Báo cáo khoa học: Mammalian initiator apoptotic caspases ppt

REVIEW ARTICLE Mammalian initiator apoptotic caspases Po-ki Ho 1,2,3 and Christine J. Hawkins 1,2,3 1 Murdoch Children’s Research Institute, Parkville, Victoria, Australia 2 Children’s Cancer Centre, Royal Children’s Hospital, Parkville, Victoria, Australia 3 Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia It has been more than a decade since the discovery of the interleukin-1b-converting enzyme (ICE) [1], the first member of a family of enzymes termed caspases. Caspases are an evolutionarily conserved family of cys- teine proteases that are responsible for diverse cellular functions including inflammation and apoptosis. Fea- tures common to all members in this family of protea- ses include the catalytic cysteine residue in the active site and the ability to cleave substrates on the carboxyl side of aspartate residues. Reflecting these properties, this family of cysteine aspartases was collectively desig- nated caspases [2]. To date, human caspases-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -12 and -14 have been cloned, and character- ized to varying extents [3,4]. Unambiguous murine homologues have been identified for most of these. However, the murine genome appears to lack clear counterparts of human caspases-4, -5 and -10. Murine caspase-11 is similar to human caspases-4 and -5. The gene originally designated human caspase-13 [5] has since been demonstrated to be a bovine gene, most clo- sely related to human caspase-4 [6]. Caspases or caspase-like enzymes have also been identified in nonmammalian metazoans and even other nonmetazoans [7]. Caspase-like activities observed in plants and some protists have been attributed to metacaspases and paracaspases, two classes of caspase- like proteins of ancient origin. While metacaspases have been observed in certain bacteria, protists, fungi and plants, paracaspases have been found in slime moulds and animals [8]. In Saccharomyces cerevisiae, the yeast caspase 1 (YCA1) has been associated with yeast cell death [9]. Other caspases have been identified in invertebrates such as the nematode Caenorhabditis elegans (CED-3, CSP-1 and CSP-2) [10,11] and the fruitfly Drosophila melanogaster (Dcp-1, Dronc, Drice, Keywords activation; apoptosis; CARD; DED; gene knockout models; human diseases; initiator caspases Correspondence C. Hawkins, Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, VIC 3052, Australia Fax: +61 3 93454993 Tel: +61 3 93455823 E-mail: chris.hawkins@mcri.edu.au (Received 24 July 2005, accepted 12 September 2005) doi:10.1111/j.1742-4658.2005.04966.x Caspases are a conserved family of cysteine proteases. They play diverse roles in inflammatory responses and apoptotic pathways. Among the casp- ases is a subgroup whose primary function is to initiate apoptosis. Within their long prodomains, caspases-2, -9 and -12 contain a caspase activation and recruitment domain while caspases-8 and -10 bear death effector domains. Activation follows the recruitment of the procaspase molecule via the prodomain to a high molecular mass complex. Despite sharing some common features, other aspects of the biochemistry, substrate specificity, regulation and signaling mechanisms differ between initiator apoptotic caspases. Defects in expression or activity of these caspases are related to certain pathological conditions including neurodegenerative disorders, auto- immune diseases and cancer. Abbreviations ALPS, autoimmune lymphoproliferative syndrome; BIR, baculoviral IAP repeat; CARD, caspase activation and recruitment domain; DD, death domain; DED, death effector domain; FADD, Fas-associated death domain; FLICE, FADD-like ICE; FLIP, FLICE-inhibitory protein; IAP, inhibitors of apoptosis; ICE, interleukin-1b-converting enzyme; TNF, tumor necrosis factor; TRADD, TNF-R1-associated death domain protein. 5436 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS Dredd ⁄ Dcp-2, Decay, Damm ⁄ Daydream and Strica ⁄ Dream) [7]. Caspases have also been recognized in nonmammalian vertebrates including zebrafish Danio rerio [12], frog Xenopus laevis [13] and chicken Gallus gallus [14–16]. Categorization of caspases based on their structure, function and substrate preference results in different classification systems. The first method, utilizing the structural characteristics of each caspase, places the mammalian family members into two main categories: long prodomain and short prodomain (Fig. 1). Casp- ases-1, -2, -4, -5, -8, -9, -10, -11 and -12 belong to the former category. Each has a long prodomain that encompasses structural motifs in the death domain superfamily including the caspase activation and recruitment domain (CARD) or death effector domains (DEDs). These motifs enable caspases to associate with other proteins via homotypic interaction mechanisms. Caspases-3, -6, -7 and -14 fall into the latter category. These caspases bear short prodomains and are activa- ted upon proteolytic cleavage by other caspases. The second method of classification divides the casp- ases into two main streams on a functional basis, distinguishing between inflammatory and apoptotic caspases (Fig. 1). Caspases-1, -4, -5, and -11 have been reported to play roles in cytokine maturation and inflammatory responses [17]. The remaining family members are primarily involved in apoptotic signaling pathways. These apoptotic caspases can be further divi- ded into ‘initiators’ (caspases-2, -8, -9, -10, -12) and ‘effectors’ (caspases-3, -6, -7, -14). Initiator caspases function upstream within apoptotic signaling pathways. They are capable of activating downstream caspases (effector caspases) either directly, through proteolysis, or indirectly via a secondary messenger mechanism. Upon activation by an initiator caspase, effector casp- ases are immediate ‘executioners’ of the apoptotic program, cleaving certain cellular substrates to cause demolition of the cell. Interestingly, these two methods of classification yield a close structure–function rela- tionship among the caspases: all initiator apoptotic caspases contain a large prodomain whereas all effector caspases have a short prodomain. In this article, we will focus on the apoptotic human caspases with long prodomains (capases-2, -8, -9, -10 and -12). The structural and biochemical characteris- tics, regulation and signaling mechanisms, and func- tional roles will be discussed. Structural comparison The protein–protein interaction domains CARD and DED are members of the death domain (DD) super- family of motifs, which play important roles in apop- totic signaling [18]. Caspases-2, -9, and -12 contain one CARD, whereas caspases-8 and -10 bear two DEDs arranged in tandem in their prodomains (Fig. 2). The CARD protein interaction motif is conserved among multiple key apoptotic regulators such as caspases, adaptor molecules RAIDD [19,20] and Apaf-1 [21], and inhibitors of apoptosis c-IAP1 and c-IAP2 [22,23]. It mediates the association of interacting partners via homodimerization through the CARD interface. The solution structure of the RAIDD CARD motif, obtained from NMR analysis, suggested that it consists of six antiparallel a-helices packed in a topological arrangement similar to that of the Fas DD [24]. More- over, modeling of the caspase-2 CARD revealed a structure highly similar to that of RAIDD CARD [24]. The biological significance of this domain is exemplified by the observation that the formation of the apop- tosome and the subsequent activation of caspase-9 rely on the CARD–CARD interaction between procaspase- 9 and Apaf-1 [25]. The DED is structurally similar to the CARD, in that its conserved backbone also compri- ses six a-helices. Like CARDs, DEDs are also present in numerous apoptotic regulatory proteins including caspases-8 and -10, the adaptor molecule Fas-associ- ated death domain (FADD), the antiapoptotic proteins FADD-like ICE (FLICE)-inhibitory proteins (FLIPs) CARD Short Prodomain Inflammatory Apoptotic 1, 4, 5, 11 3, 6, 7, 14 2, 9, 12 8, 10 DED Fig. 1. Classification of caspases. Caspases are classified based on their prodomain structure or primary function. Caspases-1, -2, -4, -5, -9, -11 and -12 contain a long prodomain with a caspase activa- tion and recruitment domain (CARD). Among these CARD-contain- ing caspases, caspase-1, -4, -5 and -11 have functions in the inflammatory response, while caspases-2, -9 and -12 play roles in apoptosis. Caspases-8 and -10 possess two death effector domains (DEDs) in their prodomains, and are also apoptotic family members. Caspases-3, -6, -7 and -14 have short prodomains and are effector caspases in the apoptotic pathway. P K. Ho and C. J. Hawkins Mammalian initiator apoptotic caspases FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS 5437 [26], and the Bcl-XL-procaspase-8-associated protein Bap31 [27]. The DED present in FADD links procasp- ases-8 and -10 to the death receptors and provokes formation of the death-inducing signaling complex (DISC). The DED-mediated oligomerization of pro- caspases-8 and -10 recruited to the DISC is pivotal in their activation and autoprocessing [28–30]. Of the initiator apoptotic caspases, the three-dimen- sional structures of caspases-2, -8, and -9 have been deduced by X-ray crystallography [31–33]. In general, the functional unit of the mature caspase is a hetero- tetramer consisting of two large and two small sub- units. The active site cysteine resides within the large subunit, while residues forming the S1 subsite are derived from both large and small subunits. Caspase-2 exists as a dimer in solution: the two monomers are covalently linked by a disulfide bridge at the dimer interface [31]. Dimer formation is possible even in the absence of substrate or inhibitor binding [31]. In con- trast, caspases-8 and -9 exist predominantly as mono- mers in solution [33,34] and dimerization occurs only upon recruitment to complexes described below. In comparison with other published caspase structures, the catalytic domains in the caspase-9 dimer are atyp- ical, in that they are nonidentical, with one catalyti- cally intact and the other in an enzymatically incompetent conformation [33]. Mechanism of activation In order to exert its catalytic function, a procaspase molecule must be activated to undergo conformational changes and (usually) cleavage to produce its mature form. The major mechanism that governs the activation of initiator caspases is oligomerization or induced prox- imity [35,36]. This mode of activation involves the recruit- ment of adaptor molecules to a death complex. This is followed by conformational changes that allow the recruitment of procaspases to these adaptor molecules, via domain-specific protein–protein interactions. Subse- quent oligomerization of procaspase molecules, due to close proximity and elevated local concentration, favors autocatalytic processing. This mechanism is mediated by the N-terminal CARD or DED motifs present in the initiator procaspases-2, -8, -9, and -10. However, when artificially induced to oligomerize by chemical means, a caspase-8 mutant lacking the DEDs is capable of auto- activation and causing apoptosis in transfected HEK 293 and HeLa cells [37,38], suggesting that activation is a consequence of induced close proximity. As mentioned above, induced proximity is preceded by the recruitment of procaspase molecules to a high molecular mass complex, which is formed in response to a death stimulus. Different caspases are recruited to different death complexes at the onset of apoptotic signaling pathways. These complexes have been desig- nated the ‘PIDDosome’, ‘DISC’ and ‘apoptosome’. Caspase-2 in the PIDDosome and other complexes RAIDD ⁄ CRADD was first cloned and identified as a caspase-2-interacting adaptor molecule [19,20]. A transfection study [20] and an in vitro translation ⁄ GST Fig. 2. Schematic representation of mam- malian apoptotic initiator procaspases. Casp- ases contain a conserved consensus active site sequence of QACXG and are expressed as inactive zymogens, or procaspases. Caspases with long prodomains contain either a CARD or two DEDs in tandem. Procaspases are proteolytically processed at specific cleavage sites during generation of the active enzyme. These cleavage events remove the prodomain and separate the enzyme proform into a large and a small subunit. A linker peptide between these subunits in procaspases-2 and -9 is also removed upon cleavage. Depicted in the dia- gram is a full-length version of human pro- caspase-12 that carries a read-through mutation at amino acid position 125, due to a natural polymorphism in some Africans [72]. Mammalian initiator apoptotic caspases P K. Ho and C. J. Hawkins 5438 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS A B C Fig. 3. Caspase activation complexes. Lines in red denote molecular interactions between domains. (A) Complex formation for caspase-2. (i) A putative complex involving caspase-2 in the TNF-R1 signaling pathway. Upon TNFa stimulation, TNF-R1 recruits the adaptor molecules TRADD, RIP1 and RAIDD, via homotypic interactions of the death domain (DD). Procaspase-2 is linked to the signaling complex through its CARD–CARD interaction with RAIDD [19,20]. (ii) Formation of the ‘PIDDosome’ which consists of the p53-induced protein PIDD, RAIDD and procaspase-2 [42]. (LRR, leucine-rich region). (iii) Together with TRAF2 and RIP1, caspase-2 has also been implicated in a complex that links it to the NF-jB pathway in a catalytic-independent manner [43]. (B) Formation of the death inducing signaling complex (DISC) via stimulation of Fas and TNF-R1 by their respective ligands. (i) The assembly of a TNF-induced caspase activation complex follows receptor endocytosis, internalization and subsequent dissociation of TRADD, TRAF2 and RIP1 from the receptor. TRADD then binds FADD via interactions between their DDs, and FADD in turn recruits procaspase-8 and ⁄ or procaspase-10 through DED–DED associations. The induced proximity forces the procaspase molecules to oligomerize and autoprocess to attain full enzymatic activity. (ii) Fas ligation triggers direct recruitment of FADD and procaspase-8 and ⁄ or procaspase-10, provoking proximity-induced caspase activation. (C) Assembly of the apoptosome. Cytoso- lic cytochrome c binds to and activates Apaf-1, permitting dATP binding. This alters the conformation of Apaf-1 to allow association with procaspase-9 molecules via their CARDs. Electron cryomicroscopy and molecular modeling has revealed that the apoptosome is a heptamer- ic arrangement of the cytochrome c-bound Apaf-1 with procaspase-9 [65]. P K. Ho and C. J. Hawkins Mammalian initiator apoptotic caspases FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS 5439 pull-down approach [19] both indicated that RAIDD and caspase-2 associate via their CARDs. This interac- tion may link caspase-2 indirectly to receptor interact- ing protein (RIP1), a RAIDD-binding serine ⁄ threonine kinase that acts as a component of tumor necrosis fac- tor (TNF) receptor signaling pathway via its interac- tion with TRADD [19,39,40] (Fig. 3A). However, direct evidence for a caspase-2-RAIDD-RIP1- TRADD-TNF-R1 pathway has been elusive. Read and coworkers reported the recruitment of caspase-2 to a large protein complex that is independent of Apaf-1 and cytochrome c and lacks RAIDD [41]. This provided the first evidence that caspase-2 engages in complex formation, which could perhaps result in its activation. Upon genotoxic stress, caspase-2 partici- pates in another activation complex which contains the adaptor RAIDD and the p53-related death domain- containing protein PIDD, hence designated the ‘PIDDosome’ [42] (Fig. 3A). Lamkanfi and colleagues subsequently postulated yet another caspase-2-contain- ing protein complex, comprising the signal transducer TRAF2 and RIP1 (Fig. 3A). Counter-intuitively, that data links caspase-2 to the activation of NF-jB and p38 MAPK in a catalytic activity-independent manner [43], and is therefore not related to caspase-2 activa- tion. It is possible that caspase-2 is recruited to differ- ent large protein complexes in response to various death stimuli, and plays differential roles in multiple signaling pathways. Caspase-8 and caspase-10 in the DISC Death receptors belong to the TNF receptor super- family. They include Fas ⁄ CD95 ⁄ Apo1, TNF-R1, TNF-R2, DR3 ⁄ WSL-1 ⁄ TRAMP, DR4 ⁄ TRAIL-R1, DR5 ⁄ TRAIL-R2, and DR6. These receptors are char- acterized by the presence of a cytoplasmic death domain (DD). The recruitment of the adaptor mole- cule FADD to the death receptors occurs via homo- typic interaction between their DDs [44,45] (Fig. 3B). Recent evidence indicates that direct contact between TRAIL receptors and FADD [46,47] involves the DD of FADD as well as residues located in its death effec- tor domain [48]. TNF-R1 DISC formation (Fig. 3B) involves an extra intermediate adaptor molecule: TNF- R1-associated death domain protein (TRADD) [49], which is recruited to the TNF-R1 via the DD and interacts with FADD [50]. It has been reported that FADD and caspase-8 are not recruited to the TNF-R1 DISC upon TNFa stimulation, despite the fact that they are crucial components of TNF-induced apoptosis [51]. In a model proposed by Micheau and Tschopp, TNF-R1-mediated apoptosis is induced by the forma- tion of two sequential signaling complexes [52]. Bind- ing of TNFa to TNF-R1 is rapidly followed by the recruitment of TRADD, RIP1 and TRAF2, thus form- ing complex I. During receptor endocytosis, modi- fication of TNF-R1, TRADD and RIP1 occurs, promoting the dissociation of TRADD, RIP1 and TRAF2 from complex I. The dissociated TRADD and RIP1 can then interact with FADD via its DD and recruit procaspase-8 and ⁄ or -10 to form complex II [52]. TNF-R1 internalization and the formation of TNF receptosomes are critical for the recruitment of TRADD, FADD and caspase-8 to the TNF-R1 DISC, as shown by Schneider-Brachert et al. [53]. The associ- ation of FADD with procaspase-8 molecules through their DEDs leads to the oligomerization and activation of caspase-8 [54]. Molecular modeling has indicated that the stoichiometry of Fas, FADD and caspase-8 is 3 : 3 : 3 [55]. Proper maintenance of this stoichiometric ratio seems to be essential for the formation of the Fas DISC. Some autoimmune lymphoproliferative syndrome (ALPS)-affected individuals exhibit defects in DISC formation and failure in apoptosis due to the expression of defective Fas chains which cannot trimerize [56]. Caspase-10 is structurally similar to caspase-8 and can be recruited to the DISC via FADD upon Fas or TRAIL ligation [29,30,57,58], however, the ability of caspase-10 to functionally replace caspase-8 is conten- tious. Recent studies tend to suggest that it may not be capable of functionally replacing caspase-8 unless very high levels are expressed [29,30,57]. The substrate specificity of caspase-10, as predicted from its cleavage of a peptide combinatorial library, differs somewhat from that of caspase-8 [59]. It is therefore possible that caspase-10 possesses distinct roles relative to caspase-8, although it should be noted that caspase-10 arose in evolution after the divergence of mice and humans, so it is clearly not critical for mammalian biology. Caspase-9 in the apoptosome The activation mechanism of caspase-9 is well docu- mented. The assembly of an activation complex is trig- gered by the intrinsic or mitochondrial pathway, which is initiated upon various forms of cell stress including the presence of the protein kinase inhibitor staurospo- rine [60] and cytotoxic compounds such as etoposide [61] and doxorubicin [62]. The Bcl-2 family members control the release from the mitochondria of proapop- totic factors including cytochrome c [63]. Cytosolic cytochrome c and dATP (or ATP) bind to Apaf-1 to promote its multimerization [21,64]. The conforma- tional changes to Apaf-1 associated with this binding Mammalian initiator apoptotic caspases P K. Ho and C. J. Hawkins 5440 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS expose the N-terminal CARD, facilitating the CARD– CARD interaction with procaspase-9 molecules. This yields a heptameric complex (termed the apoptosome) composed of seven cytochrome c-bound Apaf-1 mole- cules surrounding seven procaspase-9 molecules in the core [65] (Fig. 3C). Alternatively, death receptor liga- tion can initiate the extrinsic pathway, where caspase-8 becomes activated upon DISC formation. Caspase-8- mediated proteolytic cleavage of the proapoptotic Bcl-2 family member Bid enables the translocation of the truncated Bid to the mitochondria and the subse- quent leakage of cytochrome c into the cytosol. This pathway acts as an amplification loop for the apopto- some formation and therefore caspase-9 activation. Caspase-9 activity is weak in the absence of Apaf-1 [66], so it is likely that the primary function of the ap- optosome is to enhance the allosteric conformation of the caspase-9 zymogen rather than to drive its matur- ation by cleavage. In fact, Rodriguez and Lazebnik showed that the Apaf-1 ⁄ caspase-9 complex acts as a holoenzyme to mediate the cleavage and activation of procaspase-3 [66]. In agreement with this observation, Stennick et al. generated procaspase-9 mutants harbor- ing mutations at cleavage sites and demonstrated their abilities to activate downstream caspases in the pres- ence of Apaf-1 and cytochrome c [67]. In a recent report, Chao et al. contrasted engineered dimeric caspase-9 with apoptosome-activated caspase-9 and found a 35-fold difference in catalytic activities between the two [68]. This suggests that a unique con- formational change in caspase-9 induced upon binding to the apoptosome contributed to its higher activity which cannot be achieved by dimerization alone [68]. Caspase-12 activation Formation of a high molecular mass complex has not been reported for caspase-12. In mouse glial cells undergoing endoplasmic reticulum (ER) stress, caspase-12 zymogen cleavage is calpain-dependent [69]. The calpain-cleaved caspase-12 then autoactivates to produce the mature heterotetramer. It has also been shown that cleavage and activation of caspase-12 in cisplatin-induced apoptosis of porcine renal tubular epithelial cells is a result of the oxidative stress caused by the interaction of cisplatin and the cytochrome P450 system in the ER [70]. In humans, however, caspase-12 has been dubbed ‘pseudo-caspase-12’ in a recent review [4], as it contains coding sequence aber- rations that prevent the translation of the putative full-length enzyme [71]. This argues against the exist- ence of a functional caspase-12 in humans and its hypothesized physiological roles in ER stress response. Interestingly, it has subsequently been reported that caspase-12 is naturally polymorphic and is expressed in its full-length form in 20% of people of African descent [72]. Individuals expressing full-length caspase- 12 are more susceptible to sepsis as a result of attenu- ated inflammatory and innate immune response to endotoxins [72]. These observations suggest that caspase-12 may have dual roles in apoptotic and inflammatory functions, which may also be species- dependent. Substrate specificity Synthetic peptide substrates As mentioned above, caspases can be classified based on their substrate preferences. Studies conducted by Thornberry et al. in which the substrate specificities of caspase family members were investigated using a novel combinatorial library approach, defined three major groups of caspases [59,73]. This positional scan- ning technique uses fluorogenically labeled synthetic tetrapeptides in different amino acid combinations. The differential preference for the P4-P2 residues strongly influences target specificities. Caspase-2, like effector caspases-3 and -7, has a strict requirement for Asp in P4. On the contrary, caspases-8, -9 and -10 pre- fer a branched chain aliphatic amino acid in P4, which is similar to caspase-6. Another study, performed by Talanian et al. presented strong evidence that the opti- mal minimal substrate sequence for caspase-2 includes a residue in the P5 position (preferably a hydrophobic amino acid) [74]. The substrate specificity of caspases- 12 has not yet been deduced in a similar experimental context. Thornberry and colleagues proposed that the similarities between the optimal tetrapeptide cleavage sequence and the sequences linking the subunits in some caspases (including caspases-2, -8 and -9) may reflect their autocatalytic capabilities [73]. The short peptide screening technique was a highly innovative approach for the discovery of caspase substrates and inhibitors, and identification of preferred peptide sequences has facilitated the development of in vitro caspase activity assays. However, while this technique provided a snapshot of preferred substrate cleavage sequences for individual caspases, it cannot conclu- sively determine the suitability of a natural cellular protein as a substrate. The synthetic peptides are small in size and are readily accessible to caspases. On the contrary, cellular proteins are relatively large in size and their caspase cleavage recognition sequence can be buried as a result of natural folding. This limitation in accessibility could mean that a protein bearing the P K. Ho and C. J. Hawkins Mammalian initiator apoptotic caspases FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS 5441 optimal cleavage sequence may not necessarily be a caspase target in vivo. Natural cellular substrates During apoptosis, a vast variety of cellular proteins undergo proteolytic cleavage by caspases. Effector caspases cleave numerous proteins, including some that are responsible for the structural integrity of the cell. Other proteins, including those that have more indirect roles in the cellular morphology and metabolic architec- ture, are cleaved by initiator caspases. A caspase sub- strate can be functionally activated or inactivated upon cleavage. Conforming to their roles as initiator caspas- es, caspases-2, -8, -9, -10 and -12 can proteolytically activate effector caspases-3 or -7 [21,75–78]. These casp- ases also target other cellular proteins that play import- ant structural or signaling roles in the cell. For example, during apoptosis caspase-2 cleaves the Golgi- specific protein golgin-160, which controls the integrity of the Golgi complex [79]. The structural protein aII- spectrin is also a caspase-2 substrate, whose cleavage can destabilize the scaffolding of the membrane cyto- skeleton [80]. More recently, the caspase-2-mediated cleavage of huntingtin has been implicated in patholo- gical neuronal cell death [81]. Caspases-8 and -9 cleave structural proteins plectin [82] and vimentin [83], respectively, in cells undergoing apoptosis. The pro- apoptotic Bcl-2 family member Bid is the most notable among various caspase-8 substrates. It becomes activa- ted upon cleavage by caspase-8 and then causes the mito- chondrial release of cytochrome c which forms part of the apoptosome [84,85]. Cleavage of another caspase-8 substrate, RIP1, leads to its inactivation, blocking the NF-jB-mediated survival signals in Fas-induced apop- tosis [17]. Regulation Because caspases play vital roles in apoptotic initi- ation, their expression and activation must be tightly regulated to maintain the homeostatic balance between apoptosis and survival. This is achieved by different mechanisms which exert control at various checkpoints within the cell. Transcriptional regulation While most caspases are constitutively expressed, some are transcriptionally regulated in particular contexts. The caspase-8 promoter contains Sp1 and ETS-like motifs that control basal expression [86,87]. The pres- ence of an interferon-stimulated response element (ISRE) and p53-responsive site enables caspase-8 up- regulation by interferon-c and p53, respectively [86– 88]. Methylation of a region within the 5¢ untranslated portion of the gene has been associated with the down-regulation of caspase-8 expression in some tumor cells [89–96]. Unlike caspase-8, caspase-10 expression is predominantly regulated by post-tran- scriptional mechanisms. Expression of caspase-10 was frequently detected at the mRNA level but rarely at the protein level in various cancer cell lines [29,95]. The lack of caspase-10 expression in these cell lines results from post-transcriptional down-regulation [29]. In caspase-2-deficient mice, caspase-9 was transcrip- tionally up-regulated to provide a possible compensa- tory effect in the absence of caspase-2 [97]. A three- fold increase in caspase-9 mRNA and protein expres- sion was detected in caspase-2-null brains and cultured sympathetic neurons [97]. An analysis of the 5¢ region of the human CASP2 gene reveals that the transcripts of the two isoforms caspase-2L and caspase-2S are expressed from separate promoter regions of different strengths and the expression is initiated from differen- tial translational start sites [98]. The proximal 5¢-flank- ing region of rat CASP9 gene has no TATA-box, but contains GC-boxes and a hypoxia-inducible factor 1-binding site [99]. The presence of this cis-acting ele- ment allows the up-regulation of caspase-9 during hyp- oxia [99]. The transcriptional regulatory elements of the murine CASP12 gene have also been examined. The promoter region lacks a TATA-box, CAAT-box, or GC-box but contains a number of putative binding sites for transcription factors including AP1, Oct -1, Sp1 and NF-jB [100]. Alternative splicing Some caspases express multiple splice variants in a tis- sue- or cell line-specific manner. While some splice vari- ants encode the wild-type active species, the predicted translation products of others would lack residues essen- tial for enzymatic activity and may theoretically either act as dominant negatives in the caspase recruitment process or be nonfunctional. It should be noted that some of these splice variants have only been detected at the mRNA but not protein level. Functions of these splice variants have not been studied in detail and most data were obtained by ectopic overexpression. Four caspase-2 splice variants have been identified in humans and two reported in rats [101–103]. Of the four human splice variants, only the wild-type caspase-2L encodes an active enzyme. This is the major isoform whose protein product has been detected in most tis- sues. The alternatively spliced caspase-2S includes exon Mammalian initiator apoptotic caspases P K. Ho and C. J. Hawkins 5442 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS 9 but lacks the sequence corresponding to the small subunit in the transcript. It has been shown to inhibit apoptosis when overexpressed in cultured cells [101]. Although a small form of caspase-2 has been detected by immunoblotting, it is unclear whether this repre- sents caspase-2S or a cleavage product [104]. Thus the existence of the putative translation product of casp- ase-2S has still not been confirmed. Another splice vari- ant, casp-2L-Pro, was detected as a truncated protein in several cell lines and was also reported to act as a negative apoptotic regulator. However, the expression of a shorter variant casp-2S-Pro has not yet been detec- ted and its function has yet to be established [102]. The alternative splicing in caspase-8 has resulted in 10 transcriptionally detectable isoforms, caspase-8 ⁄ a-h, and the two lesser known isoforms procaspase-8L and caspase-8L [105–107]. Caspase-8 ⁄ a and caspase-8 ⁄ b are the two functionally active isoforms that are predomin- antly expressed at the protein level [105]. Procaspase- 8L is a version of caspase-8 ⁄ a containing an N-terminal extension domain that is required for the association with the BAP31 complex. The procaspase-8L protein was detected in various tissues, particularly abundant in the thymus and lung [106]. Expression of the ca- spase-8L protein was detectable in peripheral blood lymphocytes and it has been proposed to act as an endogenous inhibitor of caspase-8 [107]. The expres- sion, physiological functions and biological significance of caspase-8 ⁄ c-h, however, are poorly understood. Phylogenetic divergence has given rise to four cas- pase-9 alternative transcripts that differ between humans and mice. The human caspase-9b isoform lacks the central large subunit and can block the for- mation of the apoptosome and caspase-9-mediated apoptotic events [108,109]. The alternative murine transcript caspase-9S was demonstrated to be inactive upon overexpression compared with the predominantly expressed wild-type caspase-9L [110]. Four caspase-10 isoforms have been identified. Caspase-10 ⁄ a,b,d [77,111,112] are proteolytically com- petent. Caspase-10 ⁄ c, though catalytically inactive, is capable of inducing cell death in vivo [112]. Nine alternatively spliced human caspase-12 tran- scripts were reported by Fischer et al., in which the presence of a premature stop codon prevents the trans- lation of a full-length protein [71]. However, a poly- morphism yields the full-length form of caspase-12 in one-fifth of ethnic Africans [72]. Caspase inhibition Once expressed, caspases can be inhibited by other cel- lular proteins. One mechanism of caspase inhibition is via the inhibitors of apoptosis (IAPs). IAP-like proteins are encoded by many eukaryotic genomes including those of Drosophila (DIAP-1 and -2) and mammals (XIAP ⁄ MIHA ⁄ hILP ⁄ BIRC4, cIAP-1 ⁄ MIHB ⁄ HIAP2 ⁄ BIRC2, cIAP-2 ⁄ MIHC ⁄ HIAP1 ⁄ BIRC3, NAIP ⁄ BIRC1, survivin ⁄ BIRC5, LIVIN ⁄ ML-IAP ⁄ KIAP ⁄ BIRC7, ILP- 2 ⁄ BIRC8, BRUCE ⁄ Apollon ⁄ BIRC6). These proteins are characterized by the presence of one to three baculo- viral IAP repeats (BIR) and, in some, a RING-finger motif at the C-terminus [113]. Survivin is a structurally distinct member of the IAPs [114]. It contains a BIR domain but its primary function appears to be cell cycle control rather than apoptosis inhibition [115,116]. The effector caspases-3 and -7 and the initiator caspase-9 are targets of IAPs [117,118]. In contrast, caspases-1, -2, -6, -8 and -10 are immune to inhibition by XIAP, c-IAP1 and c-IAP2 [75,117,119]. IAP-mediated inhibi- tion mechanisms differ between effector and initiator caspases [120,121]. The inhibition of caspases-3 and -7 by XIAP requires the linker between BIR1 and BIR2 whereas its inhibition of caspase-9 is mediated through BIR3 [121,122]. Regulation of caspase-9 activity can also be achieved by the release of mitochondrial pro- teins Smac ⁄ DIABLO [123] and HtrA2 ⁄ Omi [124,125] which antagonize IAP inhibition by competitively bind- ing to the BIR3 and displacing the bound caspase-9 molecule [123,126,127]. More recently, it has become apparent that ubiqutin-mediated degradation is involved in the regulation of IAPs and proteins with which they interact [128]. c-IAP2 has been reported to promote the ubiquitination of caspases-3 and -7 [129] while the degradation and regulation of Smac ⁄ DIABLO and caspase-9 can be facilitated by the ubiquitination mediated by BRUCE ⁄ Apollon [130,131] and XIAP [132]. Caspase-8 activity can be regulated by a family of proteins called FADD-like ICE (FLICE)-inhibitory proteins (FLIPs). v-FLIPs were identified in several c-herpes viruses [133]. They possess antiapoptotic abil- ities which enable them to evade host antiviral apop- totic responses. Sequence analysis revealed that v-FLIPs contain two DEDs in their N-termini and share considerable homology with caspase-8. Further observations indicated that v-FLIPs are capable of binding to FADD and caspase-8 via homotypic inter- actions. This binding blocks the recruitment of cas- pase-8 to the DISC and hence prevents caspase-8 activation [133–135]. A cellular homologue of the v-FLIPs was subsequently identified and designated c-FLIP [26], also known as CASH ⁄ CASPER ⁄ CLARP ⁄ FLAME-1 ⁄ I-FLICE ⁄ MRIT ⁄ Usurpin [136–142]. Alter- native splicing produces two variants of different lengths, c-FLIP L and c-FLIP S , both of which are P K. Ho and C. J. Hawkins Mammalian initiator apoptotic caspases FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS 5443 expressed at the protein level. The short form is struc- turally and functionally similar to v-FLIP. It has a short half life, due to its destabilizing carboxyl-ter- minal tail, which prompts ubiquitin-mediated degrada- tion [143]. The more stable long form contains two DEDs and a caspase-8-like region that encompasses an inactive proteolytic site. Experimental evidence from independent studies suggested that c-FLIP L possesses dual capabilities in the regulation of DISC-mediated apoptosis. Some studies reported the antiapoptotic activities of c-FLIP L [26,139,140] while others demon- strated its proapoptotic functions [137,138,141]. The differential functionalities seem to be concentration- dependent [144]. According to this paradigm, low c-FLIP L expression promotes apoptosis by enhancing caspase-8 activation, through heterodimerization with procaspase-8 molecules [145,146]. In contrast, high lev- els of c-FLIP L compete with caspase-8 for the recruit- ment to the DISC, leading to the inhibition of apoptosis [144,147]. Functional studies Knockout mouse models Gene knockout mouse models have provided valuable information regarding the roles of individual compo- nents in the apoptotic pathway. A number of knock- out mouse models has been established, including those of adaptor molecules Apaf-1 [148,149], FADD [150,151], RIP1 [152], RAIDD [153], effector and initi- ator caspases. Caspase-2-deleted mice generated by Bergeron and colleagues [154] were grossly normal compared with wild-type mice. Deficiency in caspase-2 appeared to be nonlethal and these caspase-2 – ⁄ – mice survived to adulthood. The females showed normal fertility despite an increased number of germ cells relative to normal mice [154]. Thymocytes derived from these mice were sensitive to various cell death triggers including FasL stimulation, dexamethasone treatment and c-irradi- ation [104,154]. Caspase-2-deficient dorsal root ganglia neurons also underwent factor-withdrawal-induced cell death like their wild-type counterparts [104]. This sug- gests that caspase-2 is dispensable for normally occur- ring cell death but may play a role in gametogenesis. Interestingly, Troy et al. detected increased caspase-9 expression in caspase-2-deficient neurons, implying that caspase-9 may play a compensatory role in the absence of caspase-2 [97]. Caspase-9-deficiency causes embryonic or postnatal lethality [155,156], although the severity of this pheno- type is strain-dependent [157]. Analyses of the embryos revealed the brain was the most profoundly affected organ. Hyperplasia and enlarged proliferation zones in the brain occurred as a result of decreased apoptosis, and caused the malformation of the brain. Hence it appears that caspase-9 plays an important role in brain development. The embryonic stem cells and embryonic fibroblasts derived from caspase-9-null mice are resist- ant to a number of apoptotic stimuli including UV and c-irradiation [156]. Caspase-3 – ⁄ – mice exhibit similar phenotypes to the caspase-9- null animals [156,158], consistent with the notion that these casp- ases participate in the same or a similar pathway. In fact, caspase-3 processing was abolished in caspase-9- null brain tissue and lysates extracted from the thymo- cytes and could be restored by the addition of in vitro translated caspase-9 to the lysates [155]. These obser- vations directly suggest that, in this cellular context, caspase-3 activation is a consequence of caspase-9 activity. Data by Zheng et al. demonstrated caspase-2- dependent activation of caspase-6 in caspase-9-deficient hepatocytes upon antibody-mediated Fas oligomeriza- tion, suggesting the possible compensatory role played by caspase-2 in the absence of caspase-9 [159]. Mature B cells, T cells and T lymphoblasts derived from the caspase-2 – ⁄ – 9 – ⁄ – double knockout mice generated by Marsden and colleagues displayed normal sensitivity towards a variety of apoptotic stimuli including cyto- kine withdrawal and dexamethasone [160]. The appar- ent discrepancies in the sensitivities towards apoptotic stimuli between caspase-9 single knockout and caspase-2 – ⁄ – 9 – ⁄ – double knockout mice may be recon- ciled by the fact that different cell types were exam- ined. The sensitivity exhibited by caspase-2 – ⁄ – 9 – ⁄ – lymphocytes prompts the hypothesis that cell death can proceed via a third mechanism that is independent of caspases-2 and -9. Caspase-8 ablation is embryonically lethal, with embryos displaying a reduction in size, impaired heart development and abnormal accumulation of erythro- cytes in the trunk area at E11.5 and E12.5 [161]. Inter- estingly, these dramatic consequences of caspase-8 deficiency result from a proliferation defect which appears to be unrelated to the role played by caspase-8 in death receptor signaling. Caspase-8 deletion in par- ticular organs and tissues demonstrated the expected resistance to death ligand-induced apoptosis [161–163]. However, cell death induced by other death stimuli including ultraviolet irradiation, cytotoxic drugs, vesic- ular stomatitis virus infection and serum deprivation were unaffected by the absence of caspase-8 [161]. Human individuals whose CASP8 gene was homozy- gously inactivated manifested defective lymphocyte apoptosis and homeostasis [164]. Caspase-8 thus plays Mammalian initiator apoptotic caspases P K. Ho and C. J. Hawkins 5444 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS a nonredundant role in normal embryonic devel- opment, lymphocyte activation and death receptor- mediated apoptosis, but is dispensable for intrinsic apoptotic pathways. Mice lacking caspase-12 were developmentally normal and phenotypically indistinguishable from wild-type littermates [165]. Caspase-12-null cells were resistant to ER stress-induced apoptosis, but sensitive to selected death stimuli. For example, caspase-12 – ⁄ – cortical neurons underwent apoptosis induced by sta- urosporine and trophic factor deprivation, but were defective with regard to stimulation by amyloid-b pro- tein [165]. Embryonic fibroblasts derived from these mice were partially resistant to apoptosis induced by brefeldin A, tunicamycin and thapsigargin, but not to staurosporin, FasL or TNFa [165]. This indicates that caspase-12 is specifically involved in certain stress response pathways. The partial protection against apoptosis also suggests that while caspase-12 is import- ant in ER stress response, it is not the sole apoptotic initiation factor. Arguing against a critical role for cas- pase-12 enzymatic activity in mammalian biology, the human gene encodes a catalytically inactive protein [71]. Pathogenesis Excessive or inadequate apoptosis can lead to the development of pathological conditions. Numerous diseases have been attributed to apoptotic machinery malfunction. Altered expression and ⁄ or activity of pathway components including receptors, ligands, adaptors, caspases and substrates contribute to several neurodegenerative diseases, some types of autoimmune disorder and cancer. Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are associated with excessive cell death involving defective regulation of caspase activity. Increased activities of caspases-8 and -9 have been observed in brain tissues [166–168] and peripheral blood mononuclear cells of Alzheimer’s dis- ease patients [169]. The neurotoxicity caused by the deposition of the pathological amyloid-b peptide could induce apoptosis and neuronal loss [170], possibly mediated by these initiator caspases. Moreover, neu- rons deficient in caspases-2 and -12 were found to have reduced susceptibility to amyloid-b toxicity in murine models, suggesting these two caspases may also play a role in the neuronal cell death signaling pathway [165,171]. The activities of caspases-8 and -9 were reported to be elevated in brain tissue from Parkin- son’s disease patients [172,173]. Huntington’s disease, another neurodegenerative disorder, is caused by the abnormal expansion of polyglutamine repeats in the huntingtin protein, which recruits and activates caspase-8 [174]. The polyglutamine-induced caspase-8 aggregation is reminiscent of the ‘proximity model’, however, this is independent of receptor ligation. Caspase-10 has also been proposed to play a role in the pathological development of Huntington’s disease in a fashion similar to that of caspase-8 [175]. Recently, the involvement of caspase-2 in the onset of neuronal cell death in this disease has been recognized. Polyglutamine-induced recruitment brings caspase-2 into close proximity to huntingtin and facilitates the generation of the neurotoxic cleavage product [81]. Inadequate apoptosis can lead to the onset of other diseases. ALPS, which comprises five subtypes, is char- acterized by nonmalignant lymphadenopathies. An examination of two unrelated ALPS II patients revealed that independent single substitution mutations in the caspase-10 gene led to amino acid changes in the catalytic subunit which resulted in decreased enzy- matic activity, impairing sensitivity to death receptor- mediated apoptosis [176]. Interestingly, one of these mutations has been detected in the heterozygous state at a high frequency in the Danish population [177], prompting debate about whether this phenomenon rep- resents genetic polymorphism or the real cause of the disease. An immunological condition due to hereditary caspase-8 deficiency has been described by Chun et al. [164]. This condition is characterized by defective death receptor signaling and activation of T lympho- cytes, B lymphocytes and natural killer cells, and leads to immunodeficiency. Affected individuals carry homo- zygous CASP8 alleles encoding an R248W substitu- tion. This mutation renders the caspase-8 protein unstable and dramatically reduces its enzymatic activity. Caspase-8 has also been linked to neuroblastoma, a tumor of the peripheral nervous system most com- monly affecting children. Lack of caspase-8 expression due to methylation-induced silencing of CASP8 gene was observed in 63% of high grade neuroblastomas [89]. Low caspase-8 levels have also been reported in other tumors including medulloblastoma [90,178], small cell lung carcinomas [91,179] and bronchial car- cinoids [91]. We have recently reported low caspase-8 expression in many ex vivo gliomas. Neither CASP8 gene methylation status nor transcription factor STAT-1 expression level correlated with caspase-8 expression in glioma cells [180], suggesting caspase-8 expression in these gliomas may employ a regulatory mechanism that is different from other types of tumors [181,182]. Deletion of the CASP8 gene was found in one lung tumor [91] and one neuroblastoma [89]. P K. Ho and C. J. 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Ambrosis A, Croce M, Pagnan G, Di Vinci A, Allemanni G, Banelli B, Ponzoni M, Romani M & Ferrini S (2004) Expression of the caspase-8 gene in FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS Mammalian initiator apoptotic caspases 89 90 91 92 93 94 95 96 97 98 99 neuroblastoma cells is regulated through an essential interferon-sensitive response element (ISRE) Cell Death Differ 11, 131–134 Teitz T, Wei T,... direct isoform-specific expression Oncogene 22, 935–946 Nishiyama J, Yi X, Venkatachalam MA & Dong Z (2001) cDNA cloning and promoter analysis of rat caspase-9 Biochem J 360, 49–56 5449 Mammalian initiator apoptotic caspases 100 Oubrahim H, Wang J, Stadtman ER & Chock PB (2005) Molecular cloning and characterization of murine caspase-12 gene promoter Proc Natl Acad Sci USA 102, 2322–2327 101 Wang L,.. .Mammalian initiator apoptotic caspases Frequent inactivating mutations of caspase-8 have been reported in advanced colorectal carcinomas [183], hepatocellular carcinomas [184], gastric carcinomas [185] and in a single... factor-induced apoptosis J Biol Chem 278, 25534–25541 52 Micheau O & Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes Cell 114, 181–190 5447 Mammalian initiator apoptotic caspases 53 Schneider-Brachert W, Tchikov V, Neumeyer J, Jakob M, Winoto-Morbach S, Held-Feindt J, Heinrich M, Merkel O, Ehrenschwender M, Adam D et al (2004) Compartmentalization of TNF... of deathinducing signaling complex (DISC) components in lung cancer cell lines and the influence of MYC amplification Oncogene 21, 8510–8514 FEBS Journal 272 (2005) 5436–5453 ª 2005 FEBS Mammalian initiator apoptotic caspases 180 Ashley DM, Riffkin CR, Muscat AM, Knight MJ, Kaye AH, Novak U & Hawkins CJ (2005) Caspase-8 is absent, low or mutated in many ex vivo gliomas Cancer 104, 1487–1496 181 Banelli... JC (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases EMBO J 17, 2215–2223 Kasof GM & Gomes BC (2001) Livin, a novel inhibitor of apoptosis protein family member J Biol Chem 276, 3238–3246 Roy N, Deveraux QL, Takahashi R, Salvesen GS & Reed JC (1997) The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases EMBO J 16, 6914–6925 . in apoptotic signaling pathways. These apoptotic caspases can be further divi- ded into ‘initiators’ (caspases- 2, -8, -9, -10, -12) and ‘effectors’ (caspases- 3,. -7, -14). Initiator caspases function upstream within apoptotic signaling pathways. They are capable of activating downstream caspases (effector caspases)