InteractiveFly: GeneBrief

BIR repeat containing ubiquitin-conjugating enzyme: Biological Overview | References


Gene name - BIR repeat containing ubiquitin-conjugating enzyme

Synonyms - Bruce

Cytological map position - 86A7-86A8

Function - signaling

Keywords - autophagy, apoptosis, inhibitor of apoptosis (IAP) protein, Protein degradation

Symbol - Bruce

FlyBase ID: FBgn0266717

Genetic map position - 3R: 6,138,366..6,157,071 [-]

Classification - BIR-containing ubiquitin-conjugating enzyme E2

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

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-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 (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 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 (Juhasz, 2005). Further, expression of the constitutively active Ras form (RasV12), 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).

A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila

Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, a novel F-box protein, which was termed Nutcracker, was isolated that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process (Bader, 2010).

Most F-box proteins also possess another protein-interaction domain, usually comprising WD40 or LRR motifs, that is responsible for binding the ubiquitylation substrate. Nutcracker belongs to the class of F-box proteins that do not contain a known protein-protein interaction domain, and differs topologically from most F-box proteins in that its F-box domain is at the very C-terminus (Kirk, 2008). Sequence alignments with several F-box-only proteins revealed that Nutcracker shares some limited amino acid similarity with the mammalian FBXO7 protein, which also contains the F-box domain at the C-terminus. Although the sequence conservation is limited primarily to the F-box domain, it is possible that these two proteins share functional properties, as do other proteins that are conserved only within limited regions. For example, the C. elegans p53 protein displays less than 20% overall primary sequence similarity to the human protein, mostly in the active sites, but has been demonstrated to function in related cellular processes. Since FBXO7 has been shown to regulate the stability of cIAP1 (BIRC2) (Chang, 2006), it is possible that these two E3 ligases have a conserved function in caspase regulation (Bader, 2010).

The ubiquitin-proteasome system is implicated in regulating caspase activity. Several studies have shown that the ubiquitylation and degradation of DIAP1 is a means of displacing it from caspases when apoptosis is favored. Also, ubiquitylation of caspases themselves contributes to their regulation by preventing a critical mass of full-length caspases from auto-activation in a normal setting. The wide variety of other ubiquitin-modifying proteins that regulate apoptosis and caspase activity, including Bruce, Morgue and Uba1, imply the existence of an elaborate regulatory network that is controlled by ubiquitylation (Bader, 2010).

In the screen isolated another ubiquitin ligase, a Cullin-3-based complex, was isolated, indicating that caspase activation in this system is tightly controlled by ubiquitin modifications. These two complexes could regulate the stability of the same substrate, as is the case for regulation of Cubitus interruptus (Ci) stability in Hedgehog signaling by both Cullin-1-based and Cullin-3-based complexes, or they might target multiple important substrates. Alternatively, the E3 ligases isolated in the screen might play non-degradative roles in controlling caspase activity. For example, mono-ubiquitylation affects the targeted localization of proteins and these ubiquitin ligases might control the proper localization of caspase regulators. Another possibility is that these E3 ligases mediate the non-classical Lys63 ubiquitin chain addition that is important for protein-protein interaction. Thus, instead of degradation, these proteins might actually control interactions between caspase regulators (Bader, 2010).

Although DIAP1 is the only Drosophila BIR-containing protein that has been shown to directly inhibit caspases in vivo, Bruce has been implicated in modifying apoptosis in several death paradigms, and mutations in its mammalian homolog cause defects associated with excess cell death. This study showed that Bruce can physically bind Nutcracker, and that this interaction is independent of the F-box domain. Therefore, Bruce might be a substrate of Nutcracker. However, it was not possible to determine the steady-state levels of Bruce in nutcracker mutants, so it is as yet unclear whether it is indeed a substrate or a complex partner. The fact that Bruce also binds to another E3 ligase isolated in the screen suggests that this protein is a common regulator of caspase activation during individualization (Bader, 2010).

nutcracker mutants cause a reduction in proteasome activity. This decreased activity does not seem to be due to proteasome mislocalization or a reduction in their numbers, suggesting that Nutcracker controls proteasome activity directly. It is possible that Nutcracker modifies proteasome regulators, which could include, for example, proteins of the regulatory particle of the proteasome. An attractive model is that Nutcracker functions through proteasomes to activate caspases. Caspase activity is tightly controlled by the ubiquitin proteasome system. Therefore, it is possible that local activation of proteasomes controls localized caspase activation (Bader, 2010).

Many questions remain regarding the non-lethal role of caspases in cellular remodeling. For instance, is it a specialized activation that is governed by dedicated proteins, and to what extent are known apoptotic regulators involved in this process? Another intriguing question is how cells tolerate a certain level of caspase activation and avoid destruction by these potentially deadly proteases. Answers to these questions will not only uncover novel caspase regulators, but might also help in understanding how diseased cells, such as cancer cells, manage to escape cell death (Bader, 2010).

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. This study shows that autophagy controls DNA fragmentation during late oogenesis in Drosophila. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. These results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death (Nezis, 2010).

Dying nurse cells exhibit several markers of apoptosis during late oogenesis in Drosophila such as caspase activation, chromatin condensation, and DNA fragmentation. To address the role of autophagy in nurse cell death, transgenic flies were generated carrying a UASp-GFP-mCherry-DrAtg8a transgene. The double-tagged Atg8a protein emits yellow (green merged with red) fluorescence in nonacidic structures such as autophagosomes, and is red only in the autolysosomes due to quenching of GFP in these acidic structures. Upon expression of GFP-mCherry-DrAtg8a in the germline, several GFP-mCherry-DrAtg8a yellow puncta were detected in the cytoplasm of nurse cells during early stage 12. After the completion of transport of the majority of the nurse cell cytoplasm to the growing oocyte during late stage 12, GFP-mCherry-DrAtg8a yellow puncta remained in nurse cell cytoplasm in close proximity to the nurse cell nuclei. Ultrastructural analysis of the nurse cells at the same developmental stage also revealed the presence of autophagosomes in the remaining nurse cell cytoplasm. Interestingly, during late stage 13 when the majority of nurse cells have degenerated, a large number of red structures were observed, indicating that the majority of the autophagosomes became autolysosomes. This was confirmed by ultrastructural analysis through detection of large autolysosomes associated with the condensed and fragmented nurse cell nucleus. These autolysosomes often contained condensed material resembling the material of the fragmented nurse cell nucleus, suggesting that the nurse cell nuclear remnants are removed by autophagy. Indeed, nurse cells of late stage 13 egg chambers expressing UASp-mCherry-DrAtg8a exhibited mCherry-DrAtg8a puncta that are located either adjacent to or attached to the fragmented nucleus, indicative of nuclear autophagy. To further examine the presence of autophagy during late oogenesis in Drosophila, protein trap lines were used that express GFP-tagged Atg5 and Atg8a. Atg5-GFP and Atg8a-GFP were detected as punctae around the nurse cell nuclei during late oogenesis, revealing the presence of autophagic compartments. These findings indicate that autophagy occurs during nurse cell death and degradation in late oogenesis in Drosophila (Nezis, 2010).

To explore the potential role of autophagy in nurse cell death during late oogenesis, germline mutant cells were generated for the core Drosophila autophagy genes atg1 and atg13 and cell death was examined using the TUNEL assay to detect fragmented DNA. Interestingly, in either atg1 or atg13 germline mutants, a significant increase was observed in the number of stage 14 egg chambers that had persisting TUNEL-negative nurse cell nuclei. This phenotype differs from wild-type stage 14 egg chambers, in which nurse cell nuclei can rarely be detected, and those few that remain are exclusively TUNEL positive. TUNEL-positive nurse cell nuclei can be detected in the wild-type egg chambers in earlier developmental stages but not in autophagy germline mutants. To further examine the role of autophagy in nurse cell degeneration, germline mutants were generated for vps34, a member of the class III PI3-kinase complex that is responsible for the production of phosphatidylinositol 3-phosphate, a phosphoinositide required for autophagy. Like the other autophagy mutants, the vps34 germline mutant egg chambers displayed significant increase in the number of egg chambers that had persisting TUNEL-negative nurse cell nuclei during late oogenesis. All autophagy germline mutants exhibited accumulation of Ref(2)P, a marker for autophagic flux, in the nurse cell cytoplasm compared with the wild type, further confirming that autophagy was inhibited. Interestingly, in all the autophagy germline mutants, the persisting nurse cell nuclei exhibited condensed nuclear staining. To examine whether proteolytic processing of caspase-3 was affected by inhibition of autophagy, immunolabeling for cleaved caspase-3 was performed in the atg1, atg13, and vps34 germline mutant egg chambers. Cleaved caspase-3 levels were markedly attenuated in autophagy germline mutants compared with the wild type, with 92% cleaved caspase-3 labeling in w1118 late stage 12-14 egg chambers, 38% in atg13−/− GLCs, and 33% in vps34−/− GLCs late stage 12-14 egg chambers. Together, these data demonstrate that autophagy functions upstream of caspase processing and DNA fragmentation during late oogenesis in Drosophila (Nezis, 2010).

How can autophagy promote caspase activity, DNA fragmentation, and cell death in the same cell? It was hypothesized that proteins crucial for cell survival could be degraded by autophagy, thus promoting cell death. To test this hypothesis, the localization of Drosophila IAPs in the nurse cells was investigated during late oogenesis along with their relationship to the autophagic marker GFP-Atg8a. Three of four known Drosophila IAPs, DIAP1, DIAP2, and dBruce were investigated. DIAP1 and DIAP2 exhibit a rather diffuse cytoplasmic staining that did not colocalize with GFP-Atg8a. In contrast, dBruce exhibited an interesting localization pattern. dBruce could not be detected in stage 10B egg chambers. Interestingly, during early stage 12, colocalization of dBruce and Atg8a-GFP was observed in structures 0.5-1.5 µm in diameter resembling autophagosomes. A similar pattern of colocalization was observed during late stage 12. In contrast, in later stages when nurse cell cytoplasm was completely transferred to the oocyte, dBruce exhibited a diffuse localization pattern mainly in the follicle cells surrounding the nurse cells remnants. These data suggest that dBruce might be degraded by autophagy. To test this hypothesis, the localization of dBruce was investigated in atg1, atg13, and vps34 germline mutants. Significantly, dBruce accumulated in the remaining cytoplasm of the nurse cells of all of these autophagy mutants and formed large aggregates 5-10 µm in diameter. Western blot analyses showed that autophagy germline mutant egg chambers contain higher levels of dBruce protein than wild-type egg chambers. These observations support the hypothesis and indicate that dBruce is degraded by autophagy in the nurse cells during late oogenesis (Nezis, 2010).

It was next asked how dBruce might be targeted for autophagy. p62 is a known adaptor protein that targets substrates for autophagic degradation. It was asked whether the Drosophila orthologue of p62, Ref(2)P may target dBruce for autophagy. Immunofluorescence analysis demonstrated that Ref(2)P staining in the nurse cells of late stage egg chambers has no correlation with the autophagic marker Atg8a-GFP. Additionally, Ref(2)P mutant egg chambers exhibited a normal pattern of DNA fragmentation, cell death, and degradation in the nurse cells during late oogenesis, which suggests that targeting dBruce for autophagy does not depend on Ref(2)P function (Nezis, 2010).

dBruce belongs to the IAP protein family. It contains both BIR (baculoviral IAP repeat, which is responsible for caspase inhibition) and UBC (responsible for ubiquitin conjugation) domains in the N and C termini, respectively. The function was tested of three different dBruce mutant alleles that result in truncated proteins with deletions either in the BIR or UBC domains. Two of them (dBruceE16 and dBrucee00984) have a deletion in the UBC domain, and one of them (dBruceE81) has a deletion in the BIR domain. All dBruce mutant alleles displayed a significant increase in the number of degenerating egg chambers during mid-oogenesis compared with the wild type. To further investigate the role of autophagic degradation of dBruce in nurse cell death, double mutants were constructed for either atg1 and dBruceE81 or vps34 and dBruceE81. Both double mutant egg chambers contained persistent nurse cell nuclei that were TUNEL positive. These data indicate that autophagic degradation of dBruce controls DNA fragmentation in the nurse cells during oogenesis in Drosophila (Nezis, 2010).

The role of autophagy in cell death has been controversial. Previous studies have shown that autophagy promotes cell death in Drosophila larval salivary glands, midgut, and embryonic serosal membrane. However, the precise mechanism by which autophagy executes the death of these cells is not clear. This study has shown that autophagic degradation of the IAP dBruce controls DNA fragmentation in nurse cells during Drosophila late oogenesis. The data also demonstrate that autophagy acts genetically upstream of caspase activation and DNA fragmentation in this developmental context and indicate that autophagy directly contributes to the activation of cell death. This agrees with recent evidence from cultured mammalian cells in which autophagy appears to act upstream of caspase-3 activation under specific experimental settings (Nezis, 2010 and references therein).

dBruce has been previously shown to suppress cell death in the Drosophila eye and also has a crucial function in nuclear degeneration during sperm differentiation in Drosophila. Interestingly, dBruce was recently shown to regulate autophagy and cell death during early and mid-oogenesis in Drosophila. In this earlier study, dBruce and caspase activity were shown to influence autophagy. In contrast, this study provides the first evidence for a mechanism by which autophagy regulates dBruce and cell death. This study provides genetic evidence that dBruce is degraded by autophagy in the degenerating nurse cells during late oogenesis and that it regulates DNA fragmentation. The fact that chromatin condensation is not affected in autophagy mutants indicates that this process is regulated independently from DNA fragmentation (Nezis, 2010),

Degradation of proteins that are crucial for cell survival is one of the mechanisms by which a cell can trigger its own death. For instance, selective depletion of catalase by autophagy has been shown to promote cell death in mammalian cells in vitro. Furthermore, it was recently shown that chaperone-mediated autophagy modulates the neuronal survival machinery by regulating the neuronal survival factor MEF2D, and dysregulation of this pathway is associated with Parkinson's disease. In a recent study, it was also demonstrated that autophagy promotes synaptogenesis in Drosophila neuromuscular junction by degrading Highwire, an E3 ubiquitin ligase which limits neuromuscular junction growth. The current in vivo data further support the idea that autophagic degradation of survival factors can promote cell death and indicate that IAPs can be degraded by autophagy, thereby causing cell death. Autophagy not only functions during late oogenesis as the cause of cell death, but can also function to efficiently degrade the nurse cell nuclei remnants, as previously shown in salivary glands. It was recently reported that dying nurse cells during late oogenesis exhibit characteristics of programmed necrosis and that the lysosomal genes dor, spinster, and cathepsin D are required for this process, showing that autophagy and necrosis participate in nurse cell death and degradation during late oogenesis. In conclusion, these findings indicate that autophagy plays an important role in nurse cell death during late oogenesis in Drosophila, first by acting upstream of DNA fragmentation, thereby causing cell death, and then by scavenging nurse cell remnants (Nezis, 2010).

Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids

Caspases are executioners of apoptosis but also participate in a variety of vital cellular processes. This study has identified Soti, an inhibitor of the Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation (individualization). Evidence is provided that the giant inhibitor of apoptosis-like protein dBruce is a target for the Cullin-3-based complex, and that Soti competes with dBruce for binding to Klhl10, the E3 substrate recruitment subunit. Soti is expressed in a subcellular gradient within spermatids and in turn promotes proper formation of a similar dBruce gradient. Consequently, caspase activation occurs in an inverse graded fashion, such that the regions of the developing spermatid that are the last to individualize experience the lowest levels of activated caspases. These findings elucidate how the spatial regulation of caspase activation can permit caspase-dependent differentiation while preventing full-blown apoptosis (Kaplan, 2010).

Programmed cell death is one of the most fundamental processes in biology. A morphologically distinct form of this active cellular suicide process, dubbed apoptosis, serves to eliminate unwanted and potentially dangerous cells during development and tissue homeostasis in virtually all multicellular organisms. Members of the caspase family of proteases are the central executioners of apoptosis. Caspases start off as inactive proenzymes and are activated upon proteolytic cleavage by other caspases. Apoptotic caspases can also participate in a variety of vital cellular processes, including differentiation, signaling, and cellular remodeling. However, the mechanisms that protect these cells against excessive caspase activation and undesirable death have remained obscure (Kaplan, 2010).

In both insects and mammals, spermatids eliminate their bulk cytoplasmic content as they undergo terminal differentiation. In Drosophila, an actin-based individualization complex (IC) slides caudally along a group of 64 interconnected spermatids, promoting their separation from each other and the removal of most of their cytoplasm and organelles into a membrane-bound sack called the cystic bulge (CB), which is eventually discarded as a waste bag (WB). This vital process, known as spermatid individualization, is reminiscent of apoptosis and requires apoptotic proteins including active caspases. However, the mechanisms that restrict caspase activation in spermatids, as opposed to their full-blown activation during apoptosis, are poorly understood (Kaplan, 2010).

The isolation of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during spermatid individualization has been described (Arama, 2007). Ubiquitin E3 ligases tag cellular proteins with ubiquitin, thereby affecting protein localization, interaction, or turnover by the proteasome. The Cullin-RING ubiquitin ligases (CRLs) comprise the largest class of E3 enzymes, conserved from yeast to human. Cullin family proteins serve as scaffolds for two functional subunits: a catalytic module, composed of a small RING domain protein that recruits the ubiquitin-conjugating E2 enzyme, and an adaptor subunit which binds to the substrate and brings it within proximity to the catalytic module. In Cullin-3-based E3 ligase complexes, BTB-domain proteins interact with Cullin-3 via the eponymous domain, while they bind to substrates through additional protein-protein interaction domains, such as MATH or Kelch domains. A large body of evidence indicates that substrate specificity and the time of ubiquitination are determined by posttranslational modifications of the substrates and the large repertoire of the adaptor proteins. In addition, the Cullins themselves are subject to different types of posttranslational regulation. Most notably, they are activated by a covalent attachment of a ubiquitin-like protein Nedd8 (Kaplan, 2010).

This study has identified a small protein called Soti that specifically binds to Klhl10, the adaptor protein of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during the nonapoptotic process of spermatid individualization. Soti acts as a pseudosubstrate inhibitor of this E3 complex and inactivation of Soti leads to elevated levels of active effector caspases and progressive severity of individualization defects in spermatids. Furthermore, the giant inhibitor of apoptosis (IAP)-like protein dBruce is targeted by this E3 complex, and this effect is antagonized by Soti. Finally, immunofluorescence studies reveal that Soti is expressed in a distal-to-proximal gradient, which promotes a similar distribution of dBruce in spermatids. Consequently, activation of caspases is restricted in both space and time, displaying a proximal-to-distal complementary gradient at the onset of individualization (Kaplan, 2010).

The current study provides insight into how some cells can utilize active caspases to promote vital cellular processes but still avoid unwanted death. According to this model, during early and advanced spermatid developmental stages, a gradient of Soti is generated, allowing graded activation of the Cullin-3-based E3 ubiquitin ligase complex in the opposite direction. This E3 complex then targets dBruce, promoting its distribution in a similar gradient as that of Soti. Subsequently, caspase activation occurs in a complementary gradient descending from proximal to distal. Since the removal of the cytoplasm and caspases also occurs in the direction of proximal to distal, the regions of the developing spermatid that are the last to individualize are also those that are the most protected against activated caspases. This setting ensures that each spermatidal domain encounters similar transient levels of activated caspases throughout the process of individualization (Kaplan, 2010).

The gradual regulation of caspase activity in spermatids is attributed to the outstanding length of Drosophila spermatids (a phenomenon called sperm gigantism). Spermatozoa of Drosophila melanogaster are about 1.9 mm long and other Drosophilids can produce sperm up to 58 mm long. Spermatids in Drosophila individualize over the course of 12 hr through a constant rate of proximal-to-distal individualization complex movement and clearance of the cytoplasmic content (including the active caspases) into a cystic bulge. Since it takes a few hours for the active effector caspasesto kill a cell, spermatids had to develop an efficient mechanism to prevent prolonged exposure of the more distal cellular regions to caspase activity. A gradient of a caspase inhibitor, descending from distal to proximal, is therefore an elegant mechanism to ensure a level of caspase activity that is sufficient to drive spermatid differentiation, yet not high enough to engage an apoptotic program (Kaplan, 2010).

Ubiquitination may target dBruce for either degradation or active redistribution. Because of technical limitations of the in vivo system, the biochemical analyses were performed in a heterologous system using truncated dBruce versions, and thus, we cannot completely rule out the possibility that at least some of the ubiquitinated dBruce is degraded by the proteasome. However, the genetic data support a model where dBruce may be redistributed by an active translocation mechanism, as hyperactivation of the Cullin-3-based complex, following Soti inactivation, leads to accumulation of dBruce at tail ends of spermatids and not to its elimination. This idea is also indirectly supported from the experiment in the eye system, showing that transgenic expression of the dBruce mini-gene enhanced the small eye phenotype caused by expression of Klhl10, suggesting that the Cullin-3-based complex does not target the dBruce mini-gene for degradation in this system. Consistent with this notion, accumulating evidence indicates that the ubiquitin 'code' on target proteins can be read by a large number of ubiquitinbinding proteins, which translate the ubiquitin code to specific cellular outputs, such as protein redistribution. Interestingly, a recent report suggests that Cullin-3-based polyubiquitination of caspase- 8 promotes its aggregation, which subsequently leads to processing and full activation of this protease. Furthermore, another Cullin-3-based ubiquitin ligase complex was shown to regulate the dynamic localization of the Aurora B kinase on mitotic chromosomes. Therefore, Cullin-3-based ubiquitin ligase complexes appear to promote also nondegradative ubiquitination and redistribution of proteins (Kaplan, 2010).

Cullin-RING ubiquitin ligases (CRLs) bind to substrates via adaptor proteins. However, adaptor proteins can also bind to pseudosubstrate inhibitors in a manner which is reminiscent of an E3-substrate-type interaction. Several lines of evidence strongly suggest that Soti is a pseudosubstrate inhibitor of the Cullin-3-based E3 ubiquitin ligase complex in spermatids. (1) The interaction between Klhl10 and Soti is an E3-substrate-type interaction. (2) Soti is not a substrate for this E3 complex. (3) dBruce polypeptides can outcompete with Soti for binding to Klhl10. Finally, Soti is a potent inhibitor of this E3 complex. Therefore, the mechanism of regulation by pseudosubstrates may represent a more common mechanism for modulation of CRL activity than has been previously appreciated (Kaplan, 2010).

Two alternative protein degradation pathways were recently described: N-terminal ubiquitination (NTU) and degradation 'by default'. Whereas the former promotes degradation of proteins by ubiquitination at N-terminal residues, the latter targets proteins for degradation by a ubiquitin-independent, 20S proteasome-dependent mechanism. Although these results cannot conclusively distinguish between these two pathways, two notable mechanistic traits of degradation 'by default' can be also attributed to Soti, including the targeting of intrinsically disordered proteins and their protection by binding to other proteins ('nannies'). Using the FoldIndex tool, Soti was predicted to be intrinsically disordered, while it is stabilized by attachment of a structured Myc-tag to its N terminus. Furthermore, Soti is highly unstable in the absence of its binding partner Klhl10, suggesting that Klhl10 functions as a 'nanny' for its own inhibitor. In conclusion, this study has uncovered a mechanism that restricts caspase activation during the vital process of spermatid individualization. This process appears to be conserved both anatomically and molecularly from Drosophila to mammals (reviewed in detail in Feinstein-Rotkopf and Arama, 2009). Moreover, several recent studies suggest that a similar Klhl10-Cul3 complex is essential for late spermatogenesis in mammals. Therefore, although the mammalian sperm is about 30 times shorter than in Drosophila, similar mechanisms (albeit scaled-down) for regulation of caspase activation may also exist during mammalian spermatogenesis. Further studies of the link between the ubiquitin pathway and apoptotic proteins during sperm differentiation in Drosophila may, therefore, provide new insights into the etiology of some forms of human infertility (Kaplan, 2010).

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, a large collection of sterile male flies was screened for mutants that block effector caspase activation at the onset of spermatid individualization. This study describes the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation (Arama, 2007; full text of article).

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).


REFERENCES

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 ID: 12737804

Arama, E., Bader, M., Rieckhof, G.E., and Steller, H. (2007). A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila. PLoS Biol. 5: e251. PubMed ID: 17880263

Bader, M., Arama, E. and Steller, H. (2010). A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila. Development 137(10): 1679-88. PubMed ID: 20392747

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 ID: 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 ID: 18083103

Chang Y. F., et al. (2006). The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination. Biochem. Biophys. Res. Commun. 342: 1022-1026. PubMed ID: 16510124

Feinstein-Rotkopf, Y. and Arama, E. (2007). Can't live without them, can live with them: roles of caspases during vital cellular processes. Apoptosis 14(8): 980-95. PubMed ID: 19373560

Ferraro, E. and Cecconi, F. (2007). Autophagic and apoptotic response to stress signals in mammalian cells. Arch. Biochem. Biophys. 462: 210-219. PubMed ID: 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 ID: 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 ID: 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 ID: 18056421

Kaplan, Y., et al. (2010). Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids. Dev. Cell 19(1): 160-73. PubMed ID: 20643358

Kirk R., et al. (2008). Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor. J. Biol. Chem. 283: 22325-22335. PubMed ID: 18495667

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

Kumar, S. (2004). Migrate, differentiate, proliferate, or die: pleiotropic functions of an apical 'apoptotic caspase'. Sci. STKE pe49. PubMed ID: 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 ID: 17275304

Levine, B. and Yuan, J. (2005). Autophagy in cell death: an innocent convict? J. Clin. Invest. 115: 2679-2688. PubMed ID: 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 ID: 17717517

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

Nezis, I. P., et al. (2010). Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J. Cell Biol. 190(4): 523-31. PubMed ID: 20713604

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 ID: 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 ID: 12070099

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

Velentzas, A. D., et al. (2007). Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res. 327: 399-414. PubMed ID: 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 ID: 12121627


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