XIAP, the guardian angel

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It is now well established that cellular suicide (apoptosis or programmed cell death) is an essential part of the maintenance of homeostasis and the survival of multicellular organisms. Apoptosis is central to several physiological cellular processes, from host defence against viral infections to the sculpting of organs and tissues during embryonic development. Equally, or perhaps even more, important is the role of apoptosis in the pathogenesis of human disease. An uncontrolled rate of cellular death can have dire consequences too much cell death is often associated with the destruction of healthy cells and tissues, as exemplified in neurodegenerative disorders and autoimmune disease. Conversely, too little cell death is suspected to be partly responsible for the uncontrolled proliferation of cancer cells. It therefore follows that H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme. Cell 75, (1993). 44. Vaux, D., Weissman, I. L. & Kim, S. K. Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, (1992). 45. Nicholson, D. W. From bench to clinic with apoptosisbased therapeutic agents. Nature 407, (2000). 46. Levi-Montalcini, R. The nerve growth factor 35 years later. Science 237, (1987). 47. Cryns, V. L. & Yuan, J. Y. in When Cells Die: A Comprehensive Evaluation of Apoptosis and Programmed Cell Death (eds Lockshin, R. A., Zakeri, Z. & Tilly, J. L.) (Wiley Liss, New York, 1998). 48. Hengartner, M. O. & Horvitz, H. R. Programmed cell death in Caenorhabditis elegans. Curr. Opin. Genet. Dev. 4, (1994). 49. Hengartner, M. O. The biochemistry of apoptosis. Nature 407, (2000). 50. Meier, P., Finch, A. & Evan, G. Apoptosis in development. Nature 407, (2000). 51. Noetzel, W. Die Rückbildung der Gewebe im Schwanz der Froschlarve. Arch. Mikrosk. Anat. 45, (1895). 52. Collin, R. Recherches cytologiques dur le développement de la cellule nerveuse. Névraxe 8, (1906). Acknowledgements We thank D. Vaux for his generous sharing of his observations and material in preparation, and D. Tomei, J. Saunders, and J. Kerr for their helpful comments and photographs. The thoughts expressed here were generated under the auspices of an NIH grant to R.A.L. and one to St John s University. tight control of the apoptotic machinery is critical for cellular survival. Indeed, several decades of research into programmed cell death have identified numerous genes and pathways that control and influence, either positively or negatively, the progression of apoptosis from the initial death trigger to the final demise of the cell (see the Timeline article by Lockshin and Zakeri on page 545 for more details). One group of apoptotic regulators, the inhibitor of apoptosis (IAP) genes (BOX 1),the products of which block apoptosis, are particularly interesting as they are the most powerful intrinsic inhibitors of cell death and could potentially be used therapeutically. Among the IAPs, the X-chromosome-linked inhibitor of apoptosis (XIAP) is the most potent and versatile regulator of cell death. Furthermore, the mechanisms by which XIAP interferes with apoptotic pathways have now been worked out at the genetic, biochemical and structural levels, allowing a model to be proposed that accounts for the centrality of XIAP in the regulation of cellular suicide. Inhibition by XIAP Apoptosis is facilitated through a family of 550 JULY 2001 VOLUME 2

2 Box 1 The IAP family The inhibitor of apoptosis (IAP) proteins are intrinsic cellular regulators of programmed cell death. They were first described in baculoviruses, in which their role is to block the apoptotic response that is initiated as a defence mechanism in response to viral infection. The homologues of viral IAPs were soon discovered in metazoan species, including Caenorhabditis elegans, yeast, Drosophila melanogaster and vertebrates. Seven mammalian IAPs are known, but the list is still growing. IAPs are potent inhibitors of apoptosis induced by various triggers both in vitro and in vivo, by virtue of their ability to bind and inhibit distinct caspases. Significantly, IAPs are the only endogenous inhibitors that block both the initiator and effector caspases. The IAPs are defined by an ~80 amino-acid zinc-finger motif called the baculoviral IAP repeat (BIR; FIG. 1). The number of BIR domains in a given IAP is variable, ranging from one to three, but they are invariably present at the amino terminus of the protein and mediate the interaction with caspases. Several, but not all, IAPs also contain a carboxy-terminal RING (really interesting new gene) zinc finger, which has been recently recognized to have E3 ubiquitin ligase activity 31. specific proteases known as caspases (BOX 2) cysteine proteases that cleave their substrates after a conserved aspartate residue in their substrate target sequence (reviewed in REF. 1). Opposing the cellular destruction by caspases are two classes of cellular apoptotic inhibitor, members of the Bcl-2 and IAP gene families. Whereas the Bcl-2 proteins can block only the mitochondrial branch of apoptosis by preventing the release of cytochrome c, the IAPs block both the mitochondria- and death-receptor-(fas)-mediated pathways of apoptosis by directly binding to and inhibiting both the initiator and effector caspases. Interestingly, IAPs seem to be multifunctional proteins that, as well as regulating apoptosis, are also involved in cellular processes such as receptor-mediated signalling, cell cycle and ubiquitylation (reviewed in REF. 2). This multifunctionality is reflected in the modular structure of the IAPs, which is perhaps best characterized in XIAP (FIG. 1).The modular structure also holds true for most other IAPs (TABLE 1). The molecular mechanism by which XIAP regulates apoptosis is beginning to be unravelled. Early genetic evidence 3 indicated that baculoviral inhibitor of apoptosis repeat (BIR; BOX 1) domains might be essential for the caspase-inhibitory function of XIAP, as they bind directly to distinct caspases. XIAP inhibits both the initiator caspase-9 and the effector caspases-3 and -7. In vitro kinetic studies have shown that XIAP is the most potent caspase inhibitor in the IAP family, with K i values of 0.2 to 0.8 nm as compared with a 1 20-nM range for the other members of the IAP family 4. XIAP BIR domains BIR domains within XIAP might have distinct functions, and XIAP might therefore inhibit initiator and effector caspases in different ways. The caspase-3- and caspase-7-inhibiting activity of XIAP is localized to the second BIR domain (BIR2), but it also requires 3 residues found in the linker region that precedes BIR2 (FIG. 1). It was shown recently 5 that the isolated BIR3 domain is enough to inhibit caspase-9 potently. Furthermore, although the threedimensional structures of BIR2 and BIR3 Box 2 Caspase activation Fas Fas ligand FADD Procaspase-8 Caspase-8 Apoptosis from XIAP indicated that these two domains are very similar, different sets of amino-acid residues were found to be crucial for the inhibition of distinct caspases 5. New structural data 6 9 also support the concept that XIAP might inhibit initiator and effector caspases by different means. The XIAP BIR2 domain. The crystal structure of caspase-7 complexed with the inhibitory fragment of XIAP (amino-acid residues ) shows that the XIAP peptide binds the catalytic groove of caspase-7, completely filling the active site and thus preventing the entry of other substrates 8,9. A similar structural arrangement was found 7 between XIAP and caspase-3. This observation is not too surprising, considering that caspase-3 and caspase-7 share 54% sequence identity and have nearly identical backbone structures 9. Despite the fact that XIAP does not share any sequence motif with the natural caspase sub- Apoptosome Mitochondrion Initiator caspases Effector caspases All known apoptosis signalling pathways converge on the caspases, a family of proteases that form a self-amplifying cascade. Caspases are constitutively expressed in cells. However, to prevent them from inadvertently cleaving their targets, the caspases exist as silent (or with very low activity) pro-enzymes (procaspases). Caspase activation involves their proteolytic cleavage to liberate subunits that reconstitute an active caspase heterodimer. Caspases are activated in a hierarchical order initiator caspases (for example, caspases-6, -8, -9 and -10) are usually activated first. The initiator caspases then activate downstream effector caspases (for instance, caspases-2, -3 and -7), which propagate the caspase cascade that ultimately results in cell death. The activation of caspases can be triggered by extrinsic death stimuli (left in the figure). Specific ligands (such as FasL) cause death receptors (such as Fas) to oligomerize, which results in the recruitment of the adaptor molecules (such as FADD) and the activation of initiator caspases (such as caspase-8). Alternatively (right in the figure), intrinsic death stimuli, such as the cytosolic release of cytochrome c in response to mitochondrial damage, result in the activation of caspase-9 in the context of a holoenzyme multiprotein death complex known as the apoptosome 32. The apoptosome consists of cytochrome c, an adaptor protein Apaf-1, and procaspase-9 (FIG. 3). Once activated, the apoptosome then further recruits procaspase-3, which is subsequently processed and activated by caspase-9 (FIG. 3). NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 2 JULY

3 Receptor-mediated effects Regulation of apoptosis? Interacts with TAB1 Activation of JNK1 Binds to XAF1 Interacts with BMP receptor Inhibits caspase-3 and -7 Binds and inhibits caspase-9 Stabilizes binding to caspase-3 and -7 Binds to Smac/DIABLO E3 ubiquitin ligase XIAP binds only to processed caspase-9 not to the inactive procaspase-9 (REF. 6). Autocatalytic processing of procaspase-9 at Asp315 (after recruitment of procaspase-9 to oligomerized apoptotic protease-activating factor (Apaf-1) within the apoptosome; BOX 2) was found to liberate the p12 subunit of caspase-9 with a new free amino terminus that interacts directly with the XIAP BIR3 domain 6 (FIG. 2). Deletion or mutagenesis of the four amino-terminal residues of the p12 subunit (Ala-Thr-Pro-Phe) abolished caspase-9 binding to XIAP 6. Furthermore, when the amino-terminal sequence of processed caspase-3 (which does not normally interact with XIAP BIR3) was changed to that of processed caspase-9, it bound strongly to XIAP BIR3, showing that the tetrapeptide sequence of processed caspase-9 confers specificity for the BIR3 domain of XIAP 6. Figure 1 XIAP is a modular protein. Functional domains of the XIAP protein, the binding partners for the interactive domains, and the activity mediated by these domains are shown above and below XIAP. BMP, bone morphogenetic protein; JNK, Jun amino-terminal kinase. strates, the interactions between caspase-3/7 and XIAP are similar to that between caspases and the synthetic tetrapeptide inhibitor DEVD-CHO 9. Interestingly, the binding of XIAP is antiparallel to binding of the natural caspase substrates. Four amino-acid residues of XIAP Gly144, Val146, Val147 and Asp148 form either Van der Waals interactions or hydrogen bonds with the caspase-7 substrate-binding sites S1, S2, S3 and S4, respectively 9, with Asp148 being crucial for the docking interaction 8. The binding of XIAP to caspase-3 is mediated in a similar fashion by the same residues 7. The caspase-interacting residues, including Asp148, are conserved between XIAP and two other IAP proteins, HIAP1 (ciap2) and HIAP2 (ciap1) 8, which also inhibit 10 the effector caspases-3 and -7. However, these crucial residues are not found in the BIR2 domain itself, but in the short linker region that precedes BIR2. New data indicate that the BIR2 domain itself might be dispensable, and that it could be replaced with an unrelated protein such as glutathione S-transferase (GST) 8,9. The BIR2 domain has been proposed to stabilize the interaction between caspases and XIAP, and could mediate interactions with other IAP-binding proteins (see below). Notably, however, structural studies have focused on the isolated portion of the XIAP protein (residues ), and it is not Binds to Smac/DIABLO clear how the interactions are mediated in the context of the full-length XIAP protein containing three BIRs. The XIAP BIR3 domain. A unique feature of the IAPs is that they inhibit both the effector and the initiator caspases. The mechanism by which XIAP inhibits the initiator caspase-9 is, however, fundamentally different from its inhibition of the effector caspases-3 and -7, although both mechanisms involve the BIR domains. Genetic studies 11 identified the BIR3 domain of XIAP as being solely responsible for the inhibition of caspase-9. Interestingly, Table 1 The IAP genes inhibit distinct caspases The XIAP BIR1 domain. The function of the BIR1 domain of XIAP is still unclear, and there are no data available to indicate that BIR1 can inhibit caspases. In fact, a truncated XIAP containing only BIR1 cannot interact with caspase-3 or caspase-7, and does not inhibit Fas-induced apoptosis 3. It is possible that the BIR1 domain functions only to stabilize XIAP. An attractive hypothesis, however, is that BIR1 interacts with and inhibits as-yet-unidentified caspases. Alternatively, BIR1 could provide an interface for binding to a new class of IAP-interacting proteins (see below) that could modify the activity of XIAP against caspases. One protein, many domains Why have one protein, XIAP, with different BIR domains, inhibit many caspases? One possible explanation is that it allows simultaneous regulation of several caspase pathways. IAP gene BIR RING CARD Caspase inhibition K I (nm) References XIAP Caspase ND HIAP Caspase HIAP Caspase NAIP 3 0 0,7* ND Survivin Caspase Livin 1 1 0,7,9 ND The type and number of functional domains are indicated for each IAP gene. The inhibitory constants (K i ) for caspase inhibition are shown next to the target caspase. (ND, not determined; *J. Maier and A. E. MacKenzie, personal communication). BIR, baculoviral inhibitor of apoptosis repeat; CARD, caspase recruitment domain; IAP, inhibitor of apoptosis; RING; really interesting new gene. 552 JULY 2001 VOLUME 2

4 Recent data by Bratton and colleagues 12 indicate that XIAP associates with the apoptosome. In this complex, XIAP interacts with both caspase-3 and caspase-9, and has been suggested to prevent not only the activation of caspase-3 by caspase-9 but also the release of caspase-3 from the apoptosome. As each caspase interacts with a different BIR domain of XIAP specifically, caspase-3 and caspase-7 with BIR2, and caspase-9 with BIR3 the task of dual inhibition can be achieved more efficiently by one, rather than two proteins (FIG. 3). Alternatively, the evolution of two mechanisms of caspase inhibition by the same protein would also allow two means of regulation. For example, a hypothetical protein that could block the interaction between XIAP and caspase-9 would not affect the interaction between XIAP and caspase-3/7. Conversely, inhibiting the ability of XIAP to block caspase-3/7 would leave its caspase-9 activity intact. Is there a reason for regulating initiator and effector caspases separately? As caspases are also involved in processes other than cell suicide (for example, inflammation or differentiation; reviewed in REF. 13) the evolution of a modular caspase inhibitor such as XIAP would allow several levels of caspase regulation under various physiological conditions. Although the BIR2 and BIR3 domains of XIAP bind to their respective caspases in a structurally different way, the interaction and subsequent caspase inhibition is, in both cases, reversible. This is in stark contrast to the inhibitory activity of the broad-spectrum baculoviral caspase-inhibitor protein p35, which does not discriminate among caspases 14 and acts as a suicide inhibitor by forming a covalent thioester linkage with the target caspase 15. The structural difference between the XIAP and p35 caspase inhibition probably explains their different biological functions. The p35 protein functions as an apoptotic off switch, blocking apoptosis that was triggered in response to viral infection (and it is therefore aimed at blocking virus multiplication). By contrast, XIAP is an intrinsic caspase inhibitor that modulates (more like a throttle control) the caspase cascade in response to divergent extracellular and intracellular stimuli. Any even the slightest activation of caspases, if left uncontrolled, will rapidly lead to the amplification of the caspase cascade and, eventually, cell death. This outcome is not desirable under conditions of temporary cellular stress (such as nutrient or oxygen deprivation), and XIAP could control the initial caspase activation and prevent triggering of the caspase cascade. However, the complete inhibition of caspases is also undesirable, as cells that are damaged and dangerous to the organism would never be removed. (Indeed, higher apoptotic resistance is one of the hallmarks of cancer cells.) The ability of XIAP to release its target caspase therefore needs to be regulated, and cells have developed exquisite mechanisms to do just that. a b Death ATPF XIAP BIR3 Figure 2 BIR3 domain of XIAP binds cleaved caspase-9. a XIAP does not bind to procaspase-9. After proteolytic activation of caspase-9, however, the XIAP BIR3 domain specifically recognizes the exposed tetrapeptide sequence (Ala-Thr-Pro-Phe) in a short linker region (green) of the small subunit of caspase-9. The binding of XIAP to caspase-9 prevents the joining of large and small caspase subunits (red), inactivating caspase-9 and blocking the caspase cascade. Further processing of caspase-9 by caspase-3 removes the XIAPbinding fragment that can now function as a decoy peptide, preventing XIAP from inhibiting caspase-9 and allowing the caspase cascade to progress. The structural basis of the XIAP BIR3 and caspase-9 interaction is shown in b. The XIAP-inhibitory tetrapeptide motif is also found in several other proteins that interact with IAPs, including Smac/DIABLO. (Reproduced with permission from REF. 6 (2001) Macmillan Magazines Ltd.) Inhibiting the inhibitors Smac/DIABLO. A mitochondrial protein that negatively regulates the caspase-inhibiting activity of IAPs has been discovered recently and termed the second mitochondria-derived activator of caspase (Smac) 16 or DIABLO 17. Binding of Smac/DIABLO to IAPs disrupts the binding of IAPs to caspases and promotes apoptosis. Smac/DIABLO is synthesized as a precursor protein that is targeted into the mitochondria, where its 55- amino-acid amino-terminal targeting sequence is removed. Cleavage of the targeting sequence is essential for the role of Smac/DIABLO in apoptosis, as it exposes a tetrapeptide sequence (Ala-Val-Pro-Ile) that is crucial for the binding of Smac/DIABLO to IAPs. Mature Smac/DIABLO is released from mitochondria together with cytochrome c during mitochondria-induced apoptosis, and has been shown in vitro to interact with several of the IAP proteins including XIAP, HIAP1, HIAP2 and survivin 16,17. The structural basis of the interaction between Smac/DIABLO and XIAP has been worked out recently 18,19, and provides new insight into the regulation of the apoptotic process that is probably applicable to other IAPs as well. In addition, Smac/DIA- BLO connects, for the first time, IAPs with mitochondria-controlled cell death. Smac/DIABLO interacts with both the BIR2 and BIR3 domains of XIAP 18. The crystal structure of Smac/DIABLO reveals that it forms a homodimer through a large, hydrophobic interface, and that homodimerization is essential for its binding to the BIR2, but not BIR3, domain of XIAP 18. The four amino-terminal residues of Smac/DIABLO (Ala-Val-Pro-Ile) make specific contact with a surface groove of the BIR2 and BIR3 domains, but not with the BIR1 domain, of XIAP 19,20 (FIG. 2). Significantly, the conserved tetrapeptide motif has remarkable homology to the IAP-interacting motif found in the p12 amino-terminal sequence of caspase-9 (Ala- Thr-Pro-Phe) and the Drosophila proteins Hid (Ala-Val-Pro-Phe), Reaper (Ala-Val-Ala- Phe) and Grim (Ala-Ile-Ala-Tyr) 18. The ability of XIAP to release its target caspase needs to be regulated, and cells have developed exquisite mechanisms to do just that. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 2 JULY

5 Apaf-1 Cytochrome c Mitochondrion Apoptotic trigger XIAP Apoptosome Figure 3 XIAP associates with the apoptosome. Damage to mitochondria leads to the release of cytochrome c into the cytosol, where it promotes oligomerization of the adaptor molecule Apaf-1 and recruitment of procaspase-9 into a holoenzyme multiprotein death complex known as the apoptosome. Oligomerization of procaspase-9 within the apoptosome leads to the activation of caspase-9. Independent of this process, caspase-3 is also recruited to the apoptosome where it can be activated by either procaspase-9 or the active caspase-9. Recent evidence indicates that XIAP might be associated with the apoptosome. The proximity of XIAP to both caspase-3 and caspase-9, and the fact that BIR2 inhibits caspase-3 and BIR3 inhibits caspase-9, could account for the highly efficient and potent inhibition of these caspases by XIAP. Grim, Reaper and Hid initiate embryonic apoptosis in Drosophila by two mechanisms. The analysis of Drosophila embryos deleted for these proteins indicates that they might be directly involved in a pro-apoptotic signalling pathway. However, all three proteins also directly antagonize the Drosophila IAPs, so they can release the caspase cascade 21. This IAP-inhibiting activity is localized to the amino terminus of Grim, Reaper and Hid. Although Smac/DIABLO, by overall primary sequence structure, is not a homologue of these Drosophila proteins, it is clearly a functional analogue. Indeed, an entire new class of proteins will probably be discovered that harbours this IAP-interacting motif and could regulate IAP functions. Remarkably, the fact that the processing of caspase-9 exposes the motif that interacts with IAPs 6 suggests that this new class of proteins could be activated by caspases or other cellular proteases. If this is the case then it would add a new dimension XIAP. Unlike XIAP, however, which is found primarily in the cytoplasm, endogenous XAF1 is localized to the nucleus. The different compartmentalization of XIAP and XAF1 raises the question of how these two proteins can interact does XAF1 translocate into the cytoplasm where it inhibits XIAP, or does XIAP enter the nucleus where it is sequestered by XAF1? There is no experimental evidence to answer this question, but an attractive hypothesis is that the inhibition occurs in the nucleus. It has been suggested that caspase-3 is involved in apoptotic disruption of the nucleus because it proteolytically cleaves several nuclear substrates 25,26. However, caspase- 3 is found mainly in the cytoplasm. Faleiro and Lazebnik 27 have shown, however, that activated caspase-9 can disrupt the nuclear membrane and, by an as-yet-unknown mechanism, can increase the diffusion size of the nuclear pores. This leads to the transport of a large, caspase-3-containing protein complex inside the nucleus. As XIAP binds casto the control and regulation of apoptosis. The binding of Smac/DIABLO and caspase-9 to XIAP is mutually exclusive, with both proteins having a similar affinity for the BIR3 domain 6. This does not explain, however, how Smac/DIABLO can activate the caspase cascade, because it has to compete with caspase-9 for XIAP binding. Considering that XIAP can interact only with the activated (processed) form of caspase-9, XIAP activity can be blocked by Smac/DIABLO before this activation takes place. In addition to Smac/DIABLO peptide binding within the BIR3 groove, the second interaction interface, formed by Smac/DIABLO helices, strengthens the binding to XIAP 19. Furthermore, once the caspase cascade is activated, caspase-9 is further processed by caspase-3 at the Asp330 residue to the p10 subunit, by the removal of a short linker region. This linker region contains the IAP-interacting motif, which, at least in vitro, can bind to and inhibit XIAP 6. So, as in a positive-feedback loop, the activated caspase-9 casts off a decoy peptide that prevents XIAP from inhibiting caspase-9 activity (FIG. 2). The way that XIAP relieves caspase-3/7 inhibition is mechanistically different and has been suggested to be involved in the sensitization of type II cells, which are typically resistant to death-receptor-induced apoptosis 22.As XIAP binds caspase-3/7 through its linker region, this binding does not involve the BIR2 domain, which is then free to bind Smac/DIABLO. The binding of Smac/DIA- BLO to XIAP is proposed to destabilize the XIAP caspase interaction by steric hindrance, resulting in disruption of the XIAP caspase complex. In addition, the binding of Smac/DIABLO to the BIR2 domain of XIAP will result in a conformational change in XIAP, leading to the decreased affinity of XIAP for caspase-3/7 (REF. 8). Interestingly, Smac/DIABLO can bind to only one BIR at a time and monomeric Smac/DIABLO does not interact with BIR2 only with BIR3 (REF. 18). If these restrictions also exist in vivo, perhaps there are additional mechanisms to regulate the interactions between Smac/DIABLO and XIAP. Furthermore, Smac/DIABLO has a pro-apoptotic activity that is independent of its aminoterminal tetrapeptide sequence 23. The basis of this mechanism, which is probably independent of the binding to IAPs, is unclear. How can Smac/DIABLO induce apoptosis by binding to XIAP if the latter does not bind unactivated caspases? Given that caspases are suspected to have other important cellular functions (involving differentiation, inflammatory responses and cell-cycle effects) that are independent of apoptosis, low levels of active caspases might be present in cells. So, XIAP could bind to these activated caspases and prevent them from inadvertently starting the caspase cascade. The release of mature Smac/DIABLO into the cytoplasm would then be enough to relieve the inhibition of caspases by XIAP and activate the apoptotic machinery. XAF1. A different XIAP-interacting protein named XIAP-associated factor 1 (XAF1) has been identified by Liston and colleagues 24. XAF1 contains seven zinc fingers and antagonizes the ability of XIAP to suppress caspase activity and cell death in vitro. There are no structural data available on the mechanism of this inhibition and the interacting domains of XIAP and XAF1 have not yet been identified. In contrast to Smac/DIABLO, XAF1 does not need to be processed, and it seems to be constitutively able to interact with and inhibit The discovery of two and perhaps more cellular proteins that interact with XIAP and modulate its anti-apoptotic activity points to the critical role of XIAP in cellular homeostasis. 554 JULY 2001 VOLUME 2

6 pase-3, it is conceivable that XIAP could, in fact, be transported into the nucleus within the caspase-3 protein complex, where it would continue to inhibit caspase-3 activity. This caspase-inhibiting activity could then be relieved by nuclear XAF1, in a fashion similar to the cytoplasmic inhibition of XIAP by Smac/DIABLO. The different compartmental localizations of Smac/DIABLO and XAF1 reflect the need to control all branches of apoptosis at different cellular levels. As Smac/DIABLO is sequestered in mitochondria, it will only be functional in mitochondria-mediated apoptosis. XAF1, on the other hand, is constitutively present in the nucleus, so it can effectively promote progression of apoptosis irrespective of the type of initiator caspase involved. Alternatively, the role of XAF1 could be to regulate XIAP activity with respect to control of the cell cycle. It has been suggested 28 that XIAP is involved in cell-cycle regulation by downregulating cyclins A and D1, and inducing the cyclin-dependent kinase inhibitors p21 Cip1/Waf1 and p27 Kip1. Fas/Fas ligand Caspase-8 Cytochrome c Apaf-1 XIAP Apoptotic trigger Smac/DIABLO Mitochondrion Apoptosome Death Concluding remarks In this article, we have attempted to summarize the latest findings on the prototype of the IAP family, XIAP, and propose a central role for this apoptotic inhibitor in regulating programmed cell death (FIG. 4). The discovery of two and perhaps more cellular proteins that interact with XIAP and modulate its antiapoptotic activity points to the critical role of XIAP in cellular homeostasis. It has been observed that overexpression of XIAP protects cells from divergent apoptotic triggers, including ultraviolet irradiation, γ-irradiation and chemotherapy drugs. But mice with a targeted deletion of XIAP are normal and show no signs of misregulated apoptosis 29. The loss of XIAP, however, is accompanied by an increase in the levels of HIAP1 and HIAP2, suggesting that IAP activity is critical for cell survival and that the loss of XIAP needs to be compensated for. The survival benefits to cells that upregulate XIAP are obvious. Conversely, the ability to modulate XIAP activity through a subset of cellular proteins would be essential for maintaining tight control of the apoptotic process and, if left unchecked, could lead to cancer. Indeed, the data indicate that the XIAP antagonist, XAF1, is a tumour suppressor. XAF1 is expressed ubiquitously in all normal adult and fetal tissues, but is present at very low or undetectable levels in various cancer cell lines 24. By contrast, XIAP levels are relatively high in most cancers, suggesting that XAF1 might be critical in mediating the apoptotic Death XAF1 Figure 4 Proposed model for the centrality of XIAP in caspase regulation. The ability of XIAP to inhibit both the initiator and effector caspases places it at the centre of caspase cascade regulation. So, XIAP can intercept and regulate both pathways of apoptosis those mediated by death receptors and those mediated by mitochondria. The discovery of two classes of XIAP-binding protein, mitochondrial Smac/DIABLO and nuclear XAF1, indicates two possible mechanisms of negative regulation of XIAP. Smac/DIABLO, which is released from the mitochondria together with cytochrome c, would effectively block XIAP within the apoptosome in the cytoplasm. But, by contrast, Smac/DIABLO is not expected to be an effective inhibitor of XIAP in mitochondria-independent apoptosis pathways. The nuclear localization of XAF1, on the other hand, indicates that it might intercept all branches of apoptosis and might also modulate the role of XIAP during the cell cycle. resistance of cancer cells by failing to control the anti-apoptotic activity of XIAP 30.It remains to be determined whether there is a similar correlation for Smac/DIABLO. There are several outstanding issues that need to be addressed experimentally and will probably provide vital clues about the regulation of XIAP. The mechanism of XIAP s interaction with XAF1 has yet to be determined. We also need to establish how nuclear XAF1 could inhibit cytoplasmic XIAP. The experimental evidence indicates that overexpression of XAF1 leads to the translocation of endogenous XIAP into the nucleus 24. Interestingly, under some apoptotic conditions, endogenous XIAP can be observed in the nucleus (H. Gibson and R.G.K., unpublished observation). Accordingly, the investi- Nucleus gation of the cellular localization and redistribution of XIAP during the early stages of apoptosis would be extremely informative. Similarly, it should be determined whether nuclear XIAP co-localizes with caspase-3, as could be hypothesized from Faleiro and Lazebnik s data 27. And how does Smac/DIA- BLO interact with XIAP in vivo? Surprisingly, injection of mature Smac/DIABLO is, in some cases, not enough to trigger apoptosis (E. LaCasse and R.G.K., unpublished observation). In these cases, XIAP might not be receptive to inhibition by Smac/DIABLO. Perhaps XIAP needs to be in a state of conformational readiness, which could be achieved by a post-translational modification such as phosphorylation. Virtually nothing is known about the putative phosphorylation NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 2 JULY

7 status of XIAP, even though the XIAP sequence contains a number of consensus phosphorylation sites for several protein kinases, including Akt and Jun amino-terminal kinase (JNK). Finally, the discovery of a specific IAP-interacting motif indicates the possible existence of other proteins that interact with IAP. We predict that these proteins will have high specificity for individual BIR domains, pointing to alternative mechanisms of regulating XIAP. Given that the regulation of caspases is critical for cell survival, it is not surprising that this process is so complex. We would argue that XIAP sits at the centre of these regulatory events. Understanding the biology of XIAP not only provides the intellectual satisfaction of untangling the complex regulatory networks that control life and death, but it might also supply us with powerful therapeutic approaches for the treatment of several human diseases. Martin Holcik and Robert G. Korneluk are at the Children s Hospital of Eastern Ontario Research Institute, and Ægera Oncology Inc., 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada. Correspondence to M.H. martin@mgcheo.med.uottawa.ca Links DATABASE LINKS XIAP Bcl-2 cytochrome c Fas caspase-9 caspase-3 caspase-7 HIAP1 HIAP2 Apaf-1 p35 Hid Reaper Grim XAF1 cyclin A cyclin D1 p21 Cip1/Waf1 p27 Kip1 Akt FURTHER INFORMATION Holcik lab ENCYCLOPEDIA OF LIFE SCIENCES Apoptosis: molecular mechanisms 1. Nicholson, D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, (1999). 2. Holcik, M., Gibson, H. & Korneluk, R. G. XIAP: Apoptotic brake and promising therapeutic target. Apoptosis 6, (2001). 3. Takahashi, R. et al. A single BIR domain of XIAP is sufficient for inhibiting caspases. J. Biol. Chem. 273, (1998). 4. Deveraux, Q. L. & Reed, J. C. IAP family proteins suppressors of apoptosis. Genes Dev. 13, (1999). 5. Sun, C. et al. 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