Mechanisms of caspase activation Kelly M Boatright and Guy S Salvesen

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1 725 Mechanisms of caspase activation Kelly M Boatright and Guy S Salvesen The core effectors of apoptosis encompass proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells. 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. Although this appears to be true for the executioner caspases, recent research points to a distinct activation mechanism for the initiator caspases that trigger the apoptotic pathways. This mechanism is proximity-induced dimerization without cleavage, and its elucidation has led to the revision of concepts of feedback regulation of apoptosis. Addresses Program in Cell Death and Apoptosis Research, The Burnham Institute, North Torrey Pines Road, La Jolla, CA 92037, USA; and Department of Molecular Pathology, University of California San Diego gsalvesen@burnham.org This review comes from a themed issue on Cell division, growth and death Edited by Jonathon Pines and Sally Kornbluth /$ see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI /j.ceb Abbreviations Apaf-1 apoptotic protease activating factor CARD caspase-activation recruitment domain DED death effector domain DISC death-inducing signaling complex FADD fas-associated protein with death domain FLIP FLICE-like inhibitory protein Introduction The caspases constitute a family of cysteine proteases peptidases that use a cysteine residue as the catalytic nucleophile that share an exquisite specificity for cleaving target proteins at sites next to aspartic acid residues. The concerted action of caspases is responsible for apoptosis, a specific form of programmed cell death that is essential for embryonic development and the pathology of many diseases. In addition to apoptosis, a subgroup of the caspase family is involved in inflammation, where they act as pro-cytokine activators [1]. The apoptotic caspases are classified as initiators or executioners, depending on their point of entry into the apoptotic cascade. The initiator caspases are the first to be activated in a particular death pathway (Figure 1), and they constitute the first step in a minimal two-step cascade by activating the executioner caspases. Unregulated caspase activity would be lethal for a cell, so to prevent this the cell stores caspases as latent precursors zymogens. These procaspases require an activating event. Recent advances suggest that the activation mechanisms of initiator and executioner caspases are entirely distinct, but that the device for stabilizing the latent zymogens is fundamentally conserved. In this review we address the most well-characterized apoptotic pathways as a framework to understanding caspase activation. We consolidate recent findings of the fundamental processes, and explain how activation events triggered by seemingly disparate stimuli occur via a conserved mechanism. Initiator caspases Overwhelming structural and biochemical evidence demonstrates that active caspases are obligate dimers of identical catalytic units, with each catalytic unit containing one active site. Currently, all three-dimensional structures of caspases in their active form reveal that each catalytic unit is composed of one large and one small subunit. These subunits are derived from the same precursor molecule by an internal cleavage at a site that demarcates the subunits, known as the linker region. Given the evidence that the active form of a caspase contains large and small subunits, it was assumed that all caspases were activated by proteolytic cleavage within their linker region (reviewed in [2]). However, current work is leading to a paradigm shift in the understanding of the mechanism of caspase activation. Recent studies have revealed that cleavage is neither required nor sufficient for activation of the initiator caspases. The zymogens of the initiator caspases exist within the cell as inactive monomers. These monomeric zymogens require dimerization to assume an active conformation, and this activation is independent of cleavage (Figure 2a) [3,4,5 ]. The dimerization event occurs at multiprotein activating complexes, to which the caspase zymogens are recruited by virtue of their N-terminal recruitment domain. The activating complex involved depends on the origin of the death stimulus, which is classified as being either extrinsic or intrinsic. Extrinsic pathway (caspase-8 and -10) The extrinsic pathway is responsible for elimination of unwanted cells during development, immune system education and immune-system-mediated tumor removal (immunosurveillance). It is initiated by ligation of a trans-membrane death receptor of the tumor necrosis factor receptor type 1 superfamily; a particular member

2 726 Cell division, growth and death Figure 1 Intrinsic Mitochondrial cytochrome c release and apoptosome assembly Extrinsic Death receptor ligation and DISC assembly Mitochondrion DISC Cytochrome c release Apoptosome Procaspase-8 Procaspase-9 Active caspase-9 Active caspase-8 Procaspase-3, -7 Active caspase-3, -7 Current Opinion in Cell Biology Schematic overview of the apoptotic pathways. Engagement of either the extrinsic or the intrinsic death pathways leads to the activation of the initiator caspases by dimerization at multiprotein complexes. In the extrinsic pathway, the DISC is the site of activation for caspase-8 and, at least in humans, caspase-10. The active sites are represented by orange stars. Stimulation of the intrinsic pathway leads to activation of caspase-9 at the apoptosome. Caspase-9 is shown as having one active site as seen in its crystal structure. However, the number of active sites in vivo is unknown. Following activation, the initiator caspases then cleave and activate the executioner caspases-3 and -7. of this family, Fas (also known as CD95 or APO-1), has become the paradigm for the study of the extrinsic pathway (reviewed in [6]). Upon ligation, the Fas receptor forms microaggregates at the cell surface, allowing the adaptor molecule FADD (Fas-associated protein with death domain) to be recruited to its cytosolic tail by a multi-step mechanism [7 ]. FADD recruits caspase-8 zymogens by virtue of homophilic interaction with their N-terminal death effector domains (DEDs). It is within this death-inducing signaling complex (DISC) that the initiator caspase-8 is activated. The initial hypothesis for activation implicated an induced proximity model, where a small amount of activity inherent in the procaspase-8 allowed cleavage in trans of caspase-8 dimers recruited to the confined space of the DISC, thus generating the canonical active two-chain form (reviewed in [8]). New data now shed doubt on this mechanism [4,5 ]. Cleavage appears not to be required for the formation of an active site. Rather, this cleavage event is thought to provide stability to the dimer generated during DISC formation, and the fundamental activation event is dimerization of caspase-8 monomers. Induced proximity still applies, but in the updated version of the hypothesis it is the recruitment of monomers to allow dimer formation, not the recruitment of pre-formed dimers, that is important. Following dimerization to the catalytically active form, the N-terminal DEDs are proteolytically removed, presumably allowing the activated caspase to be released into the cytosol [9]. This activation mechanism relies on a previously unappreciated property of procaspase-8: that in its inactive state it is a monomer. This property has now been

3 Mechanisms of caspase activation Boatright and Salvesen 727 Figure 2 (a) Dimerization at a multiprotein activating complex (b) Cleavage of the interdomain linker Current Opinion in Cell Biology Cartoon representation of the two molecular mechanisms of pro-caspase activation. (a) Activation of initiator caspases. The zymogens of initiator caspases exist as latent monomers. These monomers are activated by dimerization, which allows translocation of the activation loop (depicted as a red sausage ) into the accepting pocket of the neighboring dimer. The active site is represented by an orange patch. (b) Activation of executioner caspases. The zymogens of executioner caspases exist as preformed dimers. Their zymogen latency is maintained by steric hindrances imposed by the interdomain linker (depicted as a yellow banana ). Cleavage of this linker permits translocation of the activation loop, facilitating formation of the active site. Notice that the fundamental process of activation, translocation of the activation loop, is conserved for both the initiator and the executioner caspases. exhaustively demonstrated for both recombinant material [5 ] and the natural endogenous zymogen [4 ]. This is in stark contrast to the zymogens of the executioner caspases-3 and -7, which are already dimeric in their latent forms. The explanation for this important difference, which at first glance is incongruous, is clarified below. The caspase-8 ortholog caspase-10 is also an initiator in death-receptor-mediated cell death, at least in humans (mice apparently lack a caspase-10 gene). There is controversy in the literature regarding the ability of caspase- 10 to functionally substitute for caspase-8 in death-receptor signaling. Initial work done on caspase-8 deficient cells derived from the Jurkat T-cell line concluded that caspase-8 was essential for death-receptor-mediated apoptosis [10]. However, subsequent studies revealed that this cell line also had reduced caspase-10 levels and found that transient transfection of these cells with caspase-10 was sufficient to sensitize them to deathreceptor-mediated cell death [11,12]. A contrasting study found that reconstitution of these Jurkat T cells by stable transfection with the death receptor DR4 in combination with caspase-10 did not sensitize to death-receptorinduced apoptosis [13]. Given the complications inherent in reconstitution experiments, it is useful to gain insight from the lessons learned from humans with caspase-8 and -10 deficiencies. Humans with mutant caspase-10 exhibit an autoimmune lymphoproliferative syndrome caused by defective lymphocyte apoptosis [14]. Humans with mutant caspase-8, while also exhibiting defects in lymphocyte apoptosis, have in addition pronounced defects in their ability to activate lymphocytes, with resulting immunodeficiency [15 ]. Significantly, the latter study revealed that caspase-8 deficiency is compatible with development in humans, although it is embryonic-lethal in mice [16]. Taken together, these studies suggest that, although there is some overlap, caspase-8 and -10 probably have distinct functions. An interesting addition to the mechanism of caspase-8 activation is the involvement of FLIP (FLICE-like inhibitory protein FLICE was one of the original names for caspase-8). FLIP is a caspase-8 homolog with certain important differences, most notably its lack of a catalytic cysteine, which renders it incapable of proteolytic activity. Initial reports in the literature were at odds over whether FLIP was pro- or anti-apoptotic. Overexpression of FLIP in some cell lines inhibited death-receptormediated apoptosis, presumably by blocking caspase-8 binding sites at the DISC. Work by Chang and colleagues provided a solution to this apparent conflict by showing that at low levels of expression (close to those occurring in a normal cell) FLIP enhances Fas-induced caspase-8 activation at the DISC; only at higher levels (as found in certain tumors, for example) does FLIP inhibit caspase-8 activation [17 ]. This study was complemented by studies revealing that FLIP was capable of forming heterodimers with caspase-8 that possessed catalytic activity [18 ], incidentally confirming the dimerization activation mechanism of caspase-8. Further elucidation of this exciting mechanism for protease regulation within normal and tumor cells will certainly yield important discoveries.

4 728 Cell division, growth and death Intrinsic pathway (caspases-9 and -2) The intrinsic pathway is used to eliminate cells in response to ionizing radiation, chemotherapeutic drugs, mitochondrial damage and certain developmental cues. Following the death trigger, mitochondria may become selectively permeabilized, leading to the release of cytochrome c and the recruitment and activation of the apical caspase of the intrinsic pathway, caspase-9, in a complex known as the apoptosome [19]. The central component of the apoptosome is a protein known as Apaf-1 (apoptotic protease activating factor 1), which recruits caspase-9 via its N-terminal caspase-activation recruitment domain (CARD) [20]. In its quiescent state, Apaf-1 is a compact molecule with the head (CARD domain) tucked between its feet (two b-propellers formed by sets of WD40 repeats). Cytochrome c (which is conveniently about the same size as the CARD) displaces the head, allowing the compact structure to stretch out into a more linear molecule that polymerizes upon binding ATP [21]. The electron cryomicroscopy studies show the apoptosome to be a seven-spoked wheel, with a central hub that contains the caspase-9 recruitment domain, which is provided by the CARD of Apaf-1. Regrettably, the conformation of caspase-9 was not visible in the images, but other techniques have suggested a monomer-to-dimer transition analogous to caspase-8 activation. Although a monomer at cytosolic concentrations, the three-dimensional crystal structure of caspase-9 reveals that the active form is a dimer [22]. Interestingly, this dimer contains only one active domain as a result of steric clashes at the dimer interface. The other domain of the dimer is in a zymogen-like conformation with the specificity determinants and catalytic apparatus disabled. Importantly, the conformation of the inactive domain is almost identical to that of the zymogen form of caspase-7, as described below. As with caspase-8, not only is cleavage unnecessary for activation of caspase-9, but also it is insufficient to produce an active enzyme [3,23]. Instead, caspase-9 is activated by small-scale rearrangements of surface loops that define the substrate cleft and catalytic residues [22]. In the simplest model, this is achieved by dimerization of caspase-9 monomers within the apoptosome, with the dimer interface providing surfaces compatible with catalytic organization of the active site. 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 [24] and DNA damage [25 ], a subset of intrinsic stimuli. Caspase-2 appears to be activated by interaction with a high-molecular-weight complex that requires the CARD of caspase-2 [26 ]. The components of this complex have yet to be identified, but it has been shown to be independent of Apaf-1. Similar to the other initiator caspases, the zymogen of caspase-2 is a latent monomer (F Scott, K Boatright and G Salvesen, unpublished data) and cleavage is not required for its activation [26 ]. Rather, the active form of caspase-2 exists in both cleaved and uncleaved states, in complex with a high-molecularweight activator of unknown composition. Executioner caspases-3 and -7 In stark contrast to the initiators, the executioner caspase- 3 and -7 zymogens exist within the cytosol as inactive dimers [4 ]. They are activated by limited proteolysis within their interdomain linker, which is carried out by an initiator caspase or occasionally by other proteases under specific circumstances (Figure 2b). Caspase-6 is not as extensively studied as caspases-3 and 7, but is usually classified as an executioner caspase on the basis of its lack of a long pro-domain and its presumptive cleavage downstream of the initiators. Additionally, in recombinant form its zymogen is a dimer [27]. 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 [28,29,30 ]. At cytosolic concentrations in human cells, the caspase-3 and -7 zymogens are already dimers, but cleavage within their respective linker segments is required for activation [29,30 ]. The same re-ordering of catalytic and substrate-binding residues as seen in caspase-9 occurs in caspase-7, indicating that the fundamental mechanism of zymogen activation is equivalent. Only the driving forces are distinct: the linker segment of pro-caspase-7 blocks ordering of the active site until cleavage, whereupon the new N- and C-terminal sequences aid in active site stabilization. The property that allows the distinct driving forces 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 on regions with an ordered secondary structure. Why are the executioner caspase zymogens dimeric whereas the apical caspase zymogens are monomeric at physiologic concentrations? Part of the reason for this lies the relatively weak hydrophobic character of the dimer interface in caspases-8 and -9, which contrasts strongly with extremely hydrophobic nature of the dimer interface in caspases-3 and 7. Specifically, the K d for caspase-3 dimerization is <50 nm [31], which is more than three orders of magnitude tighter than the K d for caspase-8 (50 mm) [5 ]. The executioner caspases-3 and 7 have shorter N- terminal extensions than the initiator caspases, and for some time the role of these prodomains has remained elusive. They do not participate in the inherent activation

5 Mechanisms of caspase activation Boatright and Salvesen 729 mechanism [32,33], but are apparently important for efficient activation of the executioners in vivo, possibly because of spatial sequestration or cellular compartmentalization [33,34]. Conclusions A combination of biochemistry, structural analysis and cell biology has led to rapid advances in our understanding of the caspases and has revealed an underlying conservation in caspase activation. Although the initiators and executioners possess differing mechanisms of activation dimerization for the initiators and interdomain cleavage for the executioners the fundamental mechanism of zymogen latency is conserved: activation of both types of caspases requires translocation of an activation loop. For the executioners, this translocation is blocked by steric hindrances imposed by the interdomain linker. For the initiators, dimerization must first occur to allow the activation loop to interact with the adjacent monomer. We predict that all initiator caspases should undergo the monomer dimer activation mechanism, by homodimerization or even heterodimerization (as proposed for caspase-8 and FLIP [18 ]). In support of this, there is data suggesting that proximity-induced activation may apply to the initiator caspases involved in inflammation. Recent studies reveal that in humans caspases-1 and -5 seem to assemble in the interleukin-1b activator complex called the inflammasome [35 ], whereas genetic evidence in mice implicates an interaction between the orthologous proteins caspases-1 and -11 [36]. Intriguingly, these may represent examples of heterodimerization as an important feature of cytokine activation, but this hypothesis awaits some crucial tests. The revised hypothesis for apical caspase activation has important consequences for the interpretation of experimental results relating to caspase activity. For example, most studies interpret the cleavage of a caspase as evidence of its activation. As we discuss this may be valid only for executioner caspases that are activated by such cleavage. Certainly, the cleavage of caspase-8 and -9 observed in numerous publications is usually a consequence of their activation, but it is not definitive. Cleavage of an apical caspase may not promote its activity unless it is already in a dimeric configuration. In this context, the concept of feedback activation from the executioners (caspases-3 and -7) to the initiators (caspases-8, -9 and -10) may be invalid. Studies that assume cleavage of an apical caspase indicates activation should be evaluated cautiously. It stands to reason that the initial activating event in a proteolytic pathway cannot be proteolysis itself. Indeed, most proteolytic cascades are initiated by cofactor-driven conformational changes in protease zymogens. In the case of apoptosis, the specific conformational change driven by the cofactors (DISC or apoptosome) is dimerization. Induced proximity overcomes the energetic barrier for initiator caspase dimerization to occur by converting a bimolecular interaction to a unimolecular one. Once you have generated the first proteolytic signal you can utilize specific proteolysis to drive forward the cascade. From this perspective we speculate that the activation mechanism of executioner caspases is a recent development. The primordial mechanism may therefore be proximityinduced dimerization. Update Two groups recently revealed an unexpected aspect of extrinsic pathway activation. In contrast to the simple process shown in Figure 1 for the activation of caspase-8 at the Fas-induced DISC, caspase-8 activation by the TNF pathway does not occur at a membrane-associated signalling complex [37,38 ]. Rather, caspase-8 appears to be activated by association with a cytosolic complex during TNF-induced apoptosis. On a separate front, the crystal structure of caspase-2 in complex with an aldehyde inhibitor was reported by Schweizer and colleagues [39 ]. In this structure, caspase-2 was a cleaved dimer with the interface stabilized by a disulfide bridge. Thus, this suggests a role for redox conditions in modulating the activation of caspase-2 by dimerization. We anxiously await studies of the activation of this caspase within a physiological context. Acknowledgements We would like to thank the members of the Salvesen laboratory who have contributed to the caspase activation project. This work was supported by NIH grants CA69381 and HL51399, and the California Breast Cancer Research Program Fellowship 8GB References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Tschopp J, Martinon F, Burns K: NALPs: a novel protein family involved in inflammation. 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[4 ] by demonstrating that recombinant caspase-8 is activated through dimerization rather than by cleavage.

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7 Mechanisms of caspase activation Boatright and Salvesen Harper N, Hughes M, MacFarlane M, Cohen GM: Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. J Biol Chem 2003, 278: This work nicely complements that of Micheau and Tschopp by providing clear evidence that caspase-8 is not activated at the TNFR1 signalling complex. 38. Micheau O, Tschopp J: Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 2003, 114: The authors clearly show an association of endogenous caspase-8 with a cytosolic complex. Their work suggests that this is the activating complex for caspase-8 in the TNF-induced pathway. 39. Schweizer A, Briand C, Grutter MG: Crystal structure of caspase-2, apical initiator of the intrinsic apoptotic pathway. J Biol Chem 2003, in press. This crystal structure of an inhibited two chain caspase-2 dimer reveals a novel mechanism for dimer-interface stabilization: a disulfide bond between the cysteine residues that comprise the center of two-fold symmetry. This work suggests that redox conditions may play a role in the activation of caspase-2.