Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Overexpression of Drosophila Ice is shown to sensitize Drosophila cells to apoptotic stimuli, and expression of an N-terminally truncated form of Ice rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of Ice. Ice is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and Ice is expressed at all the stages of Drosophila development at which PCD can be induced. Drosophila caspases Dcp-1, Ice, Decay, and Damm lack long prodomains and are thus similar to downstream effector caspases in mammals. Taken together, these results strongly argue that Ice is an apoptotic caspase that acts downstream of rpr. Identification of Ice should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening (Fraser, 1997a).

Generation of functional sperm in all metazoan animals requires the elimination of most of the cytoplasm to generate a highly condensed, compact cell. The molecular and cellular mechanisms that drive this process are poorly understood. Evidence is provided that the elimination of the cytoplasm during terminal differentiation of elongated spermatids involves an apoptosis-like process. However, unlike 'regular' apoptosis, this process is restricted to the cytoplasmic compartment. The Effector caspase Ice is activated during Drosophila spermatogenesis and is necessary, along with other effector caspases, for the removal of cytoplasm and the generation of functional sperm. Other key proapoptotic proteins are also expressed and become upregulated during Drosophila spermatogenesis. These observations suggest that an apoptosome-like complex is assembled prior to individualization and is important for the removal of bulk cytoplasm from spermatids (Arama, 2003).

The final stage of spermatid terminal differentiation involves the removal of their bulk cytoplasm in a process known as spermatid individualization. Apoptotic proteins play an essential role during spermatid individualization in Drosophila. Several aspects of sperm terminal differentiation, including the activation of caspases, are reminiscent of apoptosis. Notably, caspase inhibitors prevent the removal of bulk cytoplasm in spermatids and block sperm maturation in vivo, causing male sterility. Loss-of-function mutations were identified in one of the two Drosophila cyt-c genes, Cyt-c-d; these mutations block caspase activation and subsequent spermatid terminal differentiation. Finally, a giant ubiquitin-conjugating enzyme, Bruce, is required to protect the sperm nucleus against hypercondensation and degeneration. These observations suggest that an apoptosis-like mechanism is required for spermatid differentiation in Drosophila (Arama, 2003).

Spermatogenesis in Drosophila takes place within individual units known as cysts. Each cyst contains 64 spermatids that remain initially connected after meiosis via cytoplasmic bridges and differentiate synchronously. During terminal differentiation, the round-shaped spermatids are transformed into thin, approximately 2 mm long spermatozoa with highly elongated, 'needle-shaped' nuclei. In the final stage of spermatogenesis, termed individualization, the cytoplasmic bridges are disconnected and most of the cytoplasm is expelled, leading to individual sperm. The individualization process involves the assembly of a cytoskeletal-membrane complex, referred to as the 'individualization complex' (IC), which contains actin as its major cytoskeletal component. The IC can be detected by staining with phalloidin, which binds to actin. In addition, lamin Dm0 leaves the vicinity of the nucleus and translocates as a component of the IC and thus can also be a useful marker of the IC. The IC is assembled at the nuclear end of the cyst and subsequently translocates caudally along the entire length of the spermatid bundle, expelling most of the cytoplasm in the process. The discarded cytoplasm accumulates in a membrane-enclosed structure, termed the waste bag (WB). The WBs eventually undergo fragmentation and subsequent degradation (Arama, 2003).

To investigate the possible occurrence of apoptosis during Drosophila sperm differentiation, live wild-type testes were stained with the vital dye acridine orange (AO), which specifically detects apoptotic cells. AO staining was observed in intact cystic bulges (CBs) and WBs. The staining of the CB and WB with AO in spermatids undergoing individualization suggests that the apoptotic program is activated in the late stages of sperm differentiation, and that CB and WB resemble apoptotic corpses without nuclei (Arama, 2003).

In mammals, mitochondria are an important organelle for the induction of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome c, which binds to and activates Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. This study presents evidence that the cytochrome c encoded by the Cyt-c-d gene is required for the activation of the effector caspase Ice at the onset of spermatid individualization. Loss-of-function mutants for Cyt-c-d are homozygous viable but male-sterile. Significantly, these mutants are defective in Ice activation and fail to exclude the bulk cytoplasm, producing phenotypes virtually identical to the ones resulting from the application/expression of caspase inhibitors. This provides compelling evidence of a role for the Cyt-c-d gene for caspase activation during spermatogenesis in Drosophila. Interestingly, it has been suggested that only Cyt-c-p, but not cyt-c-d, functions in respiration. Consistent with an essential role of Cyt-c-p in respiration, a P element insertion into this locus results in recessive lethality. Likewise, targeted gene inactivation of the murine cytochrome c gene causes very early embryonic lethality, and this has precluded functional studies on the role of cytochrome c for caspase activation during normal development in mammals. It is proposed that the two cytochrome c genes in Drosophila fulfill distinct functions in respiration (cyt-c-p) and caspase activation/apoptosis (cyt-c-d). Previous arguments against a role of cytochrome c for caspase activation in Drosophila were largely based on the failure to detect release of cytochrome c from mitochondria. However, because cyt-c-d is expressed at much lower levels than cyt-c-p, it would be virtually impossible to detect the release of the relevant protein in the absence of highly specific antibodies. Furthermore, because cyt-c-d null flies are viable and, apart from male sterility, have no obvious anatomical defects, it is unlikely that this gene is broadly required for the activation of apoptosis. A complete block of apoptosis in Drosophila interferes with normal embryogenesis, and mutants with significantly reduced apoptosis can be viable but are phenotypically abnormal. Therefore, the loss of cyt-c-d function may affect and/or delay apoptosis in somatic tissues, the main function of this gene appears to be in caspase activation during spermatid differentiation (Arama, 2003).

Effector caspases, such as Ice, Dcp-1, and caspase-3, normally can cleave a variety of nuclear targets, including lamins, I-CAD, and PARP. Therefore, the sperm nucleus must be protected against this potentially lethal activity of Ice. The data indicate that Bruce, which encodes a giant E2 ubiquitin-conjugating enzyme, may exercise this function. Loss of Bruce function results in nuclear hypercondensation, degeneration, and male sterility, consistent with a role of dBruce to restrain or limit caspase activity. Interestingly, Bruce contains a BIR domain, a motif also found in IAPs. This suggests that Bruce may bind to either caspases or Reaper/Hid/Grim-like (RHG) proteins. Previous work has argued against RHG proteins as direct targets for Bruce. Therefore, it is attractive to speculate that Bruce functions by directly binding to and degrading caspases. Obviously, this proposed function would have to be spatially restricted during spermatogenesis, for example, by localizing Bruce to protected compartments, or by spatially limiting its E2 activity. An additional possibility is that Ice activation occurs only locally, in the affected compartment. Consistent with this idea, strong CM1 staining was only observed distal to the nuclei, in the cytoplasmic compartment that will be eliminated. One plausible mechanism for locally restricting Ice activation may be the local release of the 'minor' cytochrome c from mitochondria, which are known to undergo dramatic morphological changes only in the postindividualized portion of the cyst (Arama, 2003).

Terminal differentiation of sperm shares many morphological and biochemical features with apoptosis. However, rather than causing the death of the entire cell, in this case apoptotic proteins are used to specifically eliminate cytoplasmic components, thereby producing a highly specialized living cell. Interestingly, a similar phenomenon is observed in mammals. As in Drosophila, intracellular bridges between spermatids and the bulk of the spermatid cytoplasm need to be eliminated during mammalian spermatogenesis. In mammals, the cytoplasm collects in the residual body (RB), which is functionally homologous to the WB in Drosophila. Consistent with this idea, mammalian RBs display several features of apoptosis. Although a role of caspases for the removal of bulk cytoplasm during mammalian spermatogenesis remains to be established, preliminary data show that active caspase-3 is present in RBs in the testes of mice. Consequently, there are both anatomical and biochemical similarities between insects and mammals that warrant more detailed studies. This is not only of academic interest, since various types of caspase inhibitors are being considered as drugs for therapeutic purposes, and effects on human fertility have not been studied. Furthermore, the abnormal spermatozoa with residual cytoplasm resulting from caspase inhibition in Drosophila bear a striking resemblance to one of the most commonly seen abnormalities of human spermatozoa, known as cytoplasmic droplet sperm. Therefore, it is possible that defects in proper caspase activation may be responsible for this pathology, and further studies of apoptotic proteins may shed light on the etiology of some forms of human male infertility (Arama, 2003).

GENE STRUCTURE

cDNA clone length - 1949

Bases in 5' UTR - 245

Exons - 1

Bases in 3' UTR - 684

PROTEIN STRUCTURE

Amino Acids - 339

Structural Domains

The suppression of cell death in Drosophila by p35 expression in vivo strongly indicates a role for caspases in the cell death machinery of Drosophila. To search for these caspases, a degenerate PCR-based strategy was used. A unique band of ~200 bp was obtained after performing PCR on a 4-8 h embryonic D. melanogaster cDNA library and this was used to probe the same cDNA library. The resulting full-length cDNA was sequenced and found to contain a single ORF, encoding a protein with 38.9% identity with human CPP32 and Mch2 and 30.4% with C. elegans CED-3. The predicted protein, Ice, contains all the residues required for catalysis by caspases. The catalytic cysteine (C211) sits in a QACQG pentapeptide, as is the case for certain mammalian caspases FLICE/MACH1 and Mch4. The predicted small subunit contains a region similar to the P4 specificity loop of human CPP32beta shown to be critical in determining its substrate specificity for an aspartic acid residue at the P4 position rather than a large hydrophobic residue and Ice might therefore be predicted to share such a specificity. CED-3 also contains this P4-specificity region and shares similar substrate specificity with CPP32beta, from which it is inferred that all three proteases, from widely divergent organisms, probably share similar substrate specificity. Ice also contains an unusual N-terminal region which contains 30.8% Ser and 28.2% Gly (S32-Y69). The only other IRP known to contain a similar sequence is CED-3, whose N-terminal (S132-G206) highly Ser-rich (36.5%) region is of unknown function. Drosophila caspases Dcp-1, Ice, Decay, and Damm lack long prodomains and are thus similar to downstream effector caspases in mammals. Given the current emerging picture in which the N-terminal prodomains of caspases couple these enzymes to upstream regulators, the conservation of such a motif may have functional significance. Moreover, CED-3 is processed at D221 which is immediately C-terminal to the Ser-rich region and, since Ice also has an aspartic acid residue (D80) at the immediate C-terminal end of its Ser-rich region, this suggests that D80 might be the Ice N-terminal processing site (Fraser, 1997a).

EVOLUTIONARY HOMOLOGS

See Drosophila Death Caspase-1 for information on eukaryotic caspases

date revised: 8 August 2003

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