing? The data are consistent with at least two models. The 'backup-plan model' suggests that low levels of CED-3 caspase activity might on occasion be sufficient to activate the eat-me signal on the surface of the cell, but not enough to kill the cell. However, even if the cell does not autonomously kill itself, exposure of the eat-me signal ensures that it will be recognized and engulfed, and therefore properly removed. The alternative 'positive-feedback model' proposes that doomed cells indicate their desire to die to neighboring cells, either through cell-surface changes or secretion of a signaling molecule. Recognition of this signal results in the neighboring cells sending back pro-apoptotic signals, encouraging the doomed cell to 'go for it', thereby ensuring that the cell completes the process. Completion of this positive feedback loop would somehow require an intact engulfment pathway, possibly for transduction of the signal in the neighboring cells (Hoeppner, 2001).

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. This study describes 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 (Joshi, 2004).

Timing of the onset of a developmental cell death is controlled by transcriptional induction of the C. elegans ced-3 caspase-encoding gene

Temporal control of programmed cell death is necessary to ensure that cells die at only the right time during animal development. How such temporal regulation is achieved remains poorly understood. In some Caenorhabditis elegans somatic cells, transcription of the egl-1/BH3-only gene promotes cell-specific death. The EGL-1 protein inhibits the CED-9/Bcl-2 protein, resulting in the release of the caspase activator CED-4/Apaf-1. Subsequent activation of the CED-3 caspase by CED-4 leads to cell death. Despite the important role of egl-1 transcription in promoting CED-3 activity in cells destined to die, it remains unclear whether the temporal control of cell death is mediated by egl-1 expression. This study show shows that egl-1 and ced-9 play only minor roles in the death of the C. elegans tail-spike cell, demonstrating that temporal control of tail-spike cell death can be achieved in the absence of egl-1. The timing of the onset of tail-spike cell death is controlled by transcriptional induction of the ced-3 caspase. The developmental expression pattern of ced-3 has been characterized; in the tail-spike cell, ced-3 expression is induced shortly before the cell dies, and this induction is sufficient to promote the demise of the cell. Both ced-3 expression and cell death are dependent on the transcription factor PAL-1, the C. elegans homolog of the mammalian tumor suppressor gene Cdx2. PAL-1 can bind to the ced-3 promoter sites that are crucial for tail-spike cell death, suggesting that it promotes cell death by directly activating ced-3 transcription. These results highlight a role that has not been described previously for the transcriptional regulation of caspases in controlling the timing of cell death onset during animal development (Maurer, 2007).

Ceramide biogenesis is required for radiation-induced apoptosis in the germ line of C. elegans

Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism were tested in C. elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis (Deng, 2008).

Although studies that use genetic deficiency in ceramide production support it as essential for apoptosis in diverse models, many have questioned whether ceramide functions as a bona fide transducer of apoptotic signals. One reason for skepticism is that, despite delineation of a number of ceramide-activated proteins, no single protein has been identified as mediator of ceramide-induced apoptosis. Recent studies have suggested an alternate mode of ceramide action, based on its capacity to self-associate and locally rearrange membrane bilayers into ceramide-rich macrodomains (1 to 5 µm in diameter), which are sites of protein concentration and oligomerization. Ceramide may thus mediate apoptosis through its ability to reconfigure membranes, coordinating protein complexation at critical junctures of signaling cascades (Deng, 2008).

To establish the role of ceramide definitively, a model of radiation-induced apoptosis was used in C. elegans germ cells. Germline stem cells, located at the distal gonad tip, divide incessantly throughout adult life, with daughter cells arresting in meiotic prophase. Upon exiting prophase, germ cells become sensitive to radiation-induced apoptosis, detected morphologically just proximal to the bend of the gonadal arm. This apoptotic pathway is antagonized by the ABL-1 tyrosine kinase, requiring sequentially the cell cycle checkpoint genes rad-5, hus-1, and mrt-2; the C. elegans p53 homolog cep-1; and the genes making up the conserved apoptotic machinery, the caspase ced-3, the apoptotic protease activating factor 1-like protein ced-4, the Bcl-2 protein ced-9, and the BH3-domain protein egl-1. This pathway differs from apoptotic somatic cell death, which is not subject to upstream checkpoint regulation via the CEP-1 pathway (Deng, 2008).

This study has identified conserved genes that regulate C. elegans sphingolipid intermediary metabolism and has tested deletion alleles. Screening for mutants resistant to radiation-induced germ cell apoptosis revealed apoptosis suppression in only deletion mutants of hyl-1 and lagr-1, two of the three ceramide synthase (CS) genes. CS gene products regulate de novo ceramide biosynthesis, acylating sphinganine to form dihydroceramide that is subsequently converted to ceramide by a desaturase. CSs contain six to seven putative transmembrane domains and a Lag1p motif [which confers enzyme activity, regions conserved in the C. elegans orthologs. The deleted CS sequences in hyl-1(ok976) and lagr-1(gk327) result in frameshifts that disrupt the Lag1p motifs. An ~1.6-kb hyl-1 transcript was detected in wild-type (WT) worms and a smaller ~1.35-kb transcript in hyl-1(ok976), whereas an ~1.4-kb lagr-1 transcript was detected in WT worms and a ~1.25-kb transcript in lagr-1(gk327). In contrast, a deletion mutant of the third C. elegans CS, hyl-2(ok1766), lacking a 1626-base pair fragment of the hyl-2 gene locus that eliminates exons 2 to 5 corresponding to 74% of the coding sequence, displayed no defect in germ cell death (Deng, 2008).

In N2 WT strain young adults, apoptotic germ cells gradually increased in abundance with age from a baseline of 0.7 ± 0.1 to 1.8 ± 0.2 corpses per distal gonad arm over 48 hours. Exposure to a 120-gray (Gy) ionizing radiation dose increased germ cell apoptosis to 5.2 ± 0.3 cells 36 to 48 hours after treatment. In contrast, in hyl-1 (ok976) and lagr-1(gk327) animals, age-dependent and radiation-induced germ cell apoptosis were nearly abolished. Similar effects were observed in the lagr-1(gk327);hyl-1(ok976) double mutant. The rate of germ cell corpse removal was unaffected in CS mutants, excluding the possibility that defective corpse engulfment elevated corpse numbers. In contrast, loss-of-function (lf) mutations of hyl-1 or lagr-1 did not affect developmental somatic cell death, nor did the lf hyl-2(ok1766) mutation. These studies indicate a requirement for two C. elegans CS genes for radiation-induced germline apoptosis (Deng, 2008).

To confirm ceramide as critical for germline apoptosis, C16-ceramide was injected into gonads of young adult WT worms. C16-ceramide is the predominant ceramide species in apoptosis induction by diverse stresses in multiple organisms. C16-ceramide microinjection resulted in time- and dose-dependent increases in germ cell apoptosis, with a median effective dose of ~0.05 µM gonadal ceramide. Peak effect occurred at ~0.1 µM gonadal ceramide at 36 hours, qualitatively and quantitatively mimicking the 120-Gy effect in WT worms. In contrast, C16-dihydroceramide, which differs from C16-ceramide in a trans double bond at sphingoid base position four to five, was without effect, indicating specificity for ceramide in apoptosis induction. Furthermore, C16-ceramide microinjection into lagr-1(gk327);hyl-1(ok976) animals (~1 µM gonadal ceramide) resulted in a 5.7-fold increase in germ cell apoptosis. Note that the baseline level of apoptosis in lagr-1(gk327);hyl-1(ok976) was less than one-half that in WT worms. Moreover, ~0.005 µM gonadal ceramide, a concentration without impact on germ cell apoptosis, completely restored radiation (120 Gy)-induced apoptosis, an effect inhibitable in a lf ced-3 background. C16-ceramide's ability to bypass the genetic defect and restore the radiation-response phenotype is strong evidence that hyl-1 and lagr-1 represent legitimate C. elegans CS genes. Animals with sphk-1(ok1097), a null allele of sphingosine kinase (SPHK), which prevents conversion of ceramide to its anti-apoptotic derivative sphingosine 1-phosphate (S1P), displayed high baseline germ cell death and were hypersensitive to radiation-induced germ cell apoptosis, inhibitable (by 85 ± 9%) in a lagr-1(gk327);sphk-1(ok1097) double mutant. Collectively, these studies identify ceramide as a critical effector of radiation-induced germ cell apoptosis, although they do not define its mode of engaging the apoptotic pathway (Deng, 2008).

Inactivation of the C. elegans ABL-1 ortholog in the lf mutant abl-1(ok171) (or by RNA interference) increases baseline and post-radiation germ cell apoptosis, modeling radiation hypersensitivity phenotypes. To order CS action relative to ABL-1, hyl-1(ok976);abl-1(ok171) and lagr-1(gk327);abl-1(ok171) and a triple mutant lagr-1(gk327);hyl-1(ok976);abl-1(ok171) were generated. lf hyl-1 or lagr-1 in an abl-1(ok171) genetic background prevented the time-dependent increase in physiologic germ cell apoptosis and completely blocked radiation-induced apoptosis. Similarly, lagr-1(gk327);hyl-1(ok976);abl-1(ok171) displayed inhibition of baseline and radiation-induced germ cell apoptosis. Thus, increased germ cell apoptosis in irradiated abl-1(ok171) depends on the CS genes hyl-1 and lagr-1 (Deng, 2008).

In C. elegans, DNA damage activates the p53 homolog CEP-1, which is required for transcriptional up-regulation of the BH3-only proteins, EGL-1 and CED-13, that in turn activate the core apoptotic machinery (CED-9, CED-4, and CED-3). Exposure of hyl-1(ok976) and lagr-1 (gk327) to 120 Gy increased egl-1 transcripts four- to fivefold at 9 hours after irradiation, whereas ced-13 expression was enhanced five- to sixfold -- levels comparable to those detected in irradiated WTworms. Thus, the loss of CS did not affect CEP-1 activation upon irradiation, suggesting that ceramide and CEP-1 might function in parallel, coordinately conferring radiation-induced germ cell death (Deng, 2008).

It was reasoned that in contrast to radiation-induced germ cell apoptosis, which apparently requires increased abundance of both BH3-only proteins and ceramide, C16-ceramide provided exogenously might act independent of p53-mediated egl-1 expression by maximizing the effect of baseline EGL-1. In fact, microinjected C16-ceramide partially restored germ cell death in cep-1(gk138) from 0.4 ± 0.13 to 2.5 ± 0.32 corpses per distal gonad arm. Since C16-ceramide is inactive in the lf egl-1 mutant egl-1(n1084n3082), it appears that there is a requirement for at least a baseline level of BH3-only proteins for ceramide-induced apoptosis. Consistent with this notion, C16-ceramide administration did not increase egl-1 and ced-13 transcription. Furthermore, inactivating the core apoptotic machinery in lf ced-3(n717) and ced-4(n1162) or in gain-of-function ced-9(n1950) animals, which abolish radiation-induced germline apoptosis, similarly abolished C16-ceramide-induced death. Collectively, these data indicate that ceramide acts in conjunction with BH3-only proteins upstream of the mitochondrial commitment step of apoptosis in the C. elegans germ line (Deng, 2008).

Since these studies point to a mitochondrial site of ceramide action, an immune histochemical approach was devised to evaluate whether ceramide might increase in the mitochondria of C. elegans germ cells. Advantage was taken of the increased frequency of germ cell apoptosis in abl-1(ok171), anticipating a maximized ceramide signal upon irradiation in this strain. Gonads from unirradiated or irradiated worms were dissected, opened by freeze-cracking, and then stained with MID15B4, a specific anti-ceramide antibody. Mitochondria were localized with an antibody to the mitochondrial marker protein OxPhos Complex IV subunit I (COX-IV) or by Rhodamine B staining. COX-IV staining (green) before and after irradiation displayed a prominent perinuclear distribution reminiscent of mitochondrial topography in some mammalian cell systems. Ceramide staining (red) displayed a similar profile and at baseline was faint, increasing 2.4-fold at 24 hours post-irradiation. Merging the two signals (red and green) revealed that ceramide accumulation was distinctively mitochondrial (yellow). Radiation-induced ceramide accumulation was abrogated in lagr1(gk327);hyl-1(ok976);abl-1(ok171) animals. Similarly, ceramide increase was abrogated in irradiated lagr-1(gk327);hyl-1(ok976) as compared with WT animals. These results define ionizing radiation-induced ceramide accumulation in the C. elegans germ line as mitochondrial in origin, mediated via the classic ceramide biosynthetic pathway (Deng, 2008).

Whether mitochondrial ceramide accumulation was required for CED-4 redistribution to nuclear membranes was examined. In nonapoptotic somatic cells, CED-4 is sequestered to mitochondria by binding CED-9. When displaced by EGL-1, CED-4 targets nuclear membranes and activates caspase CED-3, necessary for the effector phase of apoptosis. For these studies, abl-1(ok171) and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) animals were exposed to 120 Gy, and germ cells were released from gonads and stained with antibodies against C. elegans CED-4 and Ce-lamin, a nuclear membrane marker. CED-4 and Ce-lamin colocalization by confocal microscopy (yellow merged signal) served as readout for nuclear CED-4 redistribution. After irradiation nuclear CED-4 staining intensity increased 4.3-fold in abl-1(ok171). Consistent with reduced germ cell apoptosis, nuclear CED-4 staining is significantly reduced in lagr-1(gk327);hyl-1(ok976);abl-1(ok171). Specifically, baseline CED-4 intensity at the nuclear membrane is lower in lagr-1(gk327);hyl-1(ok976);abl-1(ok171) than in abl-1(ok171), increasing post-irradiation only to the control level of unirradiated abl-1(ok171) worms, an effect probably of biologic relevance as the biophysical effects of ceramide on membrane structure are concentration-dependent (Deng, 2008).

This study also used opls219 worms, a strain expressing a CED-4::GFP fusion protein (where GFP is green fluorescent protein), which permits in vivo detection of CED-4 trafficking. opls219 worms were cultured on plates containing Rhodamine B to stain mitochondria (red). Merged images detect mitochondrial CED-4 as a yellow signal (red and green overlay), whereas nonmitochondrial CED-4 appears green. Although a low-intensity green CED-4 signal was detected in nuclear membranes of unirradiated germ cells, the large majority of CED-4 was present in mitochondria before irradiation. At 36 hours postradiation, the CED-4 signal was markedly reduced in mitochondria, relocalizing primarily to nuclear membranes as bright green platformlike structures. In eight worms, overall reduction in CED-4 mitochondrial colocalization upon irradiation was ~50%, abrogated in lagr-1(gk327);opls219. Consistent with the anti-CED-4 antibody staining, the loss of mitochondrial CED-4 signal in opls219 was accompanied by a twofold increase in nuclear CED-4 signal, blocked entirely in lagr-1(gk327);opls219. These results indicate that mitochondrial ceramide contributes substantively to CED-4 displacement from mitochondrial membranes during radiation-induced germ cell apoptosis (Deng, 2008).

These data indicate that the ceramide synthetic pathway is required for radiation-induced apoptosis of C. elegans germ cells. The most parsimonious molecular ordering suggests that CS (as well as its enzymatic product ceramide) functions on a pathway that is parallel to the CEP-1/p53-EGL-1 system. The coordinated function of these two pathways occurs at the mitochondrial commitment step of the apoptotic process. It is hypothesized that ceramide may recompartmentalize the mitochondrial outer membrane, yielding a permissive microenvironment for EGL-1-mediated displacement of CED-4, the trigger for the effector stage of the apoptotic process (Deng, 2008).

Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: implicated mechanisms for caspase activation

CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. CED-4 acts as a positive regulator of caspases by enhancing the processing of procaspases to their mature forms. In mammalian 293 cells and insect SF21 cells, the processing of the proform of caspase CED-3 to its mature form is facilitated by CED-4, resulting in the acceleration of CED-3-induced cell death. CED-4 can directly activate CED-3 and ATP hydrolysis associated with the ATP binding site (P-loop) of CED-4 is required for CED-3 processing. Additionally, Apaf-1, a mammalian homolog of CED-4 that also has a P-loop motif, activates caspase-9 in the presence of cytochrome c and dATP, resulting in the sequential activation of caspase-3 in vitro. The CED-3/CED-4 complex has been shown to be activated by the oligomerization of CED-4. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induces massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis has shown that in S2 cells, both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation. Furthermore, CED-4 (K165R) partially preventes cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases (Kanuka, 1999).

Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division

Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIR (baculovirus IAP repeat) motifs. In rich medium, BIR1 mutant S. cerevisiae undergo normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids form spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that do form fail to survive beyond two divisions after germination. bir1 mutant S. pombe spores also die in the early divisions after spore germination and become blocked at the metaphase/anaphase transition. These mutants are unable to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a way similar to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin (Uren, 1999).

Other IAPs bearing BIRs that resemble those from the yeasts may also have roles in spindle function. Of the vertebrate IAP genes, the sequence and exon structure of the yeast IAPs is most closely related to that of the mammalian IAP Survivin. survivin expression increases during the G2/M phase of cell cycle, and Survivin localizes to the mitotic spindle in vivo and cosediments with polymerized tubulin. RNA-mediated gene interference of one of the IAPs from C. elegans causes abnormalities in cytokinesis during embryonal cell divisions (Uren, 1999 and references therein).

Although it is not clear whether vertebrate and insect IAPs function primarily by blocking caspase activation signals or by binding directly to caspases, most are thought to inhibit a caspase-dependent apoptotic mechanism. Survivin has been reported to bind directly to, and inhibit, caspases 3 and 7, but its homologs in C. elegans, bir-1 and bir-2, appear to have no role in the control of cell death, and the phenotype caused by inactivation of one of the C. elegans IAPs can be suppressed partially by transgenic expression of survivin. Because neither S. pombe nor S. cerevisiae appears to encode caspases, and neither has been shown to use a cell suicide program, Bir1 and BIR1 are unlikely to function by inhibiting cell death mechanisms resembling those in metazoans. The structural and functional similarities between human Survivin, cerevisiae BIR1, pombe Bir1, and the BIR-bearing proteins from C. elegans suggest that they share conserved roles in cell division (Uren, 1999 and references therein).

Structural and biochemical basis of apoptotic activation by Smac/DIABLO

The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 Å resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO (Chai, 2000).

Structural basis of IAP recognition by Smac/DIABLO

Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, because mutation of the very first amino acid leads to loss of interaction with IAPs and the concomitant loss of Smac/DIABLO function. The high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP is reported in this study. These results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighboring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, these results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, this structural analysis identifies potential targets for drug screening (Wu, 2000).

Diablo promotes apoptosis by removing miha/xiap from processed caspase 9

MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. After UV radiation, MIHA prevents apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functions after release of cytochrome c and DIABLO from the mitochondria and is able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO binds to MIHA and disrupts its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis (Ekert, 2001).

Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors

The mechanism of cytochrome c release in response to apoptotic stimuli and its regulation by the Bcl2 family of proteins is unclear. Inasmuch as the structure of Bcl-xL is reminiscent of pore-forming proteins of bacterial toxins such as diphtheria toxin and colicins, it has been hypothesized that Bcl-xL may function as an ion channel that regulates the permeability of mitochondria. Such an ion channel could minimize osmotic stress, and in doing so, the release of cytochrome c would be prevented due to mitochondrial matrix swelling and outer membrane disruption. Indeed, both swelling of the mitochondrial matrix and bursting of the outer membrane were observed in cells treated with agonistic antibody against Fas. However, whether such a phenomenon is the cause of cytochrome c release or an effect of the apoptotic program is unclear. Activation of cell surface receptor Fas leads to rapid inactivation of the electron transfer activity of cytochrome c and subsequent release of cytochrome c from mitochondria. The inactivation and release of cytochrome c induced by Fas activation is sensitive to z-VAD-fmk, a broad range caspase inhibitor. Since activation of cell surface death receptor leads to rapid activation of caspase-8, the apical caspase in the Fas-induced apoptotic pathway, the loss of cytochrome c from mitochondria is likely a result of caspase-8 activation. Indeed, addition of active caspase-8 to a Xenopus cell-free system induces rapid cytochrome c release from mitochondria. The activation of caspase-8, therefore, initiates two pathways leading to the activation of downstream caspases. Caspase-8 can activate downstream caspases (like caspase-3, caspase-6, and caspase-7) by directly cleaving them. Caspase-8 activates these downstream caspases indirectly by causing cytochrome c release from mitochondria that triggers caspase activation through Apaf1. The latter pathway is regulated by Bcl2 or Bcl-xL while a caspase-8 inhibitor like CrmA blocks both pathways. The contributions of these two pathways to Fas-induced cell death vary between different cell types, presumably due to different levels of activity (Luo, 1998 and references).

Fas/Apo [Apoptosis]-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-kappaB

The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells have been localized in close proximity to Fas ligand (FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) is expressed by some Fas-expressing cells, whereas Fas-associated death domain (FADD) is undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an inhibitor of Fas activation, is also expressed in the postnatal cerebral cortex. Fas expression is more ubiquitous in embryonic cortical neuroblasts in dissociated culture, as compared to in situ, within the developing brain, suggesting that the environmental milieu partly suppresses Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP are also expressed by subsets of dissociated cortical neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induces caspase-dependent cell death in primary embryonic cortical neuroblast cultures. The activation of Fas is also accompanied by a rapid downregulation of Fas receptor expression, non-cell cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB. Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide, initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain (Cheema, 1999).

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents, it is not known whether their normal differentiation program uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly. Caspases are activated during normal human keratinocyte differentiation and that activation is apparently required for the normal loss of the nucleus. Intermediate-filament-associated protein filaggrin and its large precursor, profilaggrin normally accumulate in keratohyalin granules in the granular layer of the epidermis. Profilaggrin cleavage precedes nuclear degradation, and it plays an important part in the reorganization of the cytoskeleton that accompanies the formation of the cornified envelope. It is suggested that keratinocyte differentiation is accompanied by the activation of procaspase-3 and that the activation of at least one caspase is required for both nuclear loss and normal filaggrin processing (Weil, 1999).

Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner

The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fiber cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis; caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x[s/l]) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labeling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it has been demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brings about a 50%-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. In contrast, the inhibitors of caspases-3 and -8 are not effective in significantly reducing the number of TUNEL-labeled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors are added reveal alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fiber cell nuclei during chick secondary lens fiber development and support the proposal that this process has many characteristics in common with apoptosis (Wride, 1999).

A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevents caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).

Structural basis for the inhibition of Caspase-3 by XIAP

The molecular mechanism(s) that regulate apoptosis by caspase inhibition remain poorly understood. The main endogenous inhibitors are members of the IAP family and are exemplified by XIAP, which regulates the initiator caspase-9, and the executioner caspases-3 and -7. The crystal structure is reported of the second BIR domain of XIAP (BIR2) in complex with caspase-3, at a resolution of 2.7 Å, revealing the structural basis for inhibition. The inhibitor makes limited contacts through its BIR domain to the surface of the enzyme, and most contacts to caspase-3 originate from the N-terminal extension. This lies across the substrate binding cleft, but in reverse orientation compared to substrate binding. The mechanism of inhibition is due to a steric blockade prohibitive of substrate binding, and is distinct from the mechanism utilized by synthetic substrate analog inhibitors (Riedl, 2001).

Blocking Caspase-3-mediated proteolysis of IKKß suppresses TNF-alpha-induced apoptosis

The transcription factor NF-kappaB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-kappaB activation. It is unknown how the protection conveyed by NF-kappaB is overridden during apoptosis. IkappaB kinase (IKK) ß is specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-alpha-induced apoptosis. Proteolysis of IKKß eliminates its enzymatic activity, interfers with IKK activation, and promotes TNF-alpha killing. Point mutations that abrogate IKKß proteolysis generate a caspase-resistant IKKß mutant, which suppresses TNF-alpha-induced apoptosis. Thus, this study demonstrates that TNF-alpha-induced apoptosis requires caspase-mediated proteolysis of IKKß (Tang, 2001).

What is the mechanism by which the UC-IKKß mutant suppresses TNF-alpha-induced apoptosis? One of the possibilities is that it functions through prolonged induction of NF-kappaB-controlled antiapoptotic proteins. It is envisioned that apoptotic death of a cell requires amplification of the caspase cascade, an event that may depend on destruction of cellular survival factors. Treatment with TNF-alpha triggers rapid activation and reactivation of IKK and NF-kappaB. This results in production of antiapoptotic proteins, such as c-IAP1, which inhibit caspases. The life balance is maintained and cells survive. The addition of cycloheximide (CHX) reduces the production of antiapoptotic proteins, allowing activation of caspases such as Casp3. Activated Casp3 in turn cleaves IKKß, blunting IKK reactivation and subsequent production of antiapoptotic proteins. Activated Casp3 also cleaves antiapoptotic proteins, including c-IAP1. Therefore, the caspase cascade is amplified, switching the life balance toward death. In contrast, the uncleavable IKKß (IKK UCß) mutant is resistant to caspase-mediated proteolysis and can be reactivated over a prolonged period of time. Since 10 µg/ml CHX reduces, but does not completely block protein synthesis, activation of NF-kappaB by the UC IKKß mutant allows the accumulation of antiapoptotic proteins at a level that is sufficient to inhibit caspases. As a result, amplification of the caspase cascade and cell death is suppressed (Tang, 2001).

Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones

Axon guidance cues trigger rapid changes in protein dynamics in retinal growth cones: netrin-1 stimulates both protein synthesis and degradation, while Sema3A elicits synthesis, and LPA induces degradation. What signaling pathways are involved? These studies confirm that p42/44 MAPK mediates netrin-1 responses and further show that inhibiting its activity blocks cue-induced protein synthesis. Unexpectedly, p38 MAPK is also activated by netrin-1 in retinal growth cones and is required for chemotropic responses and translation. Sema3A- and LPA-induced responses, by contrast, require a single MAPK, p42/p44 and p38, respectively. In addition, caspase-3, an apoptotic protease, is rapidly activated by netrin-1 and LPA in a proteasome- and p38-dependent manner and is required for chemotropic responses. These findings suggest that the apoptotic pathway may be used locally to control protein levels in growth cones and that the differential activation of MAPK pathways may underlie cue-directed migration (Campbell, 2003).

These data provide evidence for the presence of caspases in growth cones and identify caspase-3 as a potential target of p38 signaling for mediating both netrin-1-induced turning and LPA-induced growth cone collapse. This suggests that, in addition to their roles in apoptosis, caspase-induced protein degradation may play a role in growth cone guidance. Previous studies identified the netrin receptor DCC in regulating cell survival via the activation of caspase-3 by caspase-9 in the absence of netrin-1 in human embryonic kidney 293T cells. By contrast, in Xenopus retinal growth cones, netrin-1 and LPA induce the rapid activation of caspase-3 independent of caspase-9 via the MAPK- and proteasome-mediated proteolysis pathways. The activation of caspase-3 in the confined cellular compartment of the growth cone might not lead to activation of the full apoptotic cascade and cell death but rather to transient, localized changes in specific proteins. The p42/p44 and PI-3 kinase pathways identified in netrin-1 signaling are known to play roles in mediating cell survival and may ensure tight regulation of caspase activity in the growth cone. A role has been identified for caspases in synaptic plasticity independent of their roles in cell death (Campbell, 2003 and references therein).

Since caspases are proteases, a key question asks which proteins do caspases degrade? Candidate proteins include known caspase substrates, such as actin, actin binding proteins, and signal transduction pathway components. For example, gelsolin, an actin severing protein, is present in growth cones and is activated by caspase-3-mediated cleavage. Netrin-1 and LPA stimulate the rapid caspase-3-dependent cleavage of PARP. In addition to its role in maintaining genomic stability, PARP is able to interact with and activate proteasome-mediated proteolysis. Cleavage of PARP may inactivate itself, providing a possible mechanism by which proteasome-mediated proteolysis may be regulated in the case of netrin-1 and LPA. The netrin-1 receptor DCC is itself a substrate of caspase-3, and caspase-mediated cleavage of DCC may potentially be involved in mediating netrin-1-induced chemotropic responses. During apoptosis, caspase-3 is also able to cleave eukaryotic initiation factor 4G (eIF-4G), a crucial protein required for binding cellular mRNA to ribosomes. This may decrease the rate of translation and provide a possible mechanism for negative regulation of netrin-1-stimulated protein synthesis in growth cones. Since the chemotropic responses of growth cones elicited by netrin-1 and LPA are essentially blocked by inhibition of caspase-3, it is likely that of the caspases, caspase-3 plays a major role in these processes (Campbell, 2003 and references therein).

The ubiquitin-proteasome system is critically involved in apoptosis and in mediating chemotropic responses of growth cones. In neuronal cells, proteasome inhibitors protect against apoptosis by acting upstream of caspase activation. These results have revealed a parallel in retinal growth cones where the activation or cleavage of caspase-3 in response to netrin-1 and LPA requires proteasome function, suggesting that caspase-mediated protein degradation lies downstream of proteasome/ubiquitin-mediated proteolysis. Candidate proteins to undergo proteasome/ubiquitin-mediated proteolysis include the inhibitor of apoptosis (IAP) family of proteins, degradation of which can result in caspase activation. IAPs can also target caspase-3 itself for proteasome/ubiquitin-mediated proteolysis, suggesting a possible mechanism for the transient and localized nature of caspase-3 activation in growth cones (Campbell, 2003 and references therein).

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. Site-directed mutagenesis of BIR2 and its linker were used to determine the mechanism of executioner caspase inhibition by XIAP. The BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, it is predicted that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP (Scott, 2005).

The fundamental mechanism of specific protein interactions is usually conserved during protein evolution. According to conservation of mechanism, the two units of XIAP that inhibit caspases should preserve a fundamental interaction strategy. On the basis of structural studies, this concept could be questioned. The key elements of caspase-9 inhibition by BIR3 are the IBM interacting groove and the C-terminal helix. In contrast, the key element of caspase-3 and -7 inhibition by BIR2 seems to be the completely nonconserved N-terminal linker region. The most conserved surface structure of BIR domains is the IBM interacting groove. It is found on many BIR domains including the BIR2 and BIR3 of XIAP, and the BIR1 and BIR2 of an ortholog in Drosophila melanogaster, DIAP1. The surface groove of DIAP1 BIR2 is involved in binding and ubiquitination of Dronc, the initiator caspase in flies. In addition, the IBM interacting groove of DIAP1 BIR1 is absolutely required for inhibition of the executioner caspase DrICE by an unknown mechanism. It is suggested that the IBM interacting groove is a conserved interaction element of BIR domains and that for XIAP BIR2 it confers tight inhibition of caspase-3 and -7 by providing a second binding site (Scott, 2005).

Both BIR2 and BIR3 inhibit their target caspases by a two-site interaction mechanism. They have conserved a functional IBM interacting groove that participates in inhibition by binding neoepitopes revealed following activation of their target enzymes. This interaction, primarily a docking contact, represents the conserved mechanism and also provides a platform for regulation by antagonists Smac/DIABLO and HtrA2. The primary inhibition site, however, is mechanistically different for each domain: blocking the active site in caspase-3 and -7, or dissociating the dimer of caspase-9. It is far from clear how the inhibitory mechanism diverged. It is much clearer that these distinct mechanisms direct the exquisite specificity that allows XIAP BIR domains to target selectively individual caspases in a way that other inhibitory strategies, both natural and artificial have yet to achieve (Scott, 2005).

Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway

Claspin is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide causes activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin is cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that each contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress (Clarke, 2005).

Bcl-2 and Bcl-XL regulate proinflammatory Caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. A mammalian analog of this caspase-regulatory system has been identified in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-XL bind and suppress NALP1, reducing caspase-1 activation and interleukin-1β (IL-1β) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1β production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1β production. The findings reveal an interaction of host defense and apoptosis machinery (Bruey, 2007).

e protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development

The precise regulation of programmed cell death is critical for the normal development of the nervous system. This study shows that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. Evidence is provided that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. DYRK1A phosphorylates caspase-9 on threonine residue 125, and this phosphorylation event is crucial to protect retina cells from apoptotic cell death. The data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome (Laguna, 2008).

Metabolic control of oocyte apoptosis mediated by 14-3-3zeta-regulated dephosphorylation of caspase-2.

Xenopus oocyte death is partly controlled by the apoptotic initiator caspase-2 (C2). Oocyte nutrient depletion activates C2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit C2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II (maintenance of NADPH levels by flux through the pentose phosphate pathway induces a suppressive phosphorylation on S135; Nutt, 2005). This study shows that C2 phosphorylated at S135 binds 14-3-3zeta, thus preventing C2 dephosphorylation. Moreover, S135 dephosphorylation is catalyzed by protein phosphatase-1 (PP1), which directly binds C2. Although C2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3zeta from C2 is controlled by metabolism and allows for C2 dephosphorylation. Accordingly, a C2 mutant unable to bind 14-3-3zeta is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, similar control of murine C2 by phosphorylation and 14-3-3 binding was found to occur in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death (Nutt, 2010).

Dynamic role of postsynaptic caspase-3 in zebra finch song-response habituation

Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, the activated enzyme was localized to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus, the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory (Huesmann, 2006; full text of article).

This study describes a series of studies designed to assess the role of caspase-3 activity in the phenomenon of song-specific habituation in adult zebra finches. This is an especially favorable model for analyzing biochemical changes associated with memory formation. A large discrete area in the forebrain mediates the representation of songs. When a bird hears the same song repeatedly in the same context, the neurophysiological response to that specific song habituates; this habituation can persist for days or even longer. Song presentation also triggers robust molecular responses in this area, which also change as the presentation is repeated. Novel songs initially activate the ERK intracellular signaling pathway, followed by a pulse of zenk gene transcription (Kruse, 2000; Mello, 1992). zenk is an immediate early gene known as zif-268, egr-1, NGFI-A, or Krox-24, referred to by the acronym 'ZENK'. When the stimulus is repeated across an hour or more, these molecular responses themselves habituate without affecting the responses to other songs. Habituation of the zenk response to a song is correlated with emergence of a persistent change in the behavioral response to that song. The zenk gene response is especially easy to measure and thus zenk gene expression in the auditory forebrain may be used as a molecular indicator of the status of a particular contextual song memory. If the song is heard as 'novel' it induces a zenk response; after the song has been entrained it no longer induces zenk (Huesmann, 2006).

This study shows novel-song exposure also triggers a rapid and transient increase in immunoreactivity for the activated form of caspase-3 and that caspase-3 activity is necessary for development of long-term habituation. The increase is specifically localized to postsynaptic terminals within the auditory forebrain, and evidence is provided for a molecular mechanism that could account for this tight temporal and anatomical control. These results establish a key role for caspase-3 in the machinery of memory consolidation (Huesmann, 2006).

Chk1 suppresses a Caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. Depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved 'Chk1-suppressed' pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy (Sidi, 2008).


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Biological Overview

date revised: 10 August 2010

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