BIOLOGICAL OVERVIEW

Sickle (Skl), a novel Drosophila cell death protein binds to inhibitors of apoptosis proteins (IAPs) and neutralizes their apoptotic inhibitory activity. IAPs interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies, relieve IAP-mediated inhibition to induce cell death. Recently, the human serine protease Omi/Htra2 has also been identified as an IAP-inhibitory protein. IAP-inhibitory proteins bind, via a conserved N-terminal IAP binding motif (IBM), to the same BIR domains of IAPs that inhibit caspases, suggesting that they relieve the caspase-inhibitory activity of IAPs by disrupting the caspase-IAP interaction. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members (Srinivasula, 2002).

To identify additional IAP-inhibitory proteins, the entire public GenBank database was searched for genes that encode proteins with a conserved IBM at their N termini. This resulted in identification of a novel Drosophila gene of unknown function mapping near the reaper/grim/hid region of chromosome 3L. This gene consists of one exon and encodes a 108 amino acid protein. Based on the ability of this protein to induce cell death and to synergize with Grim, it was named Sickle. Sequence analysis of Skl has revealed that its N-terminal 4 residues share significant homology with the N-terminal IBM of the Drosophila proteins Hid/Grim/Reaper and mature mammalian Smac/DIABLO and caspase-9-p12. Intriguingly, Skl has an overall acidic pI (pI ~4.5), which is different from those of Reaper, Hid, or Grim but very similar to that of Smac/DIABLO (Srinivasula, 2002).

The significant homology in the IBM among Skl, Reaper, Hid, and Grim, as well as the close proximity of their genes on chromosome 3L, suggests that Skl, like Reaper/Hid/Grim, might play an important role in developmental cell death in Drosophila by binding and inhibiting the activity of IAPs. To test this hypothesis, the interactions were examined of GST-tagged Skl, Grim, and Smac/DIABLO with Drosophila DIAP1Thread or DIAP2 or with their isolated BIR domains in vitro. Skl, like Grim and Smac/DIABLO, is able to interact strongly and specifically with either of the full-length proteins or with the BIR2 of DIAP1 or BIR3 of DIAP2. However, unlike Grim or Smac/DIABLO, Skl is also able to interact with the BIR1 domain of DIAP1 (Thread). Subsequent analysis of the interactions of N-terminal truncated Skl mutants with Drosophila DIAP1 or DIAP2 or with human XIAP has revealed that deletion of the first 10 N-terminal residues of Skl (DeltaN10), which contain the IBM, or mutation of the first Ala of the IBM to Met (A2M) completely prevents the interactions of Skl with these IAPs. These data indicate that Skl is a bona fide IAP binding protein and that its interactions with IAPs are mediated by its N-terminal IBM (Srinivasula, 2002).

The ability of Skl to bind to IAPs suggests that it could neutralize the caspase-inhibitory activity of IAPs by binding to free IAPs or by disrupting already formed IAP-caspase complexes. Therefore, the ability of purified Skl protein to inhibit the activity of IAPs was tested in an in vitro caspase activation system. Addition of Skl to a reaction containing DIAP1 and the Drosophila caspase DCP-1 results in almost complete inhibition of DIAP1, as measured by the restoration of DCP-1 (Death caspase-1) activity. The ability to counteract DIAP1 is completely dependent on the IBM of Skl, since deletion (DeltaN10) or point mutation of the first Ala (A2M) of this motif abolishes the caspase-promoting activity (DIAP1 inhibition of DCP-1 is unaffected by these Skl mutants). Skl is also able to promote activation of human caspases in XIAP-containing 293 cellular S100 extracts almost to the same extent as Smac/DIABLO. This activity is also dependent on the IBM of Skl, since deletion or mutation of this motif abolishes its caspase-promoting activity. These observations suggest that the activity of Skl is evolutionarily conserved and that Skl, like Smac/DIABLO, is able to disrupt the interaction of caspases with IAPs to promote caspase activation (Srinivasula, 2002).

The Drosophila Reaper, Hid, and Grim proteins are known to induce apoptosis in mammalian cells. To determine the apoptotic activity of Skl compared to that of Reaper and Grim in mammalian cells, human MCF-7 cells were transiently transfected with C-terminal GFP-tagged Skl, Reaper, or Grim or the DeltaN10 Skl mutant. As expected, WT Skl but not the DeltaN10 mutant is able to induce apoptosis in MCF-7 cells to the same extent as Reaper and Grim. This result indicates that Skl can induce apoptosis in mammalian cells, and that this activity is dependent on its ability to bind to IAPs. Like its activity in mammalian cells, Skl is also able to induce apoptosis in Drosophila S2 cells to a similar extent as Reaper. This apoptosis is inhibited by the baculovirus caspase inhibitor p35, confirming that the apoptotic activity of Skl is caspase dependent (Srinivasula, 2002).

The ability of Skl to induce apoptosis in Drosophila embryos was tested. This was achieved by ectopically expressing the skl coding region using a heat-inducible transgene. Many embryonic cells are induced to undergo apoptosis following skl expression, as visualized by acridine orange staining. Furthermore, it was found that coexpression of Skl and Grim in the eye discs of transgenic flies results in a dramatic reduction of eye size, indicating that the induction of ectopic cell death during eye development can be enhanced by coexpression of two IAP inhibitors. Taken together, the above data clearly demonstrate that Skl is an apoptotic protein, which exerts its activity by binding to IAPs and/or disrupting caspase-IAP complexes and that it can act together with other IAP inhibitors to produce a maximal apoptotic effect (Srinivasula, 2002).

In conclusion, a novel Drosophila cell death protein named Skl has been identified and characterized. Skl belongs to the growing family of IAP binding proteins and exerts its activity through a highly conserved mechanism of interaction of its N terminus with a surface groove on the BIR domain of IAPs. This interaction results in disruption of the caspase-IAP interaction, culminating in induction of apoptosis. The restricted expression of Skl at early stages of Drosophila development and its ability to induce apoptosis in transgenic Drosophila embryos suggest that Skl might be an important regulator of programmed cell death during Drosophila development. The observed synergistic effect of coexpression of Skl and Grim in the eye imaginal disc of transgenic Drosophila suggests that Skl may function together with the other Drosophila IAP-inhibitory proteins to regulate apoptosis during development. In the absence of Reaper, Hid, and Grim, physiological levels of Skl alone may not be sufficient to bind all IAPs and induce cell death during Drosophila development in most tissues. This could explain the reported lack of developmental cell death in the Drosophila H99 deletion mutant, which removes the reaper, hid, and grim genes but not the skl gene. Further, some cell death is unaffected by the H99 deficiency, and skl function may be sufficient for apoptosis in such cases (Srinivasula, 2002).

GENE STRUCTURE

cDNA clone length - 1376

Bases in 5' UTR - 110

Exons - 1

Bases in 3' UTR - 939

PROTEIN STRUCTURE

Amino Acids - 108

Structural Domains

While the grim-reaper genes have central roles in regulating cell death activation, there is evidence suggesting the existence of other crucial death activators in Drosophila. Thus, some cell deaths still occur in Df(3L)H99 mutants that lack reaper, hid, and grim. Consequently, it was reasoned that other grim-reaper genes, perhaps also located at 75C, might mediate these deaths. To test this hypothesis, BLASTP searches were performed on the Predicted Proteins Database (Release 1) using open reading frame sequences in the 75C region. One predicted gene, CG13701, was identified that encodes a small 108 amino acid protein where residues 70-102 are 35% identical to residues 32-65 of Reaper, a region including most of the Trp-block. This gene, which is referred to as sickle (skl), is located at 75C5, 40 kb proximal to reaper. Two sickle ESTs, RE33794 and RE10476, were identified, and transcription of the sickle gene is in the same direction as for reaper, hid, and grim. Further inspection of the Sickle protein sequence revealed that it contains a single Trp-block with 26.7% identity to that of Reaper and 16.7% identity to that of Grim. Lower levels of similarity are observed between the Sickle Trp-block and HID. Significantly, Sickle also contains an NH₂-terminal RHG motif which is 35.7% identical to those of HID or Grim and 28.6% identical to that of Reaper. The first half of the Sickle RHG motif, like that of Reaper and Grim, is mostly hydrophobic, while the second half of this RHG motif, like that of HID, is highly hydrophilic. The Sickle RHG motif is unique in containing three consecutive glutamates, including Glu7 and Glu8 that correspond to conserved hydrophobic residues in Reaper, HID, and Grim that participate in association to DIAP1. These sequence differences likely provide unique functional properties for Sickle, since even the closely related RHG motifs of Reaper and Grim are functionally distinct. The Sickle protein is predicted to have a pKi of 4.67; the first 2/3 of the protein is quite acidic, and the last 1/3 is basic. This contrasts to the basic or neutral pKi predicted for Reaper (pKi = 10.47), HID (pKi = 8.62), and Grim (pKi = 6.9) and further suggests that Sickle may have unique activities. Garnier secondary structure plots predict that residues 62-86 of Sickle will adopt an alpha-helical conformation. This helical region could provide a potential protein interaction interface, perhaps facilitating formation of homodimers or heterodimers with other Grim-Reaper proteins (Wing, 2002).

sickle (skl) was identified from microarray profiling studies designed to identify radiation-responsive genes. skl encodes a predicted protein of 108 amino acids and corresponds to the predicted gene CG13701 (GenBank number AF418220). This gene is located in the cytological region 75C, ~20 kb outside the H99 proximal break point and ~41 kb away (toward the centromere) from reaper. skl contains no introns and is transcribed in the same orientation as the rpr, grim, and hid genes. Alignments of the Skl open reading frame together with Rpr and Grim highlight a shared N-terminal RHG motif, which fits the IAP binding tetrapeptide consensus found among fly and mammalian IAP antagonists. A second region in common with Rpr, Grim, and Hid, referred to as the 'Trp-block', is also present in Skl. Its proximity, orientation, and amino acid sequence potentially extends the boundaries of the Reaper region beyond the H99 interval to a fourth member among a linkage group that regulates IAP function (Christich, 2002).

date revised: 1 June 2002

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