Gene name - reaper
Cytological map position - 75C1
Function - programmed cell death
Keyword(s) - programmed cell death
Symbol - rpr
Genetic map position - 3-
Classification - death domain protein
Cellular location - cytoplasmic
Programmed cell death, or apoptosis, is the regulated elimination of cells that occurs naturally during the course of development. The same process is carried out in many pathological circumstances that required cell death for the benefit of the organism. This delibrate elimination of cells occurs in a morphologically distinct manner suggesting an active, gene-directed process. Investigation of the pathways involved in apoptosis provides a fascinating exercise in unraveling complex gene interactions.
In Drosophila, the earliest normal appearance of non-pathological cell death is observed in three places in the head, in the dorsal cephalic region, within the gnathal segments, and in the clypeolabrum as the germ band begins to retract (stage 11). Thereafter, as germ band retraction [Images] proceeds (stages 12 and 13), cell death becomes widespread throughout the embryo, particularly in the ventrolateral portions and around the procephalic lobes. Cell death becomes prominent within the most posterior abdominal segments; early signs of degeneration along the ventral midline can be observed within the most anterior thoracic segments. Scattered cell deaths also begin to appear in a segmentally reiterated pattern within the lateral portions of the ventral region, and in the ventral neurogenic region. Large numbers of degenerating cells accumulate in the interstitial spaces beneath the dorsal ridge. During dorsal closure [Images], a zone of degenerating cells, organized in the shape of a ring, forms around the closing dorsal tissue (stage 14). As head involution becomes well advanced (stage 15), zones of death apparent in earlier stages subside, and scattered subepidermal death appears throughout the embryo. Eventually, prominent cell death appears throughout the CNS as the ventral nerve cord condenses (stage 16). With the exception of death in the tightly packed cell body layer of the CNS, cell death is accompanied by the engulfment of dying and dead cells by circulating phagocytic macrophages (Abrams, 1993).
As with many complex developmental processes, programmed cell death requires a large number of interacting proteins. Central to the process is the gene reaper (rpr). The intriguing aspect of reaper is its possession of a conserved sequence domain called the "death" domain, involved in the apoptosis process in many other species. What is the death domain and how does it function in apoptosis? Once the sequence of Reaper was made available in the literature, it was noted that Reaper bears homology to mammalian proteins involved in programmed cell death. The mammalian receptor Fas is involved in immune regulation. Likewise activation of the mammalian receptor Tumor necrosis factor receptor 1 (TNFR1) can lead to apoptosis. Both Fas and TNFR1 intracellular domains bear homology to Reaper (Golstein, 1995).
Two other Drosophila genes, grim and Wrinkled, also known as head involution defective (hid), are closely linked to reaper on the chomosome. Both proteins bear sequence homology to Reaper and other death domain proteins and both are involved in programmed cell death. Whereas reaper is a relatively small protein of 65 amino acids, HID is a large novel 410 amino acid protein with homology to RPR at its N-terminal region (Grether, 1995). As with rpr, ectopic expression of hid and grim is sufficient to induce apoptosis. Grim is sufficient to elicit apoptosis in at least one context, where RPR expression is not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).
Mutants of reaper contain many extra cells and fail to hatch, but many other aspects of development appear to be quite normal. One cell type that normally undergoes programmed death in insects is the abdominal neuroblast. In the fly, approximately 25 cells are born in each abdominal neuromere, but only six cells persist to eventually produce neurons in the imaginal ganglia. In rpr mutants 20 or more cells are found in some abdominal segments. A similar increase is found in the number of cells in the larval photoreceptor organ (White, 1994).
Deletion of reaper protects embryos from apoptosis caused by x-irradiation and developmental defects. Mutation of the gene crumbs leads to widespread defects in the development of the epithelial tissues, followed by massive cell death during embryogenesis. reaper deletion blocks the massive ectopic death seen in crumbs mutant embryos (White, 1994).
Reaper and other death domain proteins initiate a cascade of protein activity that includes activation of cysteine proteases known as ICE/CED-3 like proteases. The death domain is likely to be a protein interaction domain, assemblying other proteins involved in the activation of ICE proteases. Direct evidence that proteases are centrally involved in the regulation of the cell death process has come from studies on the nematode C. elegans. A series of genes that control various elements of the programmed cell death process in this worm have been identified, two of which, ced-3 and ced-4 are required for cell death during development. Subsequently, ced-3 was found to exhibit significant homology to mammalian Ice, which converts the 33 kDa protease form of Interleukin-1ß to an active 17.5 kDa form. Ectopic expression of ice in fibroblasts results in apoptosis, suggesting that Ice is both functionally as well as structurally homologous to ced-3 (Martin, 1995 and references).
Other evidence suggests that cell death induced by Reaper occurs by a mechanism distinct from that induced by mammalian death domain proteins. Transient expression of Drosophila rpr in the lepidopteran SF-21 cell line induces apoptosis displaying nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Inhibitors of ICE-family proteases p35 and crmA, as well as members of the iap class of genes, Op-iap and D-iap2, but not bcl-2 family members (see death executioner Bcl-2 homologue), block rpr-induced apoptosis. Mutational analysis of rpr provides no support for the proposed sequence similarity of Reaper and death domain proteins. Mutations in the N-terminal region of Reaper, which displays sequence similarity to Hid and Grim, other Drosophila gene products, correlate with the initiation of apoptosis, suggesting that these residues might be functionally important. The mammalian cDNA encoding FADD (Fas-associating protein with a death domain) also induces cell death in SF-21 cells, but death progresses more slowly and with features which distinguished it from rpr-induced apoptosis. Several bcl-2 family members delay or block FADD-induced cellular death in SF-21 cells (For information about FADD and bcl-2 see the Reaper Evolutionary Homologs section). Thus, apoptosis initiated by Reaper progresses by a faster path, one which appears to differ from that of FADD-induced apoptosis. Mutational analysis or residues originally proposed to be conserved between Reaper and death domain proteins fails to support a functional significance for these proposed sequence alignments. Mutations in the residues that show the strongest conservation display no observable alteration in Reaper activity. It is concluded that cell death induced by Reaper occurs by a mechanism distinct from that induced by mammalian death domain proteins. Reaper operates through a distinct alternative cell death pathway (Vucic, 1997b).
The first Drosophila caspase identified is known as Death Caspase-1. Inhibitors of ICE/CED-3 proteases have also been identified. Existence of these inhibitors, provides indirect evidence that ICE/CED-3 proteases are involved in Drosophila programmed cell death. Drosophila inhibitors of apoptosis function to block cell death induced by Reaper or Wrinkled/Head involution defective. Two proteins, Drosophila IAP1 and Drosophila IAP2 (DIAP1/Thread and DIAP2 respectively) are homologous to a baculovirus protein IAP, a protein that can block cell death induced by stimuli other than viral infection. Baculoviruses are insect viruses that infect Bombax mori, as well as other insects. Drosophila IAP1 is allelic to the gene thread (th), which when mutated gives rise to viable flies whose aristae lack normally occurring side branches. DIAPs share common domains with baculovirus IAP as well as mammalian IAPs and human apoptosis inhibitory protein. DIAPs as well as most other members of this protein family contain RING finger motifs at the C-terminus, thought to be a protein interaction motif that forms zinc-binding sites. The proteins also contain two or three tandem repeats of about 70 amino acids, termed the BIR motif. N-terminal fragments of IAPs containing the BIRs are sufficient to prevent normally occurring and ectopically induced cell death. Mammalian IAPs inactivate interleukin-1ß converting enzyme (ICE)-like cysteine proteases known to play an important evolutionarily conserved role in bringing about cell death (Hay, 1995 and Martin, 1995 and references).
The Drosophila proapoptotic proteins (Reaper, HID, and Grim) are substrates for IAP-mediated ubiquitination. Ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, regulation of Reaper by ubiquitination has been shown to be a significant factor in determining Reaper biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance (Olson, 2003).
Drosophila Reaper can induce rapid apoptosis in vitro using an apoptotic reconstitution system derived from Xenopus eggs. To directly demonstrate Reaper-induced activation of caspases in Xenopus extracts, trace amounts of various 35S-labeled substrates of these proteases were added to the extract. The substrates used were poly(ADP) ribose polymerase (PARP), the zymogens pro-caspase 3 (Yama/CPP32), pro-caspase 1 (ICE), and pro-caspase 7 (ICE-LAP3) and baculovirus p35, which acts as a competitive inhibitor of caspases while being cleaved by them. With the sole exception of pro-caspase 1, all of these substrates are cleaved to their characteristic apoptotic fragments in extracts to which Reaper has been added. Routinely, cleavage of all these proteins precedes the initial stages of nuclear fragmentation by ~10 min. It is unclear whether the failure to cleave pro-caspase 1 reflects the fact that caspase 1 is not activated during Reaper-induced apoptosis or Is due to some incompatibility between the heterologous components in this assay (Xenopus extract, Drosophila Reaper protein and human caspase 1). However, it is striking that Reaper can engage the Xenopus apoptotic machinery, triggering endogenous protease activation (Evans, 1997).
It is known that regulated release of cytochrome c from mitochondria accompanies activation of the apoptotic program, both in mammalian cells and in the Xenopus cell-free system. Bcl-2, a potent inhibitor of apoptosis, acts, at least in part, by inhibiting cytochrome c release. The absence of cytochrome c in the reconstituted Xenopus extracts (lacking mitochondria) might account for the inability of Reaper to induce apoptosis. Therefore, bovine or equine heart cytochrome c were added to the reconstituted extracts in the presence and absence of Reaper and the appearance of apoptotic changes was examined. Between 70 and 85 min after initiation of room temperature incubation the nuclei in these extracts begin to enter apoptosis, as monitored by fluorescence microscopy, regardless of whether Reaper is present. This is in marked contrast to extracts lacking cytochrome c, which show no signs of apoptotic nuclear fragmentation, even 6 h after Reaper addition. These results were confirmed at a biochemical level by analysis of baculovirus p35 degradation. Addition of cytochrome c to extracts lacking mitochondria promotes rapid apoptotic cleavage of p35 regardless of whether or not Reaper is present. Even after careful titration a concentration of cytochrome c which can accelerate apoptosis only in the presence of reaper cannot not be found. Interestingly, addition of free cytochrome c to extracts containing mitochondria also greatly accelerates apoptosis, consistent with the idea that release of cytochrome c from mitochondria is a rate limiting step in this process. Taken together these data suggest that Reaper might induce downstream release of cytochrome c from mitochondria, thereby triggering caspase activation. To determine whether Reaper can indeed induce mitochondrial cytochrome c release, Reaper was incubated in unfractionated Xenopus egg extracts. Aliquots were assayed at various times for cytochrome c release and caspase activity. Although the exact timing of apoptotic events varies between extracts, Reaper accelerates both the release of cytochrome c from mitochondria and the consequent activation of DEVD-cleaving caspases. Bcl-2 antagonizes these effects, but high levels of Reaper can overcome the Bcl-2 block. These results demonstrate that Reaper can function in a vertebrate context, suggesting that Reaper-responsive factors are conserved elements of the apoptotic program (Evans, 1997).
Many questions remain to be resolved. How is reaper transcriptions regulated, that is, what are the developmental and regulatory signals that induce apoptosis? What are the immediate targets of Reaper in the cell death regulatory hierarchy? What are the targets of ICE/CED-3 proteases, and how do these targets function to regulate apoptosis?
Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, because mutation of the very first amino acid leads to loss of interaction with IAPs and the concomitant loss of Smac/DIABLO function. The high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP is reported in this study. These results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighboring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, these results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, this structural analysis identifies potential targets for drug screening (Wu, 2000).
date revised: 21 September 2003
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