BIOLOGICAL OVERVIEW

Drosophila Nedd2-like caspase, referred to here and in the literature as Dronc (Drosophila Nedd2-like caspase, not to be confused with Death related ced-3/Nedd2-like protein), is a caspase recruitment domain-containing Drosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1 (thread). The dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Ark. Furthermore, extracts made from Ark homozygous mutant flies have reduced ability to process Dronc, showing that Ark is required for Dronc processing. Using the RNA interference technique, it has been shown that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila (Quinn, 2000).

Caspases are cysteine proteases that act as central effectors of programmed cell death. These proteases are synthesized as precursor molecules that are processed in cells undergoing apoptosis to generate two subunits that fold into a tetrameric active enzyme conformation. In mammals, 14 caspases have been described thus far, which can be grouped into two classes based upon the length of their prodomain. Caspases containing a long prodomain, such as caspase-2, -8, -9, and -10, appear to be activated first by a proximity-induced autoprocessing mechanism involving clustering of procaspase molecules, often assisted by specific adaptor molecules. These caspases contain specific protein-protein interaction domains in the prodomain region that mediate their interaction with their respective adaptors or mediate dimerization of procaspase molecules. For example, caspase-2 and caspase-9 contain caspase recruitment domains (CARDs) in their prodomain, whereas caspase-8 and caspase-10 contain two copies of death effector domains (DEDs). The CARD in caspase-2 is required for homodimerization, whereas the CARD in caspase-9 interacts with the mammalian CED-4-like adaptor Apaf-1 (Drosophila homolog, Apaf-1-related-killer). Cytochrome c- and dATP-dependent oligomerization of Apaf-1, followed by interaction of oligomerized Apaf-1 with procaspase-9, results in proximity-induced activation of caspase-9. One of the DEDs in caspase-8 interacts with the DED in the adaptor FADD, a molecule that helps recruit procaspase-8 to activated death receptors of the tumor necrosis factor receptor family. Again, this adaptor-mediated recruitment of the procaspase molecules is believed to be sufficient for caspase activation. Once the caspases containing long prodomains are activated, they are believed to activate downstream caspases that lack specific protein-protein interaction domains, thereby initiating a cascade of caspase activation (Quinn, 2000 and references therein).

In Drosophila, three proteins, Reaper, Hid, and Grim, play critical roles in apoptosis. These proteins act upstream of caspase activation and appear to be required to counteract the caspase inhibitors Diap1 and Diap2, which are Drosophila homologs of the baculovirus inhibitor of apoptosis, IAP. In Drosophila, specific mutations in diap1 (thread) show increased cell death in the embryo, and Diap1 has been shown to bind to and inhibit the activity of Drosophila effector caspases. The Drosophila Apaf-1-related-killer (Ark) and a proapoptotic CED-9/Bcl-2 homolog, Debcl/Drob-1/dBorg-1, have been described. There are seven known caspases in Drosophila, including five published ones (Dcp-1, Dredd, Drice, Dronc, and Decay, and two unpublished ones (Damm and Strica; GenBank^TM accession numbers AF240763 and AF242734, respectively). Dredd and Dronc contain long prodomains carrying DEDs and a CARD, respectively, suggesting that these two caspases may act as upstream caspases. Strica also contains a long prodomain, but it lacks any CARD/DED sequences. However, Dcp-1, Drice, Decay, and Damm lack long prodomains and are thus similar to downstream effector caspases in mammals. A dcp-1 mutation results in larval lethality and melanotic tumors. Additionally, dcp-1 mutants show a defect in transfer of nurse cell cytoplasmic contents to developing oocytes, suggesting that dcp-1 may also be required for Drosophila oogenesis. Because no mutants for other Drosophila caspases are currently available, their precise functions remain unknown. However, a number of indirect observations point to a role for other Drosophila caspases in apoptosis in vivo. For example, dredd mRNA accumulates in embryonic cells undergoing programmed cell death; in nurse cells in the ovary at a time that coincides with nurse cell death, dronc mRNA, although widely expressed during development, is up-regulated by ecdysone in larval salivary glands and midgut before histolysis of these tissues. jAntibody depletion experiments suggest that Drice is required for apoptotic activity in the S2 Drosophila cell line. Furthermore, a deficiency uncovering dredd dominantly suppresses the ablated eye phenotype due to ectopic expression of rpr, hid, or grim, and a mini-gene of dredd reverses this suppression, showing that dredd is important for PCD in vivo (Quinn, 2000 and references therein).

Overexpression of Dronc induces cell death in mammalian cells (Dorstyn, 1999). In two recent studies (Meier, 2000 and Hawkins, 2000), it has been shown that ectopic expression of dronc promotes apoptosis in the developing Drosophila eye that can be suppressed by co-expression of diap1. Furthermore, in yeast, Diap1 inhibits Dronc activity, and this inhibition is abrogated by co-expression of Hid or Grim (Hawkins, 2000). Diap1 binds to the prodomain of Dronc, and, consistent with this, expression of a truncated version of Dronc lacking the prodomain results in a more severely ablated eye phenotype that can not be rescued by co-expression of Diap1 (Meier, 2000). These studies also show that a mutated version of dronc (containing a mutation in the caspase active site) acts in a dominant negative manner to suppress rpr- and hid-induced cell death (Meier, 2000). Likewise, a deficiency removing the dronc gene is able to dominantly suppress rpr- and hid-induced cell death in the eye (Meier, 2000), showing that Dronc mediates cell death by Rpr and Hid. In addition, these studies showed that Dronc associates with the effector caspase Drice and is able to process Drice to the active form (Meier, 2000 and Hawkins, 2000). Ectopic expression of dronc at various developmental stages results in apoptosis. The dronc eye ablation phenotype can also be blocked by co-expression of the baculoviral caspase inhibitor p35. Confirming these genetic interactions, Dronc can form complexes with Diap1, Grim, Ark, and P35; however, the interaction between Dronc and Grim or P35 is indirect. Because a direct binding between Dronc and P35 could not be demonstrated in vitro, it is likely that P35 interacts with and inhibits a downstream caspase rather than Dronc itself. One possible candidate is Drice, which has been shown to interact with both P35 (19) and Dronc (Meier, 2000), or Dcp-1, which can cleave P35. Ark is required for Dronc activation because Dronc is poorly processed in extracts from Ark homozygous mutant flies. Because specific dronc mutants are currently unavailable, the technique of RNA interference (RNAi) was used to ablate dronc mRNA in embryos. Dronc is shown to be essential for cell death during embryogenesis and these results suggest a central function for Dronc in the cell death effector machinery in Drosophila (Quinn, 2000 and references therein).

Based on homology and the ability of Dronc to form a complex with Ark, Dronc is expected to be a functional homolog of CED-3/caspase-9. Therefore, Dronc is expected to be downstream of Ark and the proteins of the H99 complex, which are known to induce PCD by activating caspases. Consistent with this, a dronc deficiency or expression of the dominant negative dronc mutant is able to suppress the ablated eye phenotype of GMR-hid and GMR-rpr (Meier, 2000; Hawkins, 2000 and Quinn, 2000). Because overexpression of upstream caspases generally results in autoactivation, ectopic expression of dronc was expected to be epistatic to (downstream of) the H99 genes and Ark. However, halving the dosage of the H99 genes or Ark suppresses the GMR-dronc eye phenotype, suggesting that the H99 genes and Ark are rate-limiting for dronc function (Quinn, 2000). This may be explained by the possibility that Dronc, when overexpressed as a zymogen, is not able to self-activate very efficiently and may therefore be dependent on the dosage of upstream activating genes. Another possibility is that the suppression of GMR-dronc by halving the dosage of the H99 genes may be a result of a feedback amplification loop between active caspases and Rpr, Hid, and Grim (Quinn, 2000 and references therein).

Consistent with the genetic interaction, Dronc forms a complex with the H99 gene product Grim when co-expressed in cells. However, this interaction is not direct and may occur through Diap1, which can bind to Dronc and to Grim (Meier, 2000; Hawkins, 2000 and Quinn, 2000). The significance of the in vivo interaction between Grim and Dronc is unclear and requires further investigation. As a CED-3/caspase-9 homolog, activation of Dronc is expected to require Ark, and, consistent with this, Dronc and Ark form a complex in SL2 cells. A complex of Ark and Dronc has also been observed in 293T cells that results in generation of the cleaved, active form of Dronc. Ark mutant extracts are defective in their ability to generate the cleaved active form of Dronc and have lower levels of active caspases. These results, together with genetic data, suggest that Dronc is likely to be a functional homolog of CED-3/caspase-9 because it is activated by Ark, the CED-4/Apaf-1 homolog. Furthermore, the amino-terminal region of Ark containing the CARD and CED-4 homology domain is sufficient for Dronc binding (Quinn, 2000).

The Drosophila apoptosis inhibitor Diap1 inhibits the activity of Drice and Dcp-1 and is antagonized by Rpr, Hid, or Grim. The data showing a dose-dependent enhancement of GMR-Dronc by diap1 mutations and that Dronc and Diap1 form a complex, are consistent with Diap1 also acting as an inhibitor of Dronc. Diap1 and Dronc interact genetically and biochemically and the prodomain of Dronc is required for the Diap1 interaction (Meier, 2000). Expression of diap1 or diap2 (to a lesser extent) is able to suppress the GMR-dronc phenotype, indicating that Diap2 as well as Diap1 can prevent Dronc-mediated cell killing. However, because a diap2 deficiency does not show a dominant enhancement of GMR-dronc, and Diap2 does not form a complex with Dronc, it is likely that the suppression of GMR-Dronc by GMR-Diap2 is indirect, perhaps via the inhibition of downstream caspases. The genetic and biochemical observations for a role for Diap1, but not Diap2, in suppressing Dronc function are consistent with previous studies showing that diap1 and diap2 function differently in inhibiting cell death. Halving the dosage of diap1, but not diap2, enhances rpr-, hid-, or grim-induced cell death, whereas overexpression of diap1 or diap2 can inhibit rpr- or hid-induced death, but only overexpression of diap1 can inhibit grim-induced cell death. Additional studies are required to further explore the precise roles of Diap1 and Diap2 in the Drosophila cell death pathway (Quinn, 2000).

In summary, Dronc is essential for cell death in early embryos and ectopic expression of Dronc can induce cell death in flies. Furthermore, Dronc is likely to be a functional homolog of CED-3/caspase-9 in flies. The data showing genetic and physical interactions between Dronc and P35, Grim, Ark, and Diap1 provide a framework for further investigation of the PCD pathway in flies (Quinn, 2000).

GENE STRUCTURE

cDNA clone length - 2096 bases

Bases in 5' UTR - 368

Exons - 2

Bases in 3' UTR - 374

PROTEIN STRUCTURE

Amino Acids - 450

Structural Domains

To identify CARD-containing molecules, the GenBank database was searched by using the prodomain of Nedd2 (mouse caspase-2). By using the TBLASTN program, a Drosophila EST was identified with a small ORF that shares 29% sequence identity with the caspase-2 prodomain over a 88-aa stretch. This EST belongs to a cluster of 11 ESTs in the Berkeley Fly Database. Sequencing of two independent cDNA clones revealed the presence of a complete ORF for a caspase-like molecule that was named DRONC (for Drosophila Nedd2-like caspase). In vitro translation of mRNA generated from dronc cDNA produced a 50-kDa protein consistent with the expected size. Over the entire length of the protein, DRONC shares 25% identity (40% similarity) with caspase-2. The region downstream of the prodomain, which encodes the two subunits of DRONC, shares highest homology (27%-28% identity, 44%-48% similarity) with all CPP32-like caspases, including caspase-3, -7, -8, -9, -10, and CED-3. Interestingly, DRONC shares <20% identity with the three known Drosophila caspases. The putative prodomain of DRONC contains a CARD that is similar to the CARDs of caspase-1, -2, -9, and CED-3. A unique feature of DRONC is the sequence PFCRG that encompasses the catalytic Cys (Cys-318) residue, which is distinct from the QAC(R/Q/G)(G/E) sequence found in all other known caspases (Dorstyn, 1999).

DRONC does not contain a typical caspase active site pentapeptide QAC(R/Q/G) (G/E) but instead has the novel sequence PFCRG. Based on the X-ray crystal structure of human caspase-1, the glutamine at position 1 of the pentapeptide forms part of the substrate-binding pocket. A change at this position may therefore indicate that DRONC has a different substrate specificity from that of classical caspases. Although the physiological cellular substrate(s) for DRONC have yet to be determined, it may be of note that DRONC cleaves three ascribed caspase substrates, drICE, lamin Dm_O and DREP-1, in an in vitro assay (Meier, 2000).

date revised: 20 October 2001

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