BIOLOGICAL OVERVIEW

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).

DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. This study shows that Drosophila inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. DIAP2 was found to regulate the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. The data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, these data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles (Ribeiro, 2007).

Caspases are best known for their role in executing apoptosis, however, they also play important signaling roles in nonapoptotic processes, such as regulation of actin dynamics, innate immunity, and cell proliferation, differentiation, and survival. Under such conditions, caspases are activated without killing the cell. Given the irreversible nature of caspase-mediated proteolysis, caspases' activation and activity is subject to complex regulation. After zymogen activation, caspase activity can be controlled by various cellular and viral inhibitors. Interestingly, the viral inhibitors CrmA and p35 neutralize caspases through a fundamentally different mechanism than cellular inhibitors such as the inhibitor of apoptosis (IAP) proteins. CrmA and p35 function as 'suicide substrates,' whereby cleavage of CrmA and p35 is required to block the caspase. Initially, CrmA and p35 are cleaved through a mechanism that resembles substrate hydrolysis. However, during the reaction, the protease's catalytic machinery is trapped via covalent linkage. This strategy is referred to as 'mechanism-based' inhibition because it relies on the caspase's catalytic property. So far, cellular-derived mechanism-based caspase inhibitors have not been identified (Ribeiro, 2007).

IAPs are defined by the presence of one to three copies of the baculovirus IAP repeat (BIR) domain, which functions as a protein interaction module. In addition, certain but not all IAPs also carry a C-terminal RING finger domain, which provides these molecules with E3 ubiquitin-protein ligase activity. In Drosophila, DIAP1-mediated inhibition of caspases is indispensable for cell survival. Mutations that abrogate the physical association of DIAP1 with the effector caspases drICE or DCP-1 or the initiator caspase Dronc cause spontaneous caspase activation and cell death (Ribeiro, 2007).

The involvement of the second Drosophila IAP (DIAP2) in regulating caspases is less clear. Overexpression studies suggest that DIAP2 can suppress cell death induced by the IAP antagonists reaper (Rpr) and head involution defective (Hid) and suppress apoptosis induced by diap1-RNAi. Because diap1-RNAi-mediated death is independent of IAP antagonists, this suggests that DIAP2 can neutralize caspases. Of the seven D. melanogaster caspases, DIAP2 binds only to the effector caspase drICE and the atypical caspase Strica. In contrast, DIAP1 exhibits much broader caspase selectivity because it inhibits Dronc, drICE, and DCP-1. Based on the domain architecture and sequence alignments, DIAP2 is the closest homologue to mammalian IAPs. In contrast, DIAP1 is more related to IAPs from insect viruses. Like the mammalian x-linked IAP (XIAP), DIAP2 carries three BIR domains and a C-terminal RING finger. XIAP blocks apoptosis by acting as a potent enzymatic inhibitor of caspase-3, -7, and -9. Residues located immediately upstream of XIAP's BIR2 domain directly bind to the active site pockets of caspase-3 and -7, thereby obstructing substrate entry. Importantly, XIAP is not cleaved by caspases because it binds to the catalytic pockets of effector caspases in a reverse orientation. Thus, XIAP relies on allosteric mechanisms to block effector caspase activity. XIAP's ability to suppress effector caspases depends on the side chain of Asp148 and, to a lesser extent, Val146. It is predicted that Asp148 must be conserved in all IAPs that neutralize effector caspases. Consistently, Asp148 is conserved in cIAP1 and 2. Intriguingly, DIAP2 also shows homology to XIAP's Val146 and Asp148, as it carries equivalent residues at Val98 and Asp100. In contrast, these residues are absent in DIAP1 (Ribeiro, 2007).

Of all the IAPs, XIAP appears to be the only IAP that potently inhibits caspases in vitro. Other IAPs, such as cIAP1 and 2, are poor inhibitors of caspases under these conditions. cIAP1 and 2 may rely on their E3 ligase activity to block caspases in vivo, making them caspase regulators rather than inhibitors. Although DIAP1 is a relatively good inhibitor of D. melanogaster caspases in vitro, DIAP1-caspase physical association alone is insufficient to neutralize caspases in the fly. In addition to binding, DIAP1 uses two independent mechanisms to regulate caspases. One relies on the E3 ubiquitin protein ligase activity of DIAP1's own RING finger, whereas the other, the 'N-end' rule, functions independently of this domain. The RING finger of DIAP1 is required to target the proform of the initiator caspase Dronc for ubiquitylation and inactivation. In contrast, active effector caspases seem to be neutralized through a mechanism that involves both the RING finger domain as well as the N-end rule degradation machinery that is recruited by DIAP1 (Ribeiro, 2007 and references therein).

In Drosophila, developmentally regulated apoptosis is induced by the IAP antagonists Rpr, Grim, and Hid. Embryos lacking rpr, grim, and hid are virtually devoid of apoptosis and die at the end of embryogenesis with accumulation of supernumeral cells. The current model suggests that Rpr, Grim, and Hid induce apoptosis by binding to the BIR domains of DIAP1, thereby liberating caspases from IAP inhibition. Furthermore, IAP antagonists also deplete DIAP1 protein levels by promoting its degradation and cause mitochondrial permeability. Although Rpr, Grim, and Hid efficiently antagonize DIAP1, they also associate with DIAP2. This implies that DIAP2 contributes to the overall antiapoptotic threshold of a cell, a view that is supported by the notion that RNAi-mediated knockdown of DIAP2 sensitizes cultured cells to stress-induced death. However, analyses using diap2 mutant flies have failed to expose any involvement of DIAP2 in regulating programmed cell death. Instead, they have shown that DIAP2 is required for NF kappaB activation during the innate immune response in D. melanogaster (Ribeiro, 2007 and references therein).

This study used the fluorescence resonance energy transfer (FRET)-based caspase-3 indicator SCAT3 to examine DIAP2's role in regulating caspase activity in vivo. Using diap2 mutant animals, it was found that these animals harbor significantly increased levels of drICE activity. Consistent with higher levels of active caspases, diap2 mutant cells are sensitized to apoptosis after exposure to sublethal doses of x-ray irradiation. Furthermore, DIAP2 tightly associates with the effector caspase drICE and DIAP2-mediated drICE inhibition requires DIAP2 cleavage at Asp100. The data are consistent with the notion that after cleavage, the caspase forms a covalent adduct with DIAP2, which results in the stabilization of the DIAP2-caspase complex. This mode of caspase binding is similar to that of p35 or CrmA. However, in addition to the mechanism-based trapping, DIAP2 also requires a functional RING finger domain to neutralize drICE-mediated cell death. Consistently, it was found that DIAP2 robustly ubiquitylates drICE in vivo (Ribeiro, 2007).

The data suggest that DIAP2 is a caspase regulator that contributes to the caspase activity threshold. Several lines of evidence support this view. First, loss of diap2 causes an increase in basal levels of caspase activity in vivo. Consistent with this increase in basal levels of caspase activity, diap2 mutant cells are sensitized to sublethal doses of x-ray irradiation, and, in tissue culture cells, depletion of DIAP2 by RNAi sensitizes cells to treatment with chemotherapeutic drugs. Second, DIAP2 overexpression in the developing fly eye efficiently suppresses cell death triggered by diap1 RNAi. Importantly, diap1 RNAi causes spontaneous and unrestrained caspase activation and cell death that occurs independently of the action of IAP antagonists. The efficiency with which DIAP2 rescues the diap1 RNAi phenotype is highly reminiscent of that of p35. Both DIAP2 and p35 rescue the diap1 RNAi eye size and pigmentation to an apparent normal morphology but fail to restore the formation of bristles. The notion that DIAP2 expression phenocopies the expression of p35, in the absence of any involvement of IAP antagonists, suggests that DIAP2 can function as a direct caspase inhibitor for a p35-sensitive caspase. Third, DIAP2 physically interacts with the effector caspase drICE and suppresses drICE-mediated cell death. Fourth, endogenous DIAP2 is readily cleaved by caspases, which indicates that DIAP2 encounters caspases in vivo. Moreover, after induction of apoptosis, the timing and extent of DIAP2 cleavage is highly reminiscent to that of DIAP1, which suggests that cleavage of DIAP2 is a relatively early event during apoptosis. Finally, IAP antagonists bind to DIAP1 and 2 with similar efficiencies. Consistent with the view that the caspase inhibitory activity of both IAPs is antagonized by IAP antagonists, it was found that Hid blocks DIAP1 and DIAP2 from binding to drICE. Together, these data support a model in which DIAP1 and 2 coordinately control drICE, thereby blocking the amplification of the caspase cascade. Accordingly, DIAP2 contributes to the apoptotic threshold by regulating drICE. However, DIAP1 controls initiation of apoptosis, as it is the sole IAP that regulates the initiator caspase Dronc and, with it, the formation of the Dronc/Dark apoptosome. Ultimately, programmed cell death is induced when IAP antagonists such as Hid synchronously bind to DIAP1 and 2, allowing apoptosome formation and unhindered amplification of the caspase cascade (Ribeiro, 2007).

Although DIAP2 seems to be a bona fide regulator of drICE, its loss does not cause spontaneous apoptosis. This is most likely caused by its epistatic position in the caspase cascade and its restricted specificity for caspases (see DIAP2's epistatic position in the caspase cascade). Because DIAP2 exclusively regulates drICE and does not bind other caspases, its loss merely causes an increase in caspase activity, presumably because of a small amount of uninhibited active drICE. Under such conditions, drICE activation remains regulated because it requires proteolytic input from Dronc, which, in turn, is under the control of DIAP1. Although loss of DIAP2 results in sublethal levels of active caspases, RNAi-mediated depletion of DIAP1 causes strong, apoptosome-driven activation of downstream effector caspases, which leads to the amplification of the proteolytic signal and death of the cell. Under diap1 RNAi conditions, levels of endogenous DIAP2 seem to be insufficient to suppress the amount of active caspases generated. But, if the levels of DIAP2 are increased, it efficiently blocks diap1 RNAi-mediated cell death. However, when programmed cell death is triggered through the activation of IAP antagonists, both IAPs are targeted equally, thereby releasing the inhibition of the cell death machinery. Thus, controlled activation of caspases and cell survival are ensured by both DIAP1 and 2 (Ribeiro, 2007).

The notion that diap2 mutant animals have increased levels of caspase activity indicates that cells can generate and tolerate a certain amount of activated caspases without undergoing apoptosis. This is consistent with the notion that caspases fulfill important signaling functions independent of executing cell death. For instance, DmIKKepsilon activation was recently found to reduce DIAP1 protein levels, thereby allowing activation of sublethal levels of Dronc, which, in turn, is required for the proper development of sensory organ precursor cells. Thus, modulating caspase activity in a selective, transient, and possibly spatially restricted manner is likely to be a widespread phenomenon that allows caspases to take part in signaling functions without jeopardizing cell viability. Concerted reduction of DIAP1 and 2 protein levels, or their controlled and selective inhibition, may therefore allow controlled activation of caspases and caspase-mediated signaling. The cellular concentration of IAPs would thereby set a cell type-specific threshold for caspase activation and activity. In DIAP2 mutants, however, cells are left in an unbalanced, sensitized state as they accumulate higher than normal levels of active caspases. Accordingly, diap2 mutant third instar larvae are sensitized to apoptosis when subjected to sublethal doses of x-ray irradiation. This sensitized state is only visualized by exposing diap2 mutant animals to sublethal insults. When strong proapoptotic stimuli are used, such as 40 Gy of x-ray irradiation (Huh, 2007) or overexpression of Rpr, Hid, and Grim, diap2 mutant animals display the same cell death phenotypes as WT counterparts (Huh, 2007). Under these strong proapoptotic conditions, IAP antagonists neutralize both IAPs, causing full activation and amplification of the proteolytic caspase cascade. Thus, the contribution of the sublethal amount of active caspases in diap2 mutants seems to be masked by the higher concentration of active caspases generated under such apoptotic conditions (Ribeiro, 2007).

This molecular characterization has uncovered an unexpected mechanism through which DIAP2 restrains the target enzyme drICE. Surprisingly, DIAP2 acts as a pseudosubstrate that, after cleavage, seems to trap the active caspase via a covalent linkage between DIAP2 and the catalytic machinery of drICE. Mutation of the DIAP2 caspase cleavage site abrogates its ability to bind drICE and suppress drICE-mediated cell death. It is unusual for proteins that regulate enzymes to use a mechanism-based strategy, and most types avoid the catalytic machinery by simply blocking the substrate cleft in a lock and key strategy. The cleavage of DIAP2 distinguishes it from all natural inhibitors with the exception of p35, CrmA, serpins, and α-macroglobulins. Serpins and p35 both require cleavage to function as protease inhibitors. p35 arrests proteolysis at the thioacyl intermediate stage, which results in a covalent adduct between p35 and the caspase. In particular, the cleavage site residue Asp87 of p35 links up with the catalytic cysteine of the caspase. Thus, cleaved p35 locks the catalytic machinery of the caspase in a nonproductive inactive configuration. The exquisite sensitivity of the DIAP2-drICE complex to strong nucleophiles such as NH2OH or DTT is diagnostic of a stable thioester adduct between DIAP2 and drICE. Nevertheless, it remains possible that this complex could form because of a disulfide bridge between DIAP2 and the catalytic thiol of drICE. However, the requirement of DIAP2's Asp100 for drICE binding suggests that Asp100 is involved in forming a thioester covalent adduct with the caspase active site Cys. Ultimately, crystal structure or mass spectrometric analysis will be required to identify the chemical nature of this complex. However, the relatively harsh conditions required for sample preparations for mass spectrometric analysis combined with the labile nature of thiol esters will make it difficult to identify a fragment containing a covalent linkage between DIAP2 and drICE. Collectively, the data suggest that the DIAP2-drICE complex is stabilized by a covalent linkage (Ribeiro, 2007).

Despite the notion that p35 and DIAP2 both occupy and produce a covalent linkage with the active site of caspases, the modes of interaction and mechanism of inhibition are very different. Unlike p35, DIAP2 requires additional domains for caspase binding and inhibition. First, DIAP2 needs to associate with drICE through a bimodular interaction, whereby the BIR3 domain binds to the IBM of drICE and the 97-Ser-Val-Val-Asp-100 region of DIAP2 occupies the catalytic pocket of the caspase. Each motif on its own is insufficient for caspase binding as mutation of either the BIR3 or cleavage site interferes with complex formation. Second, physical interaction with drICE is not sufficient to block caspase activity. In addition, DIAP2 requires a functional RING finger domain to regulate drICE-mediated cell death. This is evident because a DIAP2 RING finger mutant fails to suppress cell death induced by RNAi-mediated depletion of DIAP1. Moreover, DIAP2 fails to suppress drICE in vitro. In this respect, DIAP2 functions as mechanism-based regulator (relying on the enzyme's catalytic property to trap it) rather than inhibitor. After capture, DIAP2 targets drICE for ubiquitylation (Ribeiro, 2007).

An important question is why DIAP2 does not function as an inhibitor like p35 if it also establishes a covalent linkage with the catalytic active site cysteine of the caspase. One possibility is that DIAP2 and p35 differ in their ability to protect the highly labile thioester bond from hydrolysis. This view is supported by the notion that the DIAP2-drICE interaction is significantly more sensitive to nucleophile attack than p35-drICE. Thus, in the situation of DIAP2, the thioester linkage with drICE may simply help to stabilize the caspase interaction, whereas in p35-drICE, it is used to irreversibly inhibit the protease. The more labile nature of the DIAP2-drICE complex may allow regulation, which cannot occur with p35. Moreover, p35 inhibits all effector caspases, whereas DIAP2 only regulates drICE. The observation that the nontarget caspase DCP-1 can also cleave DIAP2 but is not inhibited raises the possibility that DCP-1 controls the level of functional DIAP2 through proteolytic inactivation. Examples of such regulation are manifold and include the proteolytic inactivation of serpins by nontarget proteases. Because DIAP2 tightly interacts with drICE, it is likely to control its apoptotic and nonapoptotic functions (Ribeiro, 2007).

Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE

The Drosophila inhibitor of apoptosis protein DIAP1 exists in an auto-inhibited conformation, unable to suppress the effector caspase drICE. Auto-inhibition is disabled by caspase-mediated cleavage of DIAP1 after Asp20. The cleaved DIAP1 binds to mature drICE, inhibits its protease activity, and, presumably, also targets drICE for ubiquitylation. DIAP1-mediated suppression of drICE is effectively antagonized by the pro-apoptotic proteins Reaper, Hid, and Grim (RHG). Despite rigorous effort, the molecular mechanisms behind these observations are enigmatic. This study reports a 2.4 Å crystal structure of uncleaved DIAP1-BIR1, which reveals how the amino-terminal sequences recognize a conserved surface groove in BIR1 to achieve auto-inhibition, and a 3.5 Å crystal structure of active drICE bound to cleaved DIAP1-BIR1, which provides a structural explanation to DIAP1-mediated inhibition of drICE. These structures and associated biochemical analyses, together with published reports, define the molecular determinants that govern the interplay among DIAP1, drICE and the RHG proteins (Li, 2011).

The structural and biochemical information presented in this study gives rise to a model on the interplay of Dronc, drICE, DIAP1, and the RHG proteins. During homeostasis, DIAP1 targets the Dronc zymogen for ubiquitylation and presumably proteasome-mediated degradation. This regulation depends on the interaction between a peptide fragment of Dronc and the conserved groove on DIAP1-BIR2. DIAP1 exists in an auto-inhibited conformation. Caspase-mediated cleavage of DIAP1 after Asp20 disables auto-inhibition, allowing the resulting DIAP1 fragment to bind to and inhibit active drICE. During apoptosis, the RHG proteins use their N-terminal peptides to compete with drICE and Dronc for binding to the conserved peptide-binding grooves on the BIR1 and BIR2 domains, respectively. Such competition results in the release of drICE and Dronc from DIAP1. The freed Dronc zymogen is activated by the Dark apoptosome and the mature Dronc cleaves and activates drICE. In this regard, drICE, DIAP1 and the RHG proteins together provide a fail-safe mechanism to ensure appropriate drICE activation only under bona fide apoptotic conditions (Li, 2011).

The underpinning of this regulatory network is competition among multiple protein-protein interactions mediated by the conserved grooves of the BIR1 and BIR2 domains of DIAP1. Auto-inhibition of DIAP1-BIR1 is achieved by occupation of this groove by its own N-terminal sequence ASVV. The free peptide ASVV does not stably associate with BIR1; the covalent linkage facilitates the binding by increasing the local concentration of ASVV. This binding arrangement allows disabling of auto-inhibition upon cleavage of DIAP1 after Asp20. Inhibition of drICE by BIR1 requires occupation of this groove by the N-terminal sequences ALGS of drICE. Similar to ASVV, the free ALGS peptide exhibited no detectable binding to the BIR1 fragment. Three weak interfaces between BIR1 and drICE cooperate to yield a stable hetero-tetramer with a KD of approximately 1-2 microM. Removal of drICE inhibition by BIR1 depends on the interactions between RHG and the peptide-binding groove, with KD values of 0.12-0.76 microM. Endowing RHG with the strongest interactions ensures an apoptotic phenotype once the RHG proteins are activated in cells. It is acknowledged that the proposed model may be simplistic, as the network of protein-protein interactions and regulation is likely to be more complex in vivo. Nevertheless, the biophysical underpinnings described in this model are likely to have a role in various stages of apoptosis regulation (Li, 2011).

An IAP-binding motif, though not ostensibly abbreviated as IBM, was originally defined to contain four contiguous amino acids that resemble the Smac tetrapeptide AVPI. This structurally defined motif, with binding affinities of 0.1-1 microM, has stringent requirement for the first (P1), third (P3) and fourth (P4) amino acids. The P1 residue must be Ala, which binds to a small hydrophobic pocket on one end of the conserved groove on BIR domain. The P4 residue must be hydrophobic, preferably bulky, to occupy a greasy pocket on the other end of the groove. Deletion of P1 or P4 in a tetrapeptide results in abrogation of stable interaction with the BIR domain. The P3 residue is either Pro or Ala. Pro as P3, with its unique backbone configuration, optimizes simultaneous binding by both P1 and P4 residues for DIAP1-BIR2 or XIAP-BIR3. Ala as P3 can be better accommodated by DIAP1-BIR1 and XIAP-BIR2. In recent studies1, IBM was redefined to contain three contiguous amino acids, with P3 no longer restricted to Pro or Ala. Such tripeptides, ALG/AKG for drICE/Dcp1, or their longer variants, ALGS/AKGC for drICE/Dcp1, do not meet the structural criteria for IBM. Importantly, these peptides in isolation do not form a stable complex with any BIR domain. The definition of such motifs as IBMs insinuates the incorrect assumption that such free peptide motifs may stably interact with the BIR domain. This assumption, in turn, has engendered ample confusion in data interpretation in recent years (Li, 2011).

An IBM at the N-terminus of the caspase-9 small subunit recognizes a conserved surface groove on XIAP-BIR3; this interaction locks caspase-9 in the inhibited state. During apoptosis, Smac/Diablo uses a similar tetrapeptide motif to occupy the BIR3 groove, hence releasing caspase-9 and relieving XIAP-mediated inhibition. Caspase-3 or -7 is inhibited by an 18-residue peptide segment preceding the BIR2 domain of XIAP. Because both caspase-3 and drICE are inhibited directly at the active sites by XIAP and DIAP1, respectively, the overall appearance of the two BIR-caspase complexes is similar. It should be noted, however, that the essential interactions and key features are quite different. For example, caspase-3 or -7 can be inhibited by an isolated 18-residue peptide fused to GST; but the intact BIR1 domain of DIAP1 is absolutely required for drICE inhibition. The orientation of the BIR domain relative to the caspase is different by approximately 90 degrees between caspase-3/BIR2 and drICE/BIR1. Importantly, the peptide-binding groove of XIAP-BIR2 does not have an apparent role in the inhibition of caspase-3 and -7 (Li, 2011).

Using purified, recombinant proteins, this study showed that the BIR1 domain of DIAP1 only forms a stable complex with active drICE following caspase-mediated cleavage of DIAP1 after Asp20. The uncleaved DIAP1-BIR1 exhibited very weak binding to drICE. These observations contrast the report that both uncleaved and cleaved DIAP1-BIR1 bound to drICE similarly using coimmunoprecipitation. The cleaved DIAP1, but not the uncleaved DIAP1, potently inhibits the proteolytic activity of drICE towards both peptide and protein substrates. These observations unambiguously demonstrate that drICE sequestered by cleaved DIAP1 remains catalytically inactive. In fact, the conclusion that DIAP1-sequestered drICE was catalytically active, contradicted the biochemical observation that no protease activity was detectable towards peptide or protein substrate21. It is not uncommon for a substrate to be converted into a protease inhibitor upon cleavage, as exemplified by the pan-caspase inhibitor p35 (Li, 2011).

The key question is not whether BIR1 inhibits drICE, but why BIR1-sequestered drICE continues to exhibit proteolytic activity towards the uncleaved DIAP1. A time course analysis of DIAP1 cleavage shows that the protease activity of drICE was slowed down considerably over time, as the concentration of inhibitor -- cleaved DIAP1 -- increased. The level of drICE activity at the 15-minute time point was at least 6-fold higher than that at 90-minute point. This observation again illustrates that the cleaved DIAP1 is a bona fide inhibitor of drICE (Li, 2011).

Some of the contrasting claims about the regulation of drICE by DIAP1 might be attributable to the limitations of the investigative methods. Biochemical and biophysical investigations, employing homogeneous, recombinant proteins, usually provide mechanistic answers to questions that pertain to protein-protein interactions and enzyme activities. The caveat, however, is whether such observations are biologically relevant, and if yes, to what extent these findings are important. By contrast, investigation by cellular biochemistry, exemplified by coimmunoprecipitation, provides important clues to molecular mechanisms. For both approaches, caution must be exercised for the interpretation of results. Notably, in some cases, the contrasting claims can be reconciled by a complex system. For example, despite structural data demonstrating that the auto-inhibition of DIAP1 involves the binding of its N-terminal sequences to the BIR1 domain, it remains theoretically possible that additional interactions between the N- and C-terminal domains of DIAP1 may contribute to its auto-inhibition1. The best example is Apaf-1, whose auto-inhibition entails two elements: one by the C-terminal WD40 repeats and the other within the N-terminal half. Binding to cytochrome c relieves the auto-inhibition by the WD40 repeats31, and exchange of ADP for ATP defeats the auto-inhibition imposed by intra-domain interactions within the N-terminal-half (Li, 2011).

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: 22 November 2022

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