Caspases: pharmacological manipulation
of cell death (original) (raw)
Mechanisms of caspase activation. All caspases are produced in cells as catalytically inactive zymogens and must undergo proteolytic processing and activation during apoptosis. The effector caspases are activated by initiator or apical caspases. However, one central question of apoptosis is how initiator caspases are activated and how this activation is regulated to prevent spontaneous cell death. It is generally accepted that apical caspase activation takes place in large protein complexes that bring together several caspase zymogens (22–25, 36). All initiator caspases are characterized by the presence of a member of the DD superfamily (DED or CARD), which enables their recruitment into the initiation complex. Several activating complexes for initiator caspases have been reported so far.
The death-inducing signaling complex as an activating complex for procaspase-8 and -10. Procaspase-8 and -10 are apical caspases in apoptotic pathways triggered by engagement of death receptors. Several members of the TNF receptor (TNFR) superfamily (TNFR1, CD95 [Fas/APO-1], TRAIL-R1, TRAIL-R2, DR3, DR6) comprise DD in their intracellular domain and are, therefore, termed death receptors (3, 37). Triggering of CD95 and TRAIL-R1/R2 by corresponding ligands results in the formation of a death-inducing signaling complex (DISC) (38–42). CD95 and TRAIL-R1/R2 DISCs consist of oligomerized, probably trimerized, receptors, the DD-containing adaptor molecule FADD/MORT1 (Fas-associated death domain), 2 isoforms of procaspase-8 (procaspase-8/a [FLICE, MACHα1, Mch5] and procaspase-8/b [Machα2]), procaspase-10, and the cellular FLICE-inhibitory proteins (c-FLIPL/S/R) (Figure 2A) (43, 44). The interactions between the molecules at the DISC are based on homotypic contacts. The DD of the receptor interacts with the DD of FADD, while the DED of FADD interacts with the N-terminal tandem DEDs of procaspase-8 and -10 and FLIPL/S/R. Thus, DISC formation results in assembly of procaspase-8 and -10 molecules in close proximity to each other.
Scheme of procaspase-8 processing at the CD95 DISC. CD95 DISC formation is triggered by extracellular cross-linking with CD95L (depicted in red), which is followed by oligomerization of the receptor. FADD/MORT1 is recruited to the DISC by DD interactions (shown in red); procaspase-8 and -10 as well as c-FLIP proteins are recruited to the DISC by homophilic DED interactions (yellow). Upon recruitment to the DISC, procaspase-8 undergoes processing by forming dimers (depicted in green). (A) The first step of procaspase-8 cleavage occurs between 2 protease subunits. The site of cleavage is shown by a black arrow. As a result of the first cleavage step the p10 subunit is formed, which is not released into the cytosol but remains bound to the DISC as it is involved in the interactions with the large protease subunits. (B) The second cleavage step takes place between the prodomain and the large protease subunit at Asp216. As a result of this cleavage step the active caspase-8 heterotetramer is formed, which is then released into the cytosol. (C) Prodomain p26/p24 remains bound to the DISC.
Activation of procaspase-8 is believed to follow an “induced proximity” model in which high local concentrations and favorable mutual orientation of procaspase-8 molecules at the DISC lead to their autoproteolytic activation (31, 45–47). There is strong evidence from a number of in vitro studies that autoproteolytic activation of procaspase-8 occurs upon oligomerization at the receptor complex (45–47). Furthermore, it has been demonstrated that dimers formed by procaspase-8 molecules possess proteolytic activity, and proteolytic processing of procaspase-8 occurs between precursor dimers (45). Interestingly, it has been shown that procaspase-8 and mature caspase-8 possess different substrate specificities (45). It is likely that conformational changes in the active center of caspase-8 occur upon processing to mature caspase-8.
The processing of procaspase-8/a/b at the DISC is depicted in detail in Figure 2. According to the 2-step model, the processing of procaspase-8 includes 2 cleavage events (39, 45). The first cleavage step occurs between the protease domains, and the second cleavage step takes place between the prodomain and the large protease subunit. Correspondingly, after the first cleavage step, p43/p41 and p10 subunits are formed (Figure 2, A and B). Both cleavage products remain bound to the DISC, p43/p41 by DED interactions and p10 by interactions with the large protease domain (48). As a result of the second cleavage step, p43/41 is processed to the prodomain p26/p24 and p18 (Figure 2C). Thus, the active caspase-8 heterotetramer is formed at the DISC. Subsequently, the mature caspase-8 heterotetramer is released to the cytosol to trigger apoptotic processes.
Procaspase-10 is also activated at the DISC, forming an active heterotetramer (15, 49). However, whether caspase-10 can trigger cell death in the absence of caspase-8 in response to CD95 or TRAIL-R1/R2 stimulation is controversial. Thus, the exact role of caspase-10 remains elusive.
The apoptosome as activating complex for procaspase-9. A number of apoptotic stimuli, such as cytotoxic stress, heat shock, oxidative stress, and DNA damage, lead to the release of cytochrome c from mitochondria. The release of cytochrome c is followed by the formation of a high–molecular mass cytoplasmic complex referred to as the apoptosome (50). In mammals the central scaffold protein of the apoptosome is a 140-kDa protein known as Apaf-1 (apoptotic protease–activating factor-1), which is a homologue of CED-4, a key protein involved in programmed cell death in the nematode Caenorhabditis elegans (51, 52). In the presence of cytochrome c and dATP, Apaf-1 oligomerizes to form a very large (700–1,400 kDa) apoptosome complex. Procaspase-9 is recruited to the complex by CARD interactions, which results in its activation (53). It has been biochemically demonstrated that activation of procaspase-9 occurs by dimerization (31). Moreover, it has been shown that proteolytic activation of procaspase-9 takes place upon dimerization and subsequent cleavage within an interdomain linker, which itself is important for stabilization of caspase-9 dimers.
The inflammosome as activating complex for caspase-1 and -5. The activation of the initiator caspase-1 and -5 takes place in a complex that was named the inflammosome (54). The inflammosome comprises procaspase-1 and -5 as well as the CARD-containing protein NALP-1. The formation of this complex results in the processing and activation of the cytokines IL-1β and IL-18, which play a central role in the immune response to microbial pathogens.
Effector caspase cascade. The activation of the effector caspase cascade differs between extrinsic (death receptor–mediated) and intrinsic (mitochondria-mediated) pathways.
In death receptor–mediated apoptosis, 2 types of signaling pathways have been established (55). So-called type I cells are characterized by high levels of DISC formation and increased amounts of active caspase-8 (Figure 3). Activated caspase-8 directly leads to the activation of downstream, effector caspase-3 and -7. In type II cells, there are lower levels of DISC formation and, thus, lower levels of active caspase-8. In this case, signaling requires an additional amplification loop that involves the cleavage of the Bcl-2–family protein Bid by caspase-8 to generate tBid and a subsequent tBid-mediated release of cytochrome c from mitochondria (56). The release of cytochrome c from mitochondria results in apoptosome formation, followed by the activation of procaspase-9, which in turn cleaves downstream, effector caspase-3 and -7. Type II signaling might be blocked by Bcl-2 family members such as Bcl-2 and Bcl-xL.
Caspase signaling and its modulation. In the extrinsic pathway, DISC formation leads to caspase-8 activation. Two signaling pathways downstream from the receptor were established. In type I cells (shown in light blue) caspase-8 directly cleaves caspase-3, which starts the death cascade. In type II cells (shown in light red) an additional amplification loop is required, which involves tBid-mediated cytochrome c release from mitochondria followed by apoptosome formation. Initiation of the intrinsic pathway results in mitochondria-mediated apoptosome formation, followed by caspase-9 and -3 activation, leading to destruction of the cell. Caspase action can be modulated on several levels. Activation of caspases at the DISC is inhibited by c-FLIP proteins; activation of effector caspases is inhibited by IAPs (see text). Effector caspases are shown in light green; cellular caspase inhibitors are presented in yellow. The targets for pharmacological modulation are shown with an orange arrow.
In the intrinsic pathway, which is triggered by a number of factors, including UV or γ-irradiation, growth factor withdrawal, and chemotherapeutic drugs, the release of cytochrome c from mitochondria leads to apoptosome formation and activation of procaspase-9 (53). Subsequently, procaspase-9 cleaves effector caspase-3 and -7, which, correspondingly, initiate the death cascade. There are reports pointing toward a role of procaspase-2 in genotoxic stress acting upstream of mitochondria; however, this question requires further clarification (57, 58).
Cellular inhibitors of caspases. The action of caspases is regulated on several levels, including blockade of activation of caspases at the DISC as well as inhibition of enzymatic caspase activity (Figure 3). c-FLIP proteins are well-known inhibitors of death receptor–induced apoptosis (44, 59, 60). c-FLIPs possess 2 tandem DEDs at their N termini that facilitate their recruitment to the DISC. There are 3 c-FLIP isoforms described on the protein level, c-FLIPL, c-FLIPS, and c-FLIPR (61). Under conditions of overexpression, all isoforms inhibit activation of procaspase-8 at the DISC by blocking its processing (62) (Figure 3). At the same time, there is increasing evidence that c-FLIPL, when present at the DISC at low concentrations, facilitates the cleavage of procaspase-8 at the DISC by forming c-FLIPL–procaspase-8 heterodimers (45, 63).
The IAP (inhibitor of apoptosis) family of proteins includes 8 mammalian family members, including XIAP, c-IAP1, c-IAP2, and ML-IAP/livin (64–66). They specifically inhibit the initiator caspase-9 and the effector caspase-3 and -7 (Figure 3). The functional unit in IAP is the baculoviral IAP repeat (BIR), which contains about 80 amino acids folded around a central zinc atom. XIAP, c-IAP-1, and c-IAP2 contain 3 BIR domains each. The third BIR domain (BIR3) is involved in interactions with caspase-9 resulting in the inhibition of its activity (67). The linker region between BIR1 and BIR2 selectively targets caspase-3 and -7. The activity of IAPs is regulated by Smac/DIABLO, a structural homologue of the Drosophila proteins Reaper, Hid, and Grim (68, 69) (Figure 3). Smac/DIABLO is released from mitochondria and inhibits IAPs, which facilitates caspase activation during apoptosis. Omi/HtrA2 has been recently identified as another modulator of IAP function (70). Omi/HtrA2 is a mitochondria-located serine protease, which is released into the cytosol and inhibits IAPs by a mechanism similar to that of Smac.
IAPs are not the only natural inhibitors of caspases. The baculoviral p35 protein is a pan-caspase inhibitor, and it targets most caspases, in contrast to IAPs, which affect only caspase-3, -7, and -9 (71). The mechanism of caspase inhibition by p35 involves the formation of an inhibitory complex that is characterized by a protected thioester link between the caspase and p35 (72). Structural analysis of the inhibitory complex between p35 and caspase-8 reveals a unique active-site conformation that protects the intermediary thioester link from hydrolysis. Another pan-caspase inhibitor, serpin CrmA, is derived from the cowpox virus (73). The mechanism of CrmA inhibition is likely to involve covalent modification of the caspase active center.