A novel isoform of rat caspase-9 has been identified in which the C terminus of full-length caspase-9 is replaced with an alternative peptide sequence. Casp-9-CTD (where CTD is carboxyl-terminal divergent) is expressed in multiple tissues, with the relative highest expression observed in ovary and heart. Casp-9-CTD is found primarily in the cytoplasm and is not detected in the nucleus. Structural predictions suggest that in contrast to full-length caspase-9, casp-9-CTD is not able to be processed. This model is supported by reduced protease activity of casp-9-CTD preparations in vitro and by the lack of detectable processing of casp-9-CTD proenzyme or the induction of cell death following transfection into cells. Both neuronal and non-neuronal cell types transfected with casp-9-CTD are resistant to death evoked by trophic factor deprivation or DNA damage. In addition, cytosolic lysates prepared from cells permanently expressing exogenous casp-9-CTD are resistant to caspase induction by cytochrome c in reconstitution assays. Taken together, these observations indicate that casp-9-CTD acts as a dominant-negative variant. Its expression in various tissues indicates a physiological role in regulating cell death (Angelastro, 2001).
Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. Proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. A unified caspase activation hypothesis is proposed whereby apical caspases are activated by dimerization of monomeric zymogens (Boatright, 2003).
In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8) were compared. wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The KD for dimeric wtC8 is ~50 microM and decreases when inhibitor binds. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. These findings suggest that dimerization is the crucial factor for caspase-8 activation (Donepudi, 2003).
X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevent caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).
MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. After UV radiation, MIHA prevents apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functions after release of cytochrome c and DIABLO from the mitochondria and is able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO binds to MIHA and disrupts MIHA's association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis (Ekert, 2001).
The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 Å resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. Monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases (Shiozaki, 2003).
The hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases has been tested through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo (Kuida, 1998).
Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9-/- ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells (Hakem, 1998).
Programmed cell death is critical for normal nervous system development and is regulated by Bcl-2 and Caspase family members. Targeted disruption of bcl-x(L), an antiapoptotic bcl-2 gene family member, causes massive death of immature neurons in the developing nervous system whereas disruption of caspase-9, a proapoptotic caspase gene family member, leads to decreased neuronal apoptosis and neurodevelopmental abnormalities. To determine whether Bcl-X(L) and Caspase-9 interact in an obligate pathway of neuronal apoptosis, bcl-x/caspase-9 double homozygous mutants were generated. The increased apoptosis of immature neurons observed in Bcl-X(L)-deficient embryos is completely prevented by concomitant Caspase-9 deficiency. In contrast, bcl-x(-/-)/caspase-9(-/-) embryonic mice exhibit an expanded ventricular zone and neuronal malformations identical to those observed in mice lacking only Caspase-9. These results indicate both epistatic and independent actions of Bcl-X(L) and Caspase-9 in neuronal programmed cell death. To examine Bcl-2 and Caspase family-dependent apoptotic pathways in telencephalic neurons, the effects of cytosine arabinoside (AraC), a known neuronal apoptosis inducer, were examined on wild-type, Bcl-X(L)-, Bax-, Caspase-9-, Caspase-3-, and p53-deficient telencephalic neurons in vitro. AraC causes extensive apoptosis of wild-type and Bcl-X(L)-deficient neurons. p53- and Bax-deficient neurons show marked protection from AraC-induced death, whereas Caspase-9- and Caspase-3-deficient neurons show minimal or no protection, respectively. These findings contrast with a previous investigation of AraC-induced apoptosis of telencephalic neural precursor cells in which death was completely blocked by p53 or Caspase-9 deficiency but not Bax deficiency. In total, these results indicate a transition from Caspase-9- to Bax- and Bcl-X(L)-mediated neuronal apoptosis (Zaidi, 2001).
Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. Using an established in vivo model of Fas-mediated apoptosis, it has been demonstrated that elimination of certain caspases is compensated in vivo by the activation of other caspases. Hepatocyte apoptosis and mouse death induced by the Fas agonistic antibody Jo2 requires proapoptotic Bcl-2 family member Bid and uses a Bid-mediated mitochondrial pathway of caspase activation; deficiency in caspases essential for this pathway, caspase-9 or caspase-3, unexpectedly result in rapid activation of alternate caspases after injection of Jo2, and therefore fail to protect mice against Jo2 toxicity. Moreover, both ultraviolet and gamma irradiation, two established inducers of the mitochondrial caspase-activation pathway, also elicit compensatory activation of caspases in cultured caspase-3(-/-) hepatocytes, indicating that the compensatory caspase activation is mediated through the mitochondria. These findings provide direct experimental evidence for compensatory pathways of caspase activation. This issue should therefore be considered in developing caspase inhibitors for therapeutic applications (Zheng, 2000).
Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (Li, 1997).
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. A mammalian homolog of CED-4, Apaf-1, can associate with several death proteases in mammalian cells, including caspase-4, caspase-8, caspase-9, and nematode CED-3. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhances the killing activity of caspase-9, which requires the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promotes the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, has been shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL is mediated through both domains of Apaf-1: the CED-4-like domain and the C-terminal domain, which contains WD-40 repeats. Expression of Bcl-XL inhibits the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibits Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL fails to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans (Hu, 1998).
The reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9 is reported. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3 (Zou, 1999).
Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large (on the order of approximately 1.4 MDa). dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of approximately 1.4 MDa and approximately 700 kDa. Kinetic analysis demonstrates that in vitro the approximately 700-kDa complex is produced more rapidly than the approximately 1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the complex predominantely formed is the approximately 700-kDa Apaf-1 containing apoptosome complex. This complex processes effector caspases. Thus, the approximately 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis (Cain, 2000).
The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).
In contrast to the autoprocessing of caspase-9, little is known about the biological significance of caspase-9 processing by caspase-3 via a feedback loop in vivo. Antisera were prepared against mouse caspase-9 cleavage sites so that only the activated form of mouse caspase-9 was recognized. Using these antisera and caspase-9- and caspase-3-deficient mouse embryonic fibroblasts, it has been demonstrated that mouse caspase-9 is initially autoprocessed at D(353) and D(368) at low levels during staurosporine-induced apoptosis, whereupon the D(368) and D(168) sites are preferentially processed over D(353) by activated caspase-3 as part of a feedback amplification loop. Ac-DEVD-MCA (caspase-3-like) and Ac-LEHD-MCA (caspase-9-like) cleavage activities clearly show that caspase-9 autoprocessing is necessary for the activation of caspase-3, whereas full activation of caspase-3 and caspase-9 is achieved only through the feedback amplification loop. This feedback amplification loop also plays a predominant role during programmed cell death of dorsal root ganglia neurons at mouse embryonic day 11.5 (Fujita, 2001).
During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome has been examined. A multi-step process is proposed, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex (Bratton, 2001).
Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (1) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9; (2) a CED-4-like region enabling self-oligomerization, and (3) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Different apoptotic stimuli induce the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment is obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 is cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site is located within the WD-40 repeats and enables the oligomerization of p84 into an approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer is devoid of caspase-9 or other caspase activity. Thus, these data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis (Lauber, 2001).
Caspase-9, an initiator caspase, and caspase-3, an effector caspase, have been suggested to mediate the terminal stages of neuronal apoptosis, but little is known about their activation in vivo. Temporal and spatial aspects of caspase-9 and -3 activation have been examined in olfactory receptor neurons (ORNs) undergoing apoptosis after target removal in vivo. After removal of the olfactory bulb, enhanced expression of procaspase-9 and -3 is observed in ORNs, followed by activation initially at the level of the lesion, then in axons, and only later in the ORN soma. The amyloid precursor-like protein-2 (APLP2) is a caspase substrate that is cleaved in an identical spatiotemporal pattern, suggesting its cleavage is the result of retrograde propagation of a pro-apoptotic signal in a caudorostral wave from the synapse through the axon to the ORN cell body. A null mutation in caspase-3 causes a change in axonal patterning indicative of an overall developmental expansion of the ORN population, and mature ORNs of caspase-3 knock-outs do not undergo caspase-dependent terminal dUTP nick end labeling-positive apoptosis after olfactory bulb removal. These results demonstrate that ORNs require caspase-3 activation to undergo normal developmental and mature target-deprived apoptosis. In addition, an axonal site of action for caspase-3 and -9 has been demonstrated; regulation and activation of caspase-3 and -9 leading to apoptosis is a highly ordered process that occurs initially at the presynaptic level and only later at the cell body after deafferentation (Cowan, 2001).
Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g., during development. Vimentin has been demonstrated to be the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, shows that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. These results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death (Nakanishi, 2001).
Sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation; these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors (Troy, 2001).
Mammalian caspases are a family of cysteine proteases that plays a critical role in apoptosis. Caspase-2 processing has been analyzed in human cell lines containing defined mutations in caspase-3 and caspase-9. Caspase-2 processing, during cell death induced by UV irradiation, depends both on caspase-9 and caspase-3 activity, while, during TNF-alpha-dependent apoptosis, capase-2 processing is independent of caspase-9 but still requires caspase-3. In vitro procaspase-2 is the preferred caspase cleaved by caspase-3, while caspase-7 cleaves procaspase-2 with reduced efficiency. Caspase-2-mediated apoptosis requires caspase-9, and cells co-expressing caspase-2 and a dominant negative form of caspase-9 are impaired in activating a normal apoptotic response and release cytochrome c into the cytoplasm. These findings suggest a role played by caspase-2 as a regulator of the mitochondrial integrity and open questions on the mechanisms responsible for its activation during cell death (Paroni, 2001).
Huntington's Disease is an inherited neurodegenerative disease that affects the medium spiny neurons in the striatum. The disease is caused by the expansion of a polyglutamine sequence in the N terminus of Huntingtin (Htt), a widely expressed protein. Htt is an antiapoptotic protein in striatal cells and acts by preventing caspase-3 activity. Htt overexpression in other CNS-derived cells can protect them from more than 20 days exposure to fatal stimuli. In particular, cytochrome c continues to be released from mitochondria into the cytosol of cells that overexpress normal Htt. However, procaspase-9 is not processed, indicating that wild-type Htt (wtHtt) acts downstream of cytochrome c release. These data show that Htt inhibits neuronal cell death by interfering with the activity of the apoptosome complex (Rigamonti, 2001).
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