BIOLOGICAL OVERVIEW

Studies that have demonstrated the presence of at least four caspases and an Apaf-1 homolog (Apaf-1-related-killer) in flies strongly argue the existence of a Drosophila caspase cascade, similar to the mammalian cell death machinery. These findings have also predicted a role for the Bcl-2/CED-9 family in Drosophila apoptotic cell death. The caspases comprise a family of cysteine proteases that participate in a proteolytic cascade, cleaving downstream caspases and a number of cellular proteins that ultimately execute apoptotic biochemical events, such as DNA fragmentation and chromatin condensation. Apaf-1 functions at the initial step in this cascade to activate the initiator caspase, caspase-9, in the presence of cytochrome c and ATP/dATP. The Bcl-2 family of proteins consists of antiapoptotic and proapoptotic members, both of which control the cell-death decision by regulating such processes as mitochondrial cytochrome c release and caspase activation through adapter protein Apaf-1, and/or by neutralizing the effects of opposing Bcl-2 family members. The first Drosophila Bcl-2 protein was described simultaneously in two different laboratories: Igaki, (2000) termed the protein Rob-1, for Drosophila ortholog of the Bcl-2 family-1; Colussi, (2000) termed it Debcl (pronounced debacle) for Death executioner Bcl-2 homolog. Rob-1/Debcl was discovered on the basis of sequence homology, by the presence of expressed sequence tags (ESTs) with sequence resemblance to Bcl-2s in the EST database of the Berkeley Drosophila Genome Project. Although it seems like that the name Death executioner Bcl-2 homologue will prevail, this essay uses both names interchangeably, depending on the study cited.

To investigate whether Debcl is a pro- or anti-apoptotic protein in vivo, transgenic flies were generated with debcl cDNA under control of the yeast UAS-GAL4 promoter. Ectopic expression was then achieved by crossing these flies to various GAL4 drivers. To express debcl in all tissues at various developmental stages, UAS-debcl flies were crossed to hsp70-GAL4 flies; embryos or larvae were then heat shocked. Heat shock-induced expression of debcl results in enhanced levels of TUNEL positive cells (indicating the presence of fragmented DNA accompanying programmed cell death) in the embryo and in larval tissues (Colussi, 2000).

Tissue specific drivers were then used to express debcl during larval development. Ectopic expression of debcl in the posterior region of the eye imaginal disc, using the GMR-GAL4 driver, results in increased acridine orange staining cells in the posterior region of the eye. Similarly, expression throughout the eye imaginal disc of 2nd instar larvae, using the eyeless-GAL4 driver, results in increased TUNEL positive cells in the anterior and posterior regions of the eye. It was predicted that expression of debcl from eye specific drivers would result in adults with ablated eyes, as does expression of reaper, head involution defective, and grim from the GMR enhancer. Surprisingly, and despite the increase in apoptotic cells seen in the imaginal discs, the adult flies from these crosses exhibit only a mild rough eye phenotype, possibly because of the excess number of cells that are normally generated during eye development. However, other UAS-debcl lines, which presumably have a much higher level of expression, result in adults with severely ablated eyes when crossed to GMR-GAL4. debcl has also been expressed in the larval salivary gland using a salivary gland specific driver, 109-88-GAL4; this expression results in a massive increase in acridine orange staining cells and a reduction in the size of the salivary glands. Thus, debcl induces cell death when ectopically expressed in a number of different tissue types during Drosophila development, indicating that Debcl is a proapoptotic protein of the Bcl-2 family (Colussi, 2000).

To further characterize the biological activity of Debcl, debcl was expressed in Drosophila SL2 cells under the control of an inducible insect promoter. Within 16 h of transfection, Debcl induces apoptosis in a majority of the transfected SL2 cells. By 48 h, all debcl transfected cells have been lost. This cell death is partially inhibited by the cell permeable peptide caspase inhibitor zVAD-fmk and much more effectively by baculovirus caspase inhibitor P35, indicating that Debcl-induced apoptosis is, at least in part, mediated by caspases. While zVAD-fmk is an efficient inhibitor of many mammalian caspases, it is not known whether it can inhibit Drosophila caspases as effectively. Therefore, the partial inhibition of Debcl-induced cell death by zVAD-fmk may reflect its inability to efficiently inhibit all Drosophila caspases. To confirm that the cell killing function of Debcl is dependent on caspase activity, debcl transgenic flies were crossed with GMR-p35 flies. In the resulting flies the effect of Debcl in eye ablation is significantly reduced (Colussi, 2000).

In several proapoptotic Bcl-2 members, the BH3 domain is essential for their killing function (Adams, 1998; Gross, 1999a). To determine whether the BH3 domain in Debcl is required for its proapoptotic function, two substitution mutants (L146G and E151G) of the Debcl BH3 domain were generated and their killing activity was analyzed in SL2 cells. The ¹⁴⁶L residue is conserved in the BH3 domains of most proapoptotic Bcl-2 members, whereas ¹⁵¹E corresponds to an acidic residue in most BH3 domains. Whereas the L146G mutation partially inhibits apoptosis induction by Debcl, E151G mutation completely abrogates Debcl-mediated cell killing. Drosophila proteins Grim, Reaper, and Hid are able to induce apoptosis in mammalian cells, despite the fact that mammalian homologs of these proteins have not been found. To determine whether Debcl can also induce apoptosis in mammalian cells, debcl cDNA was cloned in a mammalian expression vector and transfected into NIH 3T3 cells. Most of the debcl-transfected cells undergo apoptosis. When Debcl is cotransfected with expression vectors carrying caspase inhibitors P35, MIHA, or IAP, a substantial decrease in apoptosis is evident. These results indicate that Debcl-induced killing is dependent on its BH3 domain and requires caspase function. In addition to caspase inhibitors, coexpression of prosurvival Bcl-2 and Bcl-x_L proteins also significantly inhibits Debcl-induced apoptosis (Colussi, 2000).

Studies indicated that Rob-1 (aka Debcl) can induce apoptosis by a caspase independent mechanism. Although Rob-1 strongly stimulates caspase activation when overexpressed in S2 cells, its ability to kill cells is barely antagonized by the caspase-related apoptosis inhibitors p35 and DIAP2, or by an active-site mutant of a Drosophila apical caspase, Dronc (Nedd2-like caspase). Furthermore, in transgenic flies, rough-eye phenotype caused by overexpression of Rob-1 is not completely suppressed by coexpression of p35. Similarly, previous studies on the proapoptotic Bax, Bak, and Mtd suggest that they all induce apoptosis in the presence of broad caspase inhibitors. Moreover, both Bax and Bak can induce mitochondrial dysfunction and also kill yeast, which lack endogenous caspases. Both Rpr- and Hid-induced cell death are blocked by coexpression of baculovirus p35. In contrast, overexpression of p35 shows no effect on Rob-1-induced apoptosis. Higher levels of p35 expression exhibit only a slight protective effect against Rob-1-induced cell killing. Similarly, DIAP2, which inhibits both Rpr- and Hid-induced apoptosis, or an active-site mutant of the CARD (caspase recruitment domain)-containing Drosophila caspase, DRONC, shows little effect on Rob-1-induced apoptosis, suggesting that the Rob-1-stimulated cell-death pathway is downstream or independent of DIAP2 and DRONC. Thus, overexpression of Rob-1 induces apoptosis, probably through a caspase-independent pathway partly distinct from that used by other known Drosophila killer proteins (Rpr, Hid, and Grim). Rob-1 overexpressed in human embryonic kidney 293T cells also exhibits a proapoptotic activity that is only slightly inhibited by p35. Thus, Rob-1, like mammalian Bax, Bak, and Mtd, may activate cell death by inducing both caspase-dependent and -independent pathways. The latter pathways probably can be antagonized by an as-yet-unidentified antiapoptotic member of the Drosophila Bcl-2 family. In the nematode C. elegans, in which two Bcl-2/CED-9 family members, CED-9 and EGL-1, have been identified, all cell deaths occur in a caspase-dependent manner. The presence of a Bax-like protein, Rob-1, in Drosophila might indicate the acquisition of a caspase-independent cell death pathway through evolution (Igaki, 2000).

GENE STRUCTURE

cDNA clone length - 1,535-bp (Colussi, 2000).

Bases in 5' UTR - 410-bp

Exons - 3

Bases in 3' UTR - 225-bp

PROTEIN STRUCTURE

Amino Acids - 300 (Colussi, 2000); 325 (Igaki, 2000)

Structural Domains

Alignment analysis reveals that Rob-1 is a Bcl-2-related protein with eight predicted alpha-helices, four Bcl-2 homology (BH) domains (BH1, BH2, BH3, and BH4), and a C-terminal hydrophobic tail. Rob-1 shows significant structural and amino acid homology with all known Bcl-2 family members including Bcl-2, Bcl-xL, Bcl-w, Bax, Bak, Mtd/Bok, and C. elegans CED-9. Rob-1 is most homologous to Mtd/Bok (Hsu, 1997; Inohara, 1998), a proapoptotic member of the mammalian Bcl-2 family, and also shows functions similar to those of this killer protein (Igaki, 2000).

Debcl, whose sequence lacks the first 25 amino acids reported for Rob-1 (Igaki, 2000), contains three of the BH domains (BH1, BH2, and BH3) but lacks the NH2-terminal BH4 domain found in some other antiapoptotic members of the Bcl-2 family (Colussi, 2000), including Rob-1. The COOH terminus of Debcl contains a putative hydrophobic membrane anchor, similar to that found in many Bcl-2-like proteins. Debcl is most similar to the other putative Drosophila Bcl-2-like protein on region 48A-E, sharing 42% identity and 62% similarity in a 169 amino acid stretch. Among the published mammalian Bcl-2 family members, the homology is mostly limited to the regions that comprise the three BH domains. In this region, Debcl shares the highest degree of homology (35% identity, 52% similarity) with Bok, a proapoptotic Bax subfamily member (Adams, 1998; Gross, 1999a). Debcl shares 20% to 30% identity with various prosurvival members of the Bcl-2 family, including A1 (30% identity, 49% similarity), Bcl-2 (25% identity, 41% similarity), Bcl-xL (25% identity, 46% similarity), Mcl-1 (26% identity, 46% similarity), and Bcl-w (21% identity, 41% similarity). The overall structure of Debcl is similar to Bax, Bak, and Bok, all of which contain BH1, BH2, and BH3 domains, a membrane anchor region, and a relatively long NH2-terminal region (Colussi, 2000).

date revised: 6 March 2000

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