InteractiveFly: GeneBrief

Bruce: Biological Overview | References

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 (for review see Klionsky, 2007). Currently, there are 31 autophagy-related (Atg) genes in yeast, and 18 Atg proteins are essential for autophagosome formation (Mizushima, 2007). 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 (for reviews see Levine, 2005; Maiuri, 2007). 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-X_L, 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 (Ferraro, 2007; Maiuri, 2007; Thorburn, 2008). 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 (Scott, 2004; Berry, 2007). 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 (Berry, 2007). In contrast, caspase activity was required for Atg1-mediated apoptotic death in the fat body (Scott, 2007). 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 (Juhasz, 2007). 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 (Velentzas, 2007), 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 LTG^high 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 (Juhasz, 2005). Further, expression of the constitutively active Ras form (Ras^V12), which has been shown to inhibit Hid activity in apoptosis (Bergmann, 1998), can also block Hid-induced autophagy (Juhasz, 2005). In Drosophila salivary glands, the Ras signaling pathway has also been shown to inhibit the autophagy process (Berry, 2007). 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 (Smith, 2002). 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 (Velentzas, 2007). 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 (Juhasz, 2007). 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, the 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).

Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death

Mammalian Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain. Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The Bruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. Drosophila Bruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1/Thread BIR2, be intact. Bruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that Bruce can regulate cell death at a novel point (Vernooy, 2002).

In Drosophila, the products of the reaper (rpr), head involution defective (hid), and grim genes are essential activators of caspase-dependent cell death. A genetic screen was carried out for suppressors of Rpr-, Hid-, and Grim-dependent cell death to identify regulators of their activity. Approximately 7000 new insertion lines of the GMREP P element transposon were generated. GMREP contains an engineered eye-specific enhancer sequence (GMR). This sequence is sufficient to drive the expression of linked genes in and posterior to the morphogenetic furrow during eye development. Thus, insertion of GMREP within a region can lead to the eye-specific expression of nearby genes. Each insertion line was crossed to flies that had small eyes due to the eye-specific expression of Rpr (GMR-Rpr flies), Hid (GMR-Hid flies), or Grim (GMR-Grim flies), and the progeny were scored for enhancement or suppression. A number of suppressors were identified. Five lines (GMREP-86A-1-5) mapped to the 86A region, and each strongly suppressed cell death induced by eye-specific expression of Rpr or Grim but not Hid. These lines mapped within a 6-kb interval. A number of other lines were obtained with P-element insertions located in the nearby region. Four of these, EP(3)0359, EP(3)0739, l(3)j8B6, and l(3)06142, mapped within six base pairs of the GMREP-86A-3-5 insertion sites. None of these, nor a fifth nearby line, l(3)06439, acted as a suppressor of GMR-Rpr-, GMR-Grim-, or GMR-Hid-dependent cell death. These results argue that the cell death suppression seen with the GMREP-86A lines was not due to a transposon-induced loss of function, but rather to the GMREP-dependent expression of a nearby gene. All of the GMREP-86A insertions were located 5' to a gene encoding the Drosophila homolog, Bruce, of murine Bruce (also known as Apollon in humans, suggesting this as an obvious candidate. The results of tissue in situ hybridizations with a Drosophila Bruce probe and immunocytochemistry with a Bruce-specific antibody support this possibility. Bruce transcript and protein are expressed at uniform low levels in wild-type eye discs. However, in the GMREP86A lines, they are expressed at high levels in and posterior to the morphogenetic furrow of the eye disc, which is where the GMR element drives expression (Vernooy, 2002).

To demonstrate that Bruce is responsible for the GMREP-86A-dependent suppression of Rpr- and Grim-dependent cell death, levels of the Bruce transcript were specifically downregulated in the eyes of flies carrying a GMR-Rpr transgene as well as a GMREP-86A element. Analysis focussed on one line, GMREP-86A-1, since all five lines behaved similarly with respect to cell death suppression and Bruce overexpression. Flies were generated that carried a Bruce RNA interference (RNAi) construct driven under GMR control (GMR-Bruce-RNAi flies). The eyes of GMR-Bruce-RNAi flies were normal. These animals were crossed to flies in which GMR-Rpr-dependent cell death was suppressed by the presence of the GMREP-86A-1 transposon and progeny from this cross were identified that carried all three transgenes, GMR-Bruce-RNAi, GMR-Rpr, and GMREP-86A-1. It was reasoned that if ectopic expression of Bruce in the eye, driven by the GMREP-86A-1 insertion, was responsible for the suppression of Rpr-dependent cell death, then expression of Bruce-RNAi should downregulate levels of the Bruce sense transcript. This should lead to an attenuation of the GMR-EP-86A-1-dependent suppression of Rpr-dependent cell death, causing a decrease in eye size. Such an attenuation was in fact observed. These observations, in conjunction with those obtained from studies with Bruce deletion mutants, argue that Bruce can suppress Rpr- and Grim-dependent cell death (Vernooy, 2002).

cDNAs encompasing the Bruce coding region were sequenced. This allowed an accurate map to be assembled of the Bruce exon-intron structure, which differs in some respects from that of the BDGP predicted gene. Overall, Bruce is 30% identical to murine Bruce. However, the Bruce N-terminal BIR domain and the C-terminal E2 domain show much higher degrees of homology, 83% and 86% identity, respectively. C. elegans homologs of Bruce were not apparent. Mutations in the Bruce gene were generated by carrying out imprecise excision of a P element, EP3731, located 3' to the Bruce transcript. Two deletions were generated that extended only in one direction, into the 3' end of the Bruce coding region. E12 deleted a relatively small region of the C terminus that includes the E2 domain, while E16 deleted approximately the C-terminal half of the Bruce coding region. Both lines were homozygous viable but male sterile. The possibility that E12 and E16 represent neomorphic mutations in Bruce cannot be excluded. However, the hypothesis that they represent hypomorphs or null mutations is favored, since they had the opposite phenotype of the GMREP-86A Bruce expression lines when in combination with GMR-Rpr, acting as enhancers rather than suppressors of Rpr-dependent cell death in the eye. E12 and E16 also enhanced GMR-Grim, but this effect is much more modest. E12 and E16 have no clear effect on cell death due to expression of Hid (Vernooy, 2002).

These results argue that endogenous Bruce levels, at least in the eye, are sufficient to act as a brake on Rpr-, and to some extent, Grim-dependent cell death. How does Bruce suppress apoptosis? A number of observations argue that Rpr- and Grim-dependent killing proceeds through distinct mechanisms and/or is regulated differently from those activities that is due to Hid. These differences are manifest at multiple points. At the level of DIAP1, point mutations of DIAP1 have effects on Rpr- and Grim-dependent cell death that are the opposite of those due to Hid. In addition, in a Drosophila extract, Hid, but not Rpr and Grim, promotes DIAP1 polyubiquitination. In contrast, in a different set of assays, Rpr and Grim, but not Hid, act as general inhibitors of protein translation. Finally, Rpr and Grim, but not Hid, show strong synergism with the effector caspase DCP-1 in terms of their ability to induce cell death in the eye. Each of these points defines a possible target for Bruce antiapoptotic action (Vernooy, 2002 and references therein).

Because Bruce strongly suppresses cell death induced by Rpr and Grim but not by Hid, one obvious possibility was that Bruce promotes Rpr and Grim ubiquitination and degradation. This hypothesis was tested by generating mutant versions of Grim and Rpr that lacked all lysines, the amino acid to which ubiquitin is added. These genes were introduced into flies under GMR control. GMR-Rpr-lys^- and GMR-Grim-lys^- flies have small eyes, indicating that these mutant proteins are effective cell death inducers. GMREP-86A-1-dependent Bruce expression suppresses this death very effectively, indicating that Bruce cannot be promoting ubiquitin-dependent degradation of Rpr or Grim. Interestingly, however, Bruce expression does not suppress cell death induced by expression of versions of Rpr (GMR-RprC) or Grim (GMR-GrimC) lacking their N termini, which are required for their IAP-caspase-disrupting interactions with the DIAP1 BIR2. This result is important because it argues that Bruce does not act to regulate this relatively uncharacterized death pathway (Vernooy, 2002).

The N-terminal Bruce BIR lacks a number of residues thought to be important for binding of Rpr, Hid, and Grim to DIAP1 BIR2. Thus, it seems unlikely that GMR-driven expression of Bruce inhibits cell death by simply titrating Rpr and Grim away from interactions with DIAP1 BIR2 as a result of similar interactions with the Bruce BIR. Nonetheless, the high degree of conservation between Bruce and mammalian Bruce in the BIR suggests that it is functionally important. To explore this role further, a fragment of Bruce that contained residues 1-531, including the BIR domain, was expressed under GMR control. Flies carrying this construct, GMR-Bruce-BIR flies, had normal appearing eyes, and in crosses to flies expressing GMR-Rpr, -Hid, or -Grim, GMR-Bruce-BIR did not enhance or suppress these eye phenotypes. These results do not rule out a role for the Bruce BIR in suppressing Rpr- and Grim-dependent cell death. However, they do suggest that the BIR alone is unlikely to mediate this inhibition (Vernooy, 2002).

Bruce overexpression in the eye also does not suppress cell death resulting from GMR-driven expression of the caspase Dronc, which is required for many apoptotic cell deaths in the fly, including those induced by expression of Rpr, Grim, and Hid. Dronc most resembles mammalian caspase-9, and its activation is likely to involve interactions with the Drosophila Apaf-1 homolog Ark. Thus, this result strongly suggests that Bruce does not block Ark-dependent Dronc activation or Dronc activity. This result is also suggested by the observation that decreasing Ark or Dronc in the eye strongly suppressed Hid-dependent cell death, which Bruce does not. A similar lack of cell death suppression is seen in the progeny of crosses between GMR-Bruce flies and flies expressing a second long prodomain caspase, Strica, whose mechanism of activation and normal functions are unknown. Finally, GMREP-86A-1 also fails to suppress the cell death due to GMR-dependent expression of the Drosophila proapoptotic Bcl-2 family member known variously as Debcl, Drob-1, dBorg-1, or Dbok (Vernooy, 2002).

Thus, the Bruce gene is found in mammals and flies, but not in the worm C. elegans. In humans, it is upregulated in some cell lines derived from gliomas and an ovarian carcinoma, and the results of antisense inhibition of Bruce suggest that it contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. The Drosophila homolog of Bruce, can potently inhibit cell death induced by Rpr and Grim but not Hid. In addition, flies with C-terminal deletions that removed the Bruce ubiquitin conjugation domain, or much larger regions of the coding region, acted as dominant enhancers of Rpr- and Grim-dependent, but not Hid-dependent, cell death. Together, these observations clearly demonstrate that Bruce can function as a cell death suppressor. The results with the deletion mutants suggest, but do not prove, that Bruce's death-inhibiting activity requires its function as a ubiquitin-conjugating enzyme. Based on the general conservation of cell death regulatory mechanisms, these results argue that mammalian Bruce is likely to facilitate oncogenesis by directly promoting cell survival in the face of specific death signals. One mechanism by which Rpr, Grim, and Hid promote apoptosis is by binding to DIAP1, thereby blocking its ability to inhibit caspase activity. It will be interesting to determine if mammalian Bruce also inhibits cell death induced by the expression of specific IAP binding proteins (Vernooy, 2002).

How does Bruce inhibit cell death? It does not promote the ubiquitination and degradation of Rpr and Grim directly. However, the possibility cannot be ruled out that Bruce somehow sequesters these proteins from their proapoptotic targets. The fact that it does not inhibit cell death due to Hid or Dronc expression argues that it is unlikely to be acting on core apoptotic regulators such as Ark, Dronc, or DIAP1, which are important for Hid-, Rpr-, and Grim-dependent cell death. An attractive hypothesis is that Bruce, perhaps in conjunction with apoptosis-inhibiting ubiquitin-protein ligases such as DIAP1 or DIAP2, promotes the ubiquitination and degradation of a component specific to Rpr- and Grim-dependent death signaling pathways. What might such a target be? Little is known about how Rpr- and Grim-dependent death signals differ from those due to Hid. However, one possibility is suggested by the recent observation that Rpr and Grim, but not Hid, can inhibit global protein translation. This creates an imbalance between levels of short-lived IAPs and the caspases they inhibit, thereby sensitizing cells to other death signals. Perhaps Bruce targets a protein(s) required for this activity (Vernooy, 2002).

Finally, Bruce is a very large protein, and thus its coding region might be expected to be subject to a relatively high frequency of mutation. Truncation of Bruce through the introduction of a stop codon or a frame shift is thus likely to be a relatively common form of Bruce mutation. The results of the deletion analysis show that C-terminal Bruce truncations act to enhance cell death in response to several different signals. Given this, it will be interesting to determine if human Bruce mutations are associated with a predisposition to pathologies that involve an inappropriate increase in cell death (Vernooy, 2002).

Search PubMed for articles about Drosophila Bruce

Arama, E., Agapite, J. and Steller, H. (2003). Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev Cell 4: 687-697. PubMed Citation: 12737804

Bergmann, A., J. Agapite, K. McCall, and H. Steller. (1998). The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 95: 331-341. PubMed Citation: 9814704

Berry, D. L. and Baehrecke, E. H. (2007). Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131: 1137-1148. PubMed Citation: 18083103

Ferraro, E. and Cecconi, F. (2007). Autophagic and apoptotic response to stress signals in mammalian cells. Arch. Biochem. Biophys. 462: 210-219. PubMed Citation: 17374522

Hou, Y. C., Chittaranjan, S., Barbosa, S. G., McCall, K. and Gorski, S. M. (2008). Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. J. Cell Biol. 182(6): 1127-39. PubMed Citation: 18794330

Juhasz, G. and Sass, M. (2005). Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues. Eur. J. Cell Biol. 84: 491-502. PubMed Citation: 15900708

Juhasz, G., Erdi, B., Sass, M. and Neufeld, T. P. (2007). Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev. 21: 3061-3066. PubMed Citation: 18056421

Klionsky, D. J. (2007). Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8: 931-987. PubMed Citation: 17712358

Kumar, S. (2004). Migrate, differentiate, proliferate, or die: pleiotropic functions of an apical 'apoptotic caspase'. Sci. STKE pe49. PubMed Citation: 15479862

Kuranaga, E. and Miura, M. (2007). Nonapoptotic functions of caspases: caspases as regulatory molecules for immunity and cell-fate determination. Trends Cell Biol. 17: 135-144. PubMed Citation: 17275304

Levine, B. and Yuan, J. (2005). Autophagy in cell death: an innocent convict? J. Clin. Invest. 115: 2679-2688. PubMed Citation: 16200202

Maiuri, M.C., Zalckvar, E., Kimchi, A. and Kroemer, G. (2007). Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 8: 741-752. PubMed Citation: 17717517

Mizushima, N. (2007). Autophagy: process and function. Genes Dev. 21: 2861-2873. PubMed Citation: 18006683

Scott, R. C., Juhasz, G. and Neufeld, T. P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17: 1-11. PubMed Citation: 17208179

Smith, J. E., Cummings, C. A. and Cronmiller, C. (2002). Daughterless coordinates somatic cell proliferation, differentiation and germline cyst survival during follicle formation in Drosophila. Development 129: 3255-3267. PubMed Citation: 12070099

Thorburn, A. (2008). Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis 13: 1-9. PubMed Citation: 17990121

Velentzas, A. D., et al. (2007). Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res. 327: 399-414. PubMed Citation: 17004067

Vernooy, S. Y. (2002). Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death. Curr. Biol. 12: 1164-1168. PubMed Citation: 12121627

date revised: 28 March 2009

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