Introduction

Conditions that drive entry into the cell cycle also prime the apoptotic machinery (Green and Evan, 2002). This ensures that cell fate is largely determined by the presence of local survival signals that hold the apoptotic response in check (Raff, 1992). In the absence of such signals cells enter apoptosis unless they have downregulated the ability to respond to the death cues. It is now clear that many cancers arise because of defects in the normal apoptosis control. The principal molecular machines that execute apoptosis, the packaging of a cell for phagocytic removal that is coincident with its demise, consist of proteolytic enzymes from the caspase family. Consequently, delineating the controls placed on the generation of caspase activity is required if we are to grasp the full view of the oncogenic process.

Caspases are highly selective proteases that have an exquisite preference for cleaving proteins after Asp residues. This fastidious specificity ensures that apoptosis is primarily a set of limited proteolytic cleavages, and not the degradative process that is often assumed to accompany proteolysis (Salvesen and Dixit, 1997). The execution phase of apoptosis is thus thought to be a result of the limited caspase-dependent cleavage of hundreds of cellular proteins (Fischer et al., 2003), the sum of which results in the morphology characterizing this form of programmed cell death (PCD).

A majority of studies on apoptosis are based on the assumption that caspase precursors are activated by cleavage, a common mechanism for most protease zymogen activations. Whereas this appears to be true for the executioner caspases, a paradigm shift is underway that points to a completely distinct activation mechanism for the initiator caspases that trigger the apoptotic pathways in humans. In contrast to the prevailing view that human caspase activation is driven mainly by caspase activation (stepping on the gas), research in drosophila suggests that apoptosis is initiated by transcriptional controls that liberate constitutively active caspases from their complex with natural inhibitors (releasing the brakes). This review considers these two fundamentally distinct mechanisms of caspase activation with implications for caspase biology.

Proteolytic cycles

Whether in coagulation, inflammation or gastrulation, protease activity is regulated by an activation/activity/inhibition cycle depicted in Figure 1. Indeed, several of these cycles can act sequentially to cause a cascade. The reason for protease cascades is still debated, but one attractive hypothesis suggests that it converts a binary event (activate/don't activate) into an analog signal where levels of downstream protease – executioner protease – can be controlled in a graded response by inhibitors.

Figure 1.

A proteolytic cycle. The fundamental mechanisms governing activity of proteases are conserved in caspase activation. Latent proteases await an activation signal. The activator may be an oligomeric activation platform, or it may be another protease. Once active, substrate and inhibitor compete for protease binding, and the outcome is defined by the local concentration of inhibitor. Significantly, active protease may be released from its inhibitory complex by factors that bind the inhibitor. This is signified by the double-headed arrow, since many cognate protease inhibitors form reversible complexes with their target proteases (Turk et al., 2002). A cascade is defined when the substrate of one protease (caspase 8 for example) is another protease zymogen (caspase 3 for example)

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Almost independent of protease type, there exist three fundamental steps at which protease activity is naturally regulated at the post-translational level in vivo: (i) zymogen activation, (ii) access to substrates, (iii) inhibition. These principal control points are beautifully exemplified by the caspases. Almost all proteases are stored as zymogens to protect sensitive cellular machinery during biosynthesis, and to allow timing and localization of the ultimate proteolytic events. Second, because most proteases show only limited degrees of stringency in substrate recognition in vitro, an important way to deliver specificity in vivo is to direct the protease to or from the cellular location of its target substrates. Third, the extent of substrate cleavage in vivo is dictated by a dynamic competition between substrate and natural inhibitors for the active site of the protease, and so the concentration of the inhibitors demonstrates the final regulation point in the cycle.

Caspase types

The human genome encodes 11 caspases, and these can be divided into subgroups depending on inherent substrate specificity (Thornberry et al., 1997), domain composition (Denault and Salvesen, 2002) or presumed roles in vivo (Nicholson, 1999). Table 1 relates them on the basis of domain composition, as this is currently the only way to compare human and fly caspases. Long prodomain apoptotic initiator types or long prodomain cytokine activator types contain Caspase Recruitment Domains (CARD) or Death Effector Domains (DED) preceding the catalytic domain. These domains are 6–7 helix bundles that direct the caspases to their activation platforms (Fesik, 2000). The short prodomain, apoptotic effector type caspases contain short prodomains of unknown structure. The fly genome encodes seven members of the caspase gene family (Kumar and Doumanis, 2000). Based on prodomain structure, two of these correspond to initiator caspases and four resemble effector-type or executioner caspases. The seventh member, Strica, bears a long and unusual amino-terminal prodomain with no homologies to other proteins (Kumar and Doumanis, 2000). Their functional order has been largely inferred through shared phylogenetic relationships and, hence, critical aspects of the currently predicted pathways await validation through direct biochemical analyses. Likewise, much remains to be learned regarding the developmental functions of these genes, since most of the current genetic evidence relies on multigenic deficiencies, dominant negatives (Meier et al., 2000) or injected dsRNAs (Quinn et al., 2000).

Table 1 - Categories of human and Drosophila caspases.

Full table

The human cytokine activators

The first caspase, caspase 1, was discovered as an aspartic-specific cysteine protease participating in the proteolytic maturation of pro-IL-1 (Cerretti et al., 1992; Thornberry et al., 1992). In agreement with this proposed role, ablation of the caspase 1 gene results in mice that cannot process pro-IL-1, or the related proinflammatory cytokine pro-IL-18 (Kuida et al., 1995). Interestingly, a similar defect is seen in mice ablated in caspase 11, and the suggestion has been made that mouse caspases 1 and 11 collaborate in proinflammatory cytokine activation (Wang et al., 1998). These mice are highly resistant to septic shock and have lower production of a variety of other cytokines in response to experimental LPS infusion. Significantly, no overt apoptotic phenotype or developmental defect has been observed demonstrating that caspases 1 and 11 are not involved in apoptosis, except possibly in a paracrine manner by sensitizing cells to apoptosis via cytokine activation (Friedlander et al., 1997). Human caspases 4 and 5 are less studied enzymes, and they are almost certainly cytokine activators because of the high sequence similarity, domain organization and comparable substrate specificity they share with caspase 1. Presumably, caspases 4 and 5, which seem to be orthologs of mouse caspase 11, process cytokines in response to different inflammatory stimuli or pathological situations than caspase 1, or cooperate with caspase 1 to produce an adequate inflammatory response. With respect to this, caspase 5 has been found to be associated with caspase 1 under specific conditions in vitro (Martinon et al., 2002).

The human apoptotic initiators

Initiator caspases constitute the point at which cell signals are converted to proteolytic activity, probably one of the most important decisions a cell can make during its life. The need for initiator caspases may be threefold. First, they permit sensing and integration of different inputs, transmitting to a common execution phase. Second, they enforce amplification of the apoptotic system by generating substantial amounts of active executioner caspases. Third, they allow for a point of regulation before the final commitment to death. In humans, we recognize two distinct initiation points: the extrinsic pathway and the intrinsic pathway (Figures 2 and 3). Recruitment and activation of the initiator caspases is achieved by adapter molecules that bridge to death receptors via homophilic DEDs – for caspases 8 and 10, or via CARDs – between caspase 9 and the cofactor Apaf 1. In many cultured cellular models in which caspases 8 and 10 are expressed, they seem redundant probably owing to the high identity (48%), similar substrate specificity and domain organization.

Figure 2.

Apoptotic caspase pathways in humans and flies – conservation. The centrally conserved intrinsic pathway in humans and flies is shown by the dark shading, and involves caspase 9 or Dronc as the initiating caspase. Upstream decisions are integrated by members of the Bcl-2 family, and signals are transduced to the activation platforms of Apaf-1 or Dark. All intrinsic caspase components are under the control of IAPs, which in turn can be derepressed – to release caspase activity – by the species-specific antagonists in the ovals. In the lighter shading are the extrinsic activation pathways. In mammals, the caspase 8 pathway plays a profound role in initiating apoptosis, but may also paradoxically provide cell activation stimuli. In contrast, the extrinsic route in flies plays a minor role in apoptosis, being more involved in innate immunity. The figure highlights the forward drive by zymogen activation (the Gas) and control by IAPs (the Brakes) that dominate the intrinsic pathway

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Figure 3.

The basis of IAP antagonists as proapoptotic proteins – variation between humans and flies. In flies there is evidence for a continuous low-level production of caspases, which are neutralized by Drosophila IAP-1 (Rodriguez et al., 2002). In this scenario, simple upregulation of one or more fly IAP antagonists could ignite the system into apoptosis, and to this extent specification of apoptosis in transcriptionally regulated (White et al., 1994). In contrast to flies, the currently known mammalian IAP antagonists are mitochondrial proteins and require translocation before they can influence the inhibitory activity of IAPs. This implies a distinct point of impact for the regulation of IAP antagonists in mammals since both positive initiator caspase signaling and mitochondrial fluxes would presumably be required, and the system may be less reliant upon transcriptional regulation. Input from the left of the diagram may be the most important event in flies, whereas in mammals, input from the top of the diagram could be more important. The shaded area expands the zymogen activation phase, in which apical (initiator) caspases acquire the ability to cleave downstream targets following dimerization at activator platforms, mediated by DED or CARD recruitment domains. The prime targets of the apoptotic initiator caspases are the executioner caspases, which pre-exist as dimers requiring simply limited proteolysis for activation

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Humans with mutant caspase 10 display an autoimmune lymphoproliferative syndrome caused by defective lymphocyte apoptosis (Wang et al., 1999a). Humans with mutant caspase 8, while also exhibiting defects in lymphocyte apoptosis, have pronounced defects in their ability to activate lymphocytes, with resulting immunodeficiency (Chun et al., 2002). Importantly, the latter study revealed that caspase 8 deficiency is compatible with development in humans, although it is embryonic lethal in mice (Varfolomeev et al., 1998), probably because mice lack caspase 10 (Reed et al., 2003). Moreover, the results of analysis of disease suffered by humans with defects in caspase 8 reveal a role for this caspase in T-cell activation, clearly distinct from its associated proapoptotic role.

The human apoptotic executioners

In contrast to the initiators, caspases 3, 6, and 7 each possess a short distinct N-terminal peptide (23–28 residues). The reason for having three executioner caspases is not clear, and indeed the evidence for the importance of caspase 6 is lacking. Whereas caspase 3 is essential for normal embryonic development, the phenotype of mice ablated in caspase 6 appears to be normal (Zheng et al., 2000). The phenotype of mice ablated in caspase 7 has yet to be reported, yet it is evident that caspases 3 and 7 are almost synonymous in their substrate and inhibitor specificity. Nevertheless, biochemical experiments place the activation of caspases 6 (Orth et al., 1996) and 7 (Yang et al., 1998; Denault and Salvesen, 2003) downstream of caspase 3 and so we will consider them as executioner caspases.

The fly apical caspases

Dronc is the sole 'CARD-carrying' caspase in the fly genome (Kumar and Doumanis, 2000) and, in this respect, the enzyme is most homologous to caspase 9. Expression of this locus is ubiquitous but acutely responsive to the steroid hormone ecdysone (Dorstyn et al., 1999a). When overexpressed, this gene provokes apoptosis in both cultured cells and in the animal (Dorstyn et al., 1999a; Meier et al., 2000; Quinn et al., 2000), which requires the participation of 'downstream' caspases (Dorstyn et al., 1999a). Although single gene mutations at this locus have not been characterized, studies with multigenic deficiencies, dominant negatives (Meier et al., 2000), and injected dsRNAs (Quinn et al., 2000) suggest that this caspase is a central player in the apoptotic programmed cell death pathway. Interestingly, in addition to Asp, Dronc may cleave following Glu residues in some proteins (including its own precursor) implying a more tolerant specificity (Hawkins et al., 2000). Notably, bacterially produced Dronc was found to be a particularly poor catalyst with kinetics 40–180-fold less than caspase 9, itself a rather inefficient enzyme when expressed in bacteria. Hence, like caspase 9, efficient Dronc activity might require the formation of a holoenzyme complex involving intimate associations with Dark, the Drosophila ortholog of Apaf-1 (Kanuka et al., 1999; Rodriguez et al., 1999; Zhou et al., 1999).

Another apical caspase in flies, Dredd (Chen et al., 1998), most closely resembles mammalian caspases 8 and 10. The long prodomain found in Dredd includes significant sequence similarities spanning the DEDS of these mammalian counterparts. Hu and Yang (2000) noted that a region of Drosophila Fadd (dFadd) binds to and shares substantial similarity with this same portion of the Dredd prodomain, and referred to this shared motif as the 'death-inducing domain'. Single gene mutations at Dredd are viable and recessive (Leulier et al., 2000). Although dredd^- animals are cell death defective when examined in sensitizing backgrounds, the absence of Dredd did not cause global defects in PCD. Instead, genetic analyses established that Dredd plays a fundamental role in the innate immune response toward bacterial pathogens (Leulier et al., 2000). When Dredd mutants were challenged by microbial pathogens, induction of several antimicrobial genes failed. Consistent with these observations, Dredd activational processing was responsive not only to apoptotic signals (Chen et al., 1998), but to LPS (Georgel et al., 2001; Stoven et al., 2003a, 2003b) and coexpression of dFadd (Hu and Yang, 2000) as well. Collectively, the findings established a limited role for this caspase in apoptosis but uncovered an obligatory role for this apical caspase in transducing responses to microbial pathogens. In flies, as in mammals, innate immunity is governed by Toll receptors that propagate signals through members of the NFB family to induce specific sets of antimicrobial peptides (reviewed in Hoffmann and Reichhart, 2002). Since the function of Dredd maps upstream of the Drosophila NFB protein, RELISH (Stoven et al., 2003a, 2003b), which itself requires proteolytic cleavage for activation, the findings illuminate a compelling new link between signal-dependent activation of NFB proteins and the action of a caspase. As Dredd and Relish can physically interact, a likely scenario proposes that Dredd directly cleaves Relish, perhaps in a phosphorylation-dependent manner (Stoven et al., 2000, 2003a, 2003b).

The third long prodomain caspase encoded in the fly genome, Strica, bears an unusual N-terminal prodomain with no homologies to other proteins (Kumar and Doumanis, 2000). Likewise, the putative prodomain exhibits no matches to previously characterized motifs (e.g. CARD, DED) but it is distinctly rich in serines and threonines. Little functional data on this enzyme currently exists, but the few studies that have been performed on this protein failed to detect activational processing of, nor proteolytic activity against, commercial substrates. However, if overexpressed, Strica can trigger apoptosis and was able to associate with DIAP1 and DIAP2 (Doumanis et al., 2001). Mutations in Strica have not been isolated and so the physiologic function of this enzyme is currently not known.

The fly executioner caspases

Dcp1, Drice, Damm, and Decay lack extensive prodomains and hence qualify as effector caspases. Single gene mutations do not yet exist in any of these genes and so the requirements for these proteins in development are not yet known (previously described DCP1 alleles are now known to be compound mutations that also affect a flanking gene (McCall et al., 2003)). The substrate specificities determined for Dcp1 and Drice are notably similar to human caspase 3, as well as nematode Ced-3, and forced expression of truncated variants can trigger apoptosis (Fraser and Evan, 1997; Song et al., 2000). In addition, although both enzymes are able to cleave Drosophila lamin their substrate specificities are not identical, as evidenced by distinct activities of these enzymes for human lamins (Song et al., 2000). Evidence from immunodepletion studies on cultured Drosophila cells implicates Drice as the predominant (if not the sole) effector caspase responsible for apoptosis at least in S2 cells (Fraser et al., 1997).

Ectopic expression of the two less well-characterized enzymes, Decay and Damm, also triggers apoptosis (Dorstyn et al., 1999b; Harvey et al., 2001). These proteins are most closely aligned with human caspases 3 and 7 (Decay) and caspase 6 (Damm), respectively. In the former case (Dorstyn et al., 1999b), this resemblance extended to enzymatic activity (Decay was active against a caspase 3 substrate) while, in contrast, Damm was not active against a synthetic substrate preferred by caspase 6 (Harvey et al., 2001).

As mentioned earlier, while the functional order of these caspases programmed cell death is largely inferred, there is broad consensus that Dronc is an important initiator caspase (Dorstyn et al., 1999a, 2002; Meier et al., 2000; Muro et al., 2002; Yu et al., 2002). The most rigorous biochemical demonstration that Dronc activates Drice comes from in vitro studies where Dronc was shown to process Drice (Hawkins et al., 2000). From other in vitro studies, there is also evidence that reciprocal 'cross-cleavage' between Dcp1 and Drice may occur (Fraser et al., 1997; Song et al., 2000) but whether this cleavage relationship occurs in vivo is not yet known.

Caspase zymogen activation: stepping on the gas

Most biochemical and structural work on caspase activation has been performed with human caspases 3, 7, 8, and 9, and a reasonably clear picture has emerged to demonstrate variation and conservation in their activation mechanisms. The executioner caspases 3 and 7 are activated by direct proteolysis at internal sites that generate the large and small subunits of their catalytic domains. In contrast, the initiator caspases 8 and 9 do not require cleavage for activation, but are activated within polymeric activation platforms.

The zymogens of the initiator caspases exist within the cell as inactive monomers. These monomeric zymogens require dimerization in order to assume an active conformation, and this activation is independent of cleavage (Stennicke et al., 1999; Boatright et al., 2003; Donepudi et al., 2003). The dimerization event occurs at multiprotein activating complexes to which the caspase zymogens are recruited to by virtue of their N-terminal recruitment domain. The activating complex involved depends on the origin of the death stimulus: in the intrinsic pathway it occurs within the Apaf-1 containing apoptosome and in the extrinsic pathway it occurs within the polymeric DISC.

Extrinsic pathway activation: caspase 8 and Dredd

In mammals, the DISC activates the extrinsic cell death pathway and is recruited to the cytoplasmic portion of death receptors. Within this complex, the adaptor protein FADD forms the essential link to apical caspases 8 and 10 via homotypic interactions involving DED domains. The fly genome clearly encodes an ortholog of Fadd, designated Dfadd, and, like its mammalian counterpart, this protein binds to and regulates an apical caspase, in this case Dredd (Hu and Yang, 2000; Naitza et al., 2002). However, while flies do express a TNF-like axis, current evidence argues that it probably does not engage the Dfadd/Dredd module to launch an apoptotic caspase cascade (Igaki et al., 2002; Kanda et al., 2002; Moreno et al., 2002; Kauppila et al., 2003). Instead, the Dfadd/Dredd module predominantly (and perhaps exclusively) regulates innate immune responses triggered by proteins known as peptidoglycan-recognition proteins, some of which encode transmembrane bacterial sensors (Hoffman, 2003). Transduction of signals by these nonself sensors is propagated through Dredd to the NFB protein, Relish (orthologous to the mammalian NFB proteins, p100, and p105) and it has been proposed that Relish is in fact a substrate for the Dredd caspase. Moreover, because Relish activation also requires IB kinases together with cleavage at an aspartate (Silverman et al., 2000; Stoven et al., 2000, 2003a, 2003b; Lu et al., 2001), the findings raise the possibility that Dredd might directly cleave RELISH in a phosphorylation-dependent manner. This scenario contrasts sharply with mechanisms proposed for mammalian NFB counterparts (p100 and p105), where proteolytic cleavage is thought to occur via the proteasome (Silverman and Maniatis, 2001). However, evidence arguing for orthologous physiologic relationships between caspase 8 and NFB proteins also exists (Chaudhary et al., 2000) and it therefore seems probable that this ancient innate immune response pathway is well conserved, at least in some mammalian cell types.

An interesting addition to the mechanism of human caspase 8 activation is the involvement of FLIP (FLICE-like inhibitory protein – FLICE being one of the original names for caspase 8). FLIP is a caspase 8 homolog with crucial differences, notably its lack of a catalytic cysteine that renders it incapable of proteolytic activity. At low levels of expression (close to those occurring in a normal cell), FLIP enhances Fas-induced caspase 8 activation at the DISC. However, at higher levels (such as that found in certain tumors) FLIP inhibits caspase 8 activation (Chang et al., 2002), presumably by saturating available recruitment sites on the DISC and preventing caspase 8 recruitment. This study was complimented by studies revealing that FLIP was capable of forming heterodimers with caspase 8 that possessed catalytic activity (Micheau et al., 2002), incidentally confirming the dimerization activation mechanism of caspase 8.

Intrinsic pathway activation: caspase 9 and Dronc

The objective of the intrinsic pathway is to integrate developmental and stress cues into activation of the apical caspase 9 or Dronc. In humans, a key component of the integrator is the mitochondrion. Importantly, oligomerization of Apaf-1 into a functional apoptosome requires release of cytochrome c from mitochondria, and is therefore clearly a post-translational event (Liu et al., 1996; Kluck et al., 1997; Li et al., 1997). In stark contrast, a parallel apoptotic role for cytochrome c in flies is controversial. Prior to overt signs of apoptosis, an otherwise hidden epitope on Dark, the fly counterpart of Apaf-1, is exposed (Varkey et al., 1999; Arama et al., 2003) and exogenous cytochrome c weakly enhanced apoptosome-like activities in lysates from fly cells (Kanuka et al., 1999; Dorstyn et al., 2002). However, most studies in fly systems find no evidence for release of cytochrome c from mitochondria during apoptosis (Varkey et al., 1999; Dorstyn et al., 2002) and, in gene silencing experiments, dsRNAs targeting DARK suppressed apoptosis but dsRNAs silencing cytochrome c RNAs showed no effect (Zimmermann et al., 2002). Hence, while a vital role for Dark in PCD is firmly established, important questions regarding how its activity is regulated and whether cytochrome c plays a role remain to be answered. This introduces the possibility of a fundamental distinction in the caspase pathway of flies and humans, since it suggests the probability that human caspase activation is regulated by activation, but that fly caspase activation is constitutive (Rodriguez et al., 2002).

Although caspase 9 is the common initiator of the intrinsic pathway, recent work demonstrates that caspase 2 is required for an apoptotic response to neurotrophic deprivation (Troy et al., 2001) and DNA damage (Lassus et al., 2002), a subset of intrinsic stimuli, in certain primary cells or cell lines. Caspase 2 appears to be activated by interaction with a high molecular weight complex that requires the CARD of caspase 2 (Read et al., 2002). The components of this complex have yet to be identified, but they are independent of Apaf-1. Similar to the other initiator caspases, the zymogen of caspase 2 is a latent monomer and cleavage is not required for its activation (Read et al., 2002). Rather, the active form of caspase 2 exists as both cleaved and uncleaved, in complex with a high molecular weight activator of currently unknown composition. Significantly, a unified model for apical (initiator) caspase activation suggests that all long prodomain caspases may be recruited as monomers to activation platforms that function to dimerize them to the active form (Boatright et al., 2003). Once the active form is achieved, downstream caspase activation takes place strictly by limited proteolysis.

Execution phase activation: caspases 3 and 7

In apparent incongruity to the initiators, the executioner caspase 3 and 7 zymogens pre-exist within the cytosol as inactive dimers (Boatright et al., 2003). They are activated by limited proteolysis within their interdomain linker carried out by an initiator caspase, and occasionally by other proteases under specific circumstances. The crystal structures of zymogen caspase 7, active caspase 7, and inhibitor-bound caspase 7 serve as a model with which to rationalize the apparent conflict between the cleavage mechanism for executioner caspase activation, and the dimerization mechanism for apical caspase activation (Wei et al., 2000; Chai et al., 2001b; Riedl et al., 2001a).

At cytosolic concentration in human cells, the caspase 3 (Bose and Clark, 2001; Pop et al., 2001) and 7 zymogens are already dimers (Boatright et al., 2003), but cleavage within their respective linker segments is required for activation (Chai et al., 2001b; Riedl et al., 2001a). The same reordering of catalytic and substrate binding residues occurs in caspase 7 as seen in caspase 9, so the fundamental mechanism of zymogen activation is equivalent. Only the driving forces are distinct since the inter-subunit linker segment of procaspase 7 blocks ordering of the active site, and upon cleavage the new N- and C-terminal sequences so generated aid in active site stabilization. The property that allows the distinct driving forces (dimerization of monomers or cleavage of pre-existing dimers) to converge on the same activation mechanism seems to be the unusual plasticity of the residues constituting the caspase active site, which rather unusually for proteases are predominantly placed on flexible loops and not ordered secondary structure.

Caspase-inhibiting IAPs: the brakes

Although the first level of regulating proteolytic pathways is by zymogen activation, an equally important level is achieved by specific inhibitors that can govern the activity of the active components. The endogenous inhibitors of caspases, those present in mammals and flies, are members of the inhibitor of apoptosis (IAP) family. IAPs contain one, two, or three baculovirus IAP repeat (BIR) domains, which represent the defining characteristic of the family. The best-characterized endogenous human caspase inhibitor is the X-linked IAP (XIAP), a member of the IAP family. The IAPs are broadly distributed and, as their name indicates, the founding members are capable of selectively blocking apoptosis, having initially been identified in baculoviruses (reviewed in Verhagen et al., 2001a). Eight distinct IAPs have been identified in humans. XIAP (which is the human family paradigm) has been found by multiple research groups to be a potent but restricted inhibitor targeting caspase 3, 7, and 9 (reviewed in Deveraux and Reed, 1999). Similarly, evidence implicates human cIAPs 1 and 2, ML-IAP, ILP-2, and Drosophila DIAP-1 as caspase inhibitors (reviewed in Salvesen and Duckett, 2002).

The second BIR domain (BIR2) of XIAP specifically target caspases 3 and 7 (K_i0.1–1 nM), and regions closely related to the third BIR domain (BIR3) specifically target caspase 9 (K_i10 nM). This led to the general assumption that the BIR domain itself was important for caspase inhibition. Surprisingly, the recent structures of BIR2 in complex with caspases 3 and 7 have revealed the BIR domain to have almost no direct role in the inhibitory mechanism. All the important inhibitory contacts are made by the flexible region preceding the BIR domain (Chai et al., 2001a; Huang et al., 2001; Riedl et al., 2001b). Interestingly, the mechanism of inhibition of caspase 9 by the BIR3 domain requires cleavage in the intersubunit linker to generate the new sequence NH₂-ATPF (Srinivasula et al., 2001). In part, this explains the cleavage of caspase 9 during apoptosis, which as described above is not required for its activation. Paradoxically, it seems required for its inactivation by XIAP, which then inhibits caspase 9 by reversing the dimeric activation process (Shiozaki et al., 2003). The importance of IAPs in cancer progression is underscored by observations of tumor-associated elevations in their levels. For example, upregulation of IAP family members is common in prostate cancers of both humans and mice (Krajewska et al., 2003). Moreover, IAP expression shows a positive correlation with chemoresistance and poor treatment outcome (Tamm et al., 2000).

Encoded within the fly genome are four BIR containing genes, DIAP1, DIAP2, dBruce, and Deterin. Among these, DIAP1 is the best studied and transgenic analyses, as well as gain-of-function and loss-of-function mutations, clearly demonstrate a central, apoptotic role for this protein (reviewed in Hay, 2000). When overexpressed, DIAP2, dBruce, and Deterin can also suppress death in certain contexts and, notably, deletion alleles at dBruce enhanced killing by Rpr and Grim (Vernooy et al., 2002). DIAP1 binds and inhibits the action of Drice, Dcp1, and Dronc (Kaiser et al., 1998; Wang et al., 1999b; Hay, 2000; Meier et al., 2000) but similar activities for other BIR containing proteins have not been reported.

IAPs clearly have functions in addition to caspase inhibition because they have been found in organisms such as yeast, which neither contain caspases nor undergo apoptosis (Uren et al., 1998). Moreover, though most of the mechanistic studies on IAPs relate to their binding and direct inhibition of caspase catalytic activity, it is likely that some of the IAPs function to downregulate caspases not by inhibiting them, but by acting as E3 ligases for their rapid removal via the proteasomal route (Huang et al., 2000). Irrespective of the actual mechanism for terminating caspase activity, the IAPs set the scent for the final phase of caspase control – derepression as a method of activation.

IAP antagonists: releasing the brakes

Reaper proteins act by derepressing IAPs. The four known activators of apoptosis in the Reaper region (RPR, GRIM, HID, and SKL) are all transcribed in the same orientation, encode partially redundant functions and share an N-terminal amino-acid motif referred to as either the RHG motif (Wing et al., 2001; Christich et al., 2002; Srinivasula et al., 2002; Wilson et al., 2002), or the IAP-binding motif (IBM) (Salvesen and Duckett, 2002; Tenev et al., 2002). How do these proteins elicit the death of a cell? One likely explanation focuses on derepression functions that liberate caspases from IAPs, which themselves act as constitutive caspase inhibitors. Among the fly IAP proteins, DIAP1 is thought to act as a central, rate-limiting brake upon caspases through direct physical contact with these enzymes (Meier and Evan, 1998; Goyal et al., 2000; Hay, 2000; Lisi et al., 2000; Wu et al., 2001). While the precise mechanisms are not completely understood, Reaper proteins and a recently identified RHG containing protein, Jafrac2, (Tenev et al., 2002) are thought to antagonize DIAP1 through direct interactions (Wilson et al., 2002) involving contacts formed by residues in the RHG motif and the BIR domains of DIAP1 (Shi, 2001). Elegant structural studies, recently reported by Shi and colleagues, have further elucidated the precise binding surfaces for this association and mapped these relative to the site on DIAP1 that recognizes Dronc (Chai et al., 2003). Their studies nicely explain how associations between DIAP1/Dronc or DIAP1/RHG motif of Reaper proteins are mutually exclusive since the same binding pocket with the second BIR repeat of DIAP1 can accommodate only one but not both proteins (Chai et al., 2003). The ultimate outcome of these interactions promotes displacement of activate caspases via mechanisms linked to: (1) autoubiquitination of DIAP1 (Ryoo et al., 2002), (2) suppression of DIAP1-mediated ubiquitination of Dronc (Meier et al., 2000; Chai et al., 2003) and (3) N-end rule degradation of DIAP1 (Ditzel et al., 2003).

Numerous observations are consistent with a caspase/DIAP1 liberation model for cell killing by Reaper-like proteins. First, the apoptotic action of these proteins is effectively suppressed by caspase inhibitors and by DIAP1 in both cultured cells and in the animal (Abrams, 1999; Hay, 2000). Second, removal of DIAP1 from cells (Igaki et al., 2002; Zimmermann et al., 2002) or embryos (Wang et al., 1999b; Rodriguez et al., 2002) promotes rapid apoptotic death. Third, DIAP1 binds and inhibits at least two of the fly caspases, Drice and Dronc (Kaiser et al., 1998; Wang et al., 1999b; Meier et al., 2000). Fourth, RPR, GRIM, and HID can bind to IAPs through their common N-terminal RHG motif and also antagonize IAP function in heterologous, yeast-based assays (Vucic et al., 1997; Kaiser et al., 1998; Vucic et al., 1998; Wang et al., 1999b; Hay, 2000; Wu et al., 2001). Fifth, the RHG motif is sufficient to elicit apoptosis (Vucic et al., 1998; Claveria et al., 2002) and flies carrying DIAP1 mutations that selectively impair binding to RPR and HID exhibit resistance to apoptosis by these proteins (Goyal et al., 2000; Lisi et al., 2000). Collectively, these observations strongly favor models whereby Reaper proteins function, at least in part, to release active caspases from inhibitory IAP complexes. Additional support for the liberation model comes from structural studies that highlighted shared features among the RHG motif and the N-termini of two mammalian proteins, Smac (Diablo in mice) and Omi/HtrA2, which also bind and antagonize IAP function (Hegde et al., 2001; Martins et al., 2001; Verhagen et al., 2001b; van Loo et al., 2002). Comparative analyses of BIR domains that were complexed with N-terminal peptides from HID, GRIM (Wu et al., 2001), and Smac (Chai et al., 2000; Wu et al., 2000), described minimal surfaces required for binding to BIRs with common contacts shared between the fly and mammalian peptides.

Although attractive and compelling, other lines of evidence are inconsistent with strict 'liberation' models that presume DIAP1 is the sole effector of RPR proteins. First, while the RHG motif is sufficient to evoke apoptosis, RPR and GRIM also exert potent killing activities distinct from this IAP binding domain (Chen et al., 1996; Vucic et al., 1998; Wing et al., 1998, 2001). Intriguing parallels to recent studies on SMAC are worth noting here, as this mammalian IAP antagonist also provokes apoptosis without its IAP binding domain (Roberts et al., 2001). Likewise, in vitro studies have uncovered significant biochemical activities for RPR proteins that do not require the RHG domain and appear to be independent of DIAP1 activity. For instance, in heterologous systems, apoptogenic activity of RPR required a ubiquitin-like domain containing protein, SCYTHE, which binds to RPR outside of the N terminal IBM (Thress et al., 1998, 1999). More recently, both RPR and GRIM were found to repress translation of proteins in vitro and, where tested, this activity was observed in the absence of the N terminal IBM (Holley et al., 2002; Yoo et al., 2002). Finally, epistasis studies in cultured cells (Zimmermann et al., 2002) and in the fly (Rodriguez et al., 2002) indicated that cells are not 'preloaded' with sufficient levels of IAP-inhibited processed caspases to achieve cell killing. Instead, the results favored a 'gas and brake' model whereby concurrent input from Dark, together with removal of IAP inhibition, drives caspase activation to levels that exceed a threshold necessary for apoptosis. Taken together, the collective body of research shows that RPR proteins cause cell death by relieving caspases from the antagonistic action of IAPs and might, concurrently, also engage other death-associated signals, perhaps through unknown effectors.

Gas, brakes, and the control of cancer

The above discussion has been used to focus readers on the apparent differences in caspase control in humans and flies. Current evidence suggests that human caspase activation is a post-translational pathway that must be triggered by specific initiators. Of course, transcription sets the scene – the level of the pro- and antiapoptotic proteins, but the stringent controls on caspase zymogen activation restrain a constant low level activation, or 'tick-over', of caspase activity. In contrast, flies may possess this tick-over, much as has been suggested for complement activation – another proteolytic cascade (Manderson et al., 2001). Are these different modes of regulation minor peculiarities, or do they provide more profound variations in the design of the intrinsic cell death machinery? Are we all blindly feeling different parts of the same elephant?

The problem in interpretation may be inherent in the relative ease of studying pathways in flies by epistatic genetics, and can be confounded in humans (or mice) by redundancy in IAPs. Nevertheless, fundamental differences in IAP antagonists suggest basic differences in caspase regulation. Paramount among these is that in Drosophila the death-inducing proteins Hid, Grim, Reaper, and Sickle all contain homologous IAP binding motifs at their N-terminus (sometimes called the RHG or IBM motif). In distinction to the known mammalian IAP antagonists, the Drosophila ones do not have transit peptides, and are therefore fully functional translation synthesis and removal of their initiator methionine. In contrast, the known human IAP antagonists Smac and Omi/HtrA2 are not active following translation since they are housed in mitochondria awaiting a signal for their release. This supports the transcription/translation driven control of apoptosis in flies, and the post-translational driven activation in humans.

Given that transformed cells invariably must lose their ability to die when out of context, it is now fairly certain that part of this oncogenic transformation results from inactivation of key proapoptotic components or upregulation of antiapoptotic ones (Reed, 1999). Among these are mutations in death receptors and adaptors, mutations in caspases 8 and 10, and upregulation of IAPs (reviewed in Kaufmann and Vaux, 2003). Paradoxically, or maybe essentially (Green and Evan, 2002), cancer cells become sensitive to apoptosis-inducing chemotherapy. In the very nice review in Oncogene that should be read in conjunction with this one, Kaufmann and Vaux (2003) pointed out that most chemotherapeutic agents act by triggering the intrinsic (caspase 9) pathway at a level upstream on mitochondria.

But toxicity raises its head with chemotherapy, and so adjuvant cancer therapy is clearly a current theme. With respect to this, we may learn a lot by studying the mechanisms of caspase control. If the objective is to activate caspases in cells that have switched off parts of the pathway, then attacking two or more points in the apoptotic pathways to rekindle death is a valuable strategy. Success in combined TRAIL/Apo2L (caspase 8 activation) and IAP antagonist experimental therapy demonstrates the lowering of the threshold of TRAIL induced death by pretreatment with IAP antagonists (Fulda et al., 2002). Small molecules that lower the kinetic barrier for apical caspase activation can now be considered, and a possible example of this is seen in the discovery of a small molecule that influences apoptosome function (Jiang et al., 2003). Exploration of nonpeptidic IAP antagonists is producing results, at least in experimental cell culture paradigms (Wu et al., 2003), and more significantly in mouse models of tumor regression (Schimmer et al., 2003).

Flies are indispensable for understanding basic mechanisms and it is unlikely that the importance of IAP antagonists would have been appreciated without the fly paradigm. We also note that in development and in cancers, pressures driving the cell cycle may be universally coupled to those which sensitize cells toward apoptosis (Abrams, 2002; Nahle et al., 2002). In other words, early staged neoplastic cells probably exist in a constitutively idling state, with elevated flux operating through pathways that provide 'the gas'. Accordingly, early staged neoplastic cells must also exist under intense selective favoring repression of caspase activity through genetic and/or epigenetic means. Hence, while flies may be an imperfect model for understanding the late stages of human cancers, they could represent an ideal model for the early phases of oncogenesis, where fundamental properties of caspase repression by IAPs can be easily accessed.

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Acknowledgements

GSS was supported by NIH Grants CA69381 and AG15402; JMA was financially supported by NIH Grant AG12466 and the American Cancer Society.