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Oncogene volume 27, pages6194–6206(2008)Cite this article
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Abstract
The demonstration of protein sequence and functional homology of the Caenorhabditis elegans programmed cell death gene product, CED-3, with human caspase-1 in 1993 triggered an explosion of research activities toward the understanding of molecular mechanisms of apoptosis.
During the past 15 years, a plethora of knowledge has been obtained on the mammalian caspases, the homologs of CED-3, with regard to their distinct physiological functions, their substrates, different activation mechanisms, the signal transduction pathways that lead to their activation as well as their involvement in the pathogenesis of diseases. Such knowledge is beginning to be translated into new therapies for the treatment of human diseases.
Overview of apoptosis
The term ‘apoptosis’ was coined by Kerr, Wyllie and Currie in 1972 to describe a mode of cell death associated with fragmentation of genomic DNA (Kerr et al., 1972). In addition to DNA fragmentation, apoptosis is morphologically characterized by the cytoplasmic condensation, nuclear pyknosis, chromatin condensation, cell rounding, membrane blebbing, cytoskeletal collapse and the formation of membrane-bound apoptotic bodies that are rapidly phagocytosed and digested by macrophages or neighboring cells without activating immune response (Wyllie et al., 1980).
Apoptosis, however, could have remained as a descriptive cell biology phenomenon for a considerably longer period of time, if without the knowledge obtained from the studies of programmed cell death in the nematode Caenorhabditis elegans first initiated by the cell lineage study by Sulston and Horvitz in 1977 and subsequently followed up by genetic and molecular studies by Horvitz and colleagues in early 1990s. As demonstrated by Sulston and Horvitz in 1977, 131 out of 1090 somatic cells invariantly undergo programmed cell death during normal development of C. elegans (Sulston and Horvitz, 1977; Sulston et al., 1983). The discovery of cell lineage-dependent programmed cell death paved the way for genetic characterization of its molecular machinery in C. elegans (Ellis and Horvitz, 1986; Hengartner et al., 1992).
The central components of the programmed cell death machinery in C. elegans are three CED (cell death abnormal) proteins, namely CED-3, CED-4 and CED-9. Since the homologs of CED-3, CED-4 and CED-9 in mammals have been found to regulate mammalian apoptosis, it is likely that the cellular machinery that regulates programmed cell death in C. elegans shares the evolution origin as that of mammalian apoptosis. Thus, programmed cell death in C. elegans represents a primordial type of apoptosis. CED-9 functions as an inhibitor of apoptosis by preventing CED-4 from interacting with CED-3 (Hengartner et al., 1992; Hengartner and Horvitz, 1994), whereas CED-4 is a pro-apoptotic adapter molecule required for the activation of CED-3, a cysteine protease responsible for the execution of cell death program (Yuan and Horvitz, 1992; Yuan et al., 1993). In mammals, the homologs of CED-9, CED-4 and CED-3 are present in the Bcl-2 family, Apaf-1/NOD-like receptor family and caspase (cysteinyl aspartate proteinases) family, respectively (Miura et al., 1993; Zou et al., 1997; Gross et al., 1999; Fritz et al., 2006). The mechanisms and functions of caspase activation remain as the central issue in apoptosis. Here, we provide an updated review on the current knowledge of caspase family in apoptosis and other physiological processes and diseases.
Basic features and classification of caspases
In 1993, C. elegans ced-3 gene was demonstrated to encode a cysteine protease similar to the independently identified human interleukin-1β-converting enzyme (Cerretti et al., 1992; Thornberry et al., 1992; Yuan et al., 1993). Overexpression of interleukin-1β-converting enzyme (later renamed caspase-1) was shown to be sufficient to induce apoptosis in mammalian cells (Miura et al., 1993). The structural and functional similarity of mammalian caspase-1 and CED-3 of C. elegans led to a burst of research activities and the discoveries of multiple caspase family members in mammals. To date, 11 genes were found in the human genome to encode 11 human caspases, caspase-1 to caspase-10 and caspase-14, whereas 10 genes were found in the mouse genome to encode 10 murine caspases, caspase-1, 2, 3, 6, 7, 8, 9, 11, 12 and 14. The human caspase-4 and -5 are functional orthologs of mouse caspase-11 and -12, whereas human caspase-10 is absent in the mouse genome. The remaining caspases with the same numbers in human and mouse are functional orthologs of each other. The protein initially named caspase-13 was later found to be a bovine homolog of caspase-4.
Caspases share a number of features distinguishable from other proteases. Caspases are synthesized as inactive zymogens containing a prodomain, a p20 large subunit and a p10 small subunit (Figure 1a). Activation of the zymogens by proteolytic cleavages separates the large and small subunits and removes the prodomain. The catalytic dyad residues in the p20 subunit consist of the active site Cys285, which is a part of the conserved ‘QACXG’ pentapeptide sequence, and His237 (caspase-1 numbering) (Fuentes-Prior and Salvesen, 2004). Caspases recognize at least four contiguous amino acids in their substrates, P4–P3–P2–P1, and cleave after the C-terminal residue (P1), usually an Asp residue. The structure feature of caspase active site and the mechanism of substrate recognition will be discussed below.
Figure 1
Structure and domain organization of mammalian caspases. (a) Domain organization of caspases and the location of catalytic center loops (L1–L4). Initiator caspases have long prodomains, CARD or DED, whereas executioner caspases have short prodomains. Loops are shown in gray. The active site Cys is shown by a red line. Processing that separates p20 and p10 subunits occurs in L2. The resulting large subunit potion of the L2 loop of one monomer and small subunit portion of the L2 loop of the neighboring monomer (L2′) are involved in loop bundle formation (b and c). (b) Ribbon representation of the active caspase-3 structure showing the positions of the active center loops (L1-L4, L2′) based on the crystal structure of the complex of caspase-3 with peptide inhibitor (in pink). Reproduced with permission from Shi (2002). (c) The active site conformations of the caspases with known structures. Loops L1 and L3 are highly conserved, whereas L2 and L4 are responsible for the differences in substrate binding specificity. Reproduced with permission from Shi (2002). CARD, caspase recruitment domain; DED, death effector domain.
Caspases can be classified by two alternative methods. On the basis of their known major functions, caspases are grouped into two subfamilies, pro-apoptotic and pro-inflammatory subfamilies. Pro-apoptotic caspases (caspase-2, -3, -6, -7, -8, -9, -10) are known to be mainly involved in mediating cell death signaling transduction, whereas pro-inflammatory caspases (caspase-1, -4, -5, -11, -12) regulate cytokine maturation during inflammation. This classification, however, can be overly simplistic, as increasing evidences indicate that caspases have roles in multiple other cellular processes that cannot be classified into pro-apoptotic or pro-inflammatory (see below).
Furthermore, the activation of ‘pro-inflammatory’ caspases can clearly induce apoptosis. An alternative classification is to divide caspases according to the lengths of their prodomains, which also correspond to their positions in the apoptotic signaling cascade. On the basis of this classification, caspases are divided into initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10, -11, -12) and effector caspases (caspase-3, -6, -7). Initiator caspases possess long prodomains that contain one of the two characteristic protein–protein interaction motifs: the death effector domain (DED) or the caspase recruitment domain (CARD) (Figure 1a) and are involved in interacting with the upstream adapter molecules. The effector caspases with short prodomains perform downstream execution steps of apoptosis by cleaving multiple cellular substrates and are typically processed and activated by upstream caspases. It should be noted that this upstream and downstream relationship is not absolute and may only exist transiently during very early phases of apoptosis. An important role of downstream caspases in the execution of apoptosis was demonstrated by caspase-3−/− and caspase-7−/− double-mutant mice, which exhibit strong apoptotic deficiency, including immediate death after birth, exencephaly and resistance of double-mutant mouse embryonic fibroblasts (MEFs) to both mitochondrial and death receptor-mediated apoptoses (see below) (Lakhani et al., 2006). Different from other caspases, the expression of caspase-14 is confined to the epithelium and it is involved in keratinocyte differentiation (Denecker et al., 2008).
Thornberry et al. used a positional scanning synthetic combinatorial library approach with the general substrate structure Ac-X-X-X-Asp-AMC to define the cleavage specificities of different caspases in vitro. The preferred tetrapeptide substrate sequences for caspase-1- to -11 were determined (Thornberry et al., 1997; Kang et al., 2000). To date, almost 400 substrates for mammalian caspases have been reported in the literature, which are compiled into an online database ‘CASBAH’ (http://www.casbah.ie) (Luthi and Martin, 2007). The fraction of the proteome that is degraded by caspases during the demolition phase of apoptosis is substantial. Reported caspase substrates include structure proteins, regulators of transcription/translation, kinases and signaling intermediaries, and so on. Caspases are not equal in terms of their ability to cleave cellular substrates. Initiator caspases appear to be more specific proteases that cleave few substrates apart from their own precursors and other caspases downstream. Effector caspases are responsible for most of the substrate proteolysis seen during the demolition phase of apoptosis. Considering this profusion of caspase substrates, it is not easy to identify the functionally important targets from many ‘innocent bystanders’ that are simply caught up in the proteolytic maelstrom during apoptosis. Nonetheless, a blueprint for how caspases regulate cell death has emerged from the dense thicket of caspase substrates that have been identified.
It is worth to point out that pro-IL-1β and pro-IL-18 are the only two cytokines known to be cleaved by pro-inflammatory caspases under non-apoptotic conditions. As increasing evidence suggests roles of caspases in non-apoptotic cellular processes (see below), additional insights with regard to the caspase substrates under non-apoptotic conditions may be an area of importance for future.
Caspase structure and mechanism of substrate recognition
The structures of active caspase-1, -2, -3, -7, -8 and -9 have been solved, providing valuable insight into the basis of caspase specificity and catalytic mechanisms (Riedl and Shi, 2004). The active caspase is a homodimer, with each monomer consisting of a large and a small subunit (Figure 1b). Six anti-parallel β-strands of each monomer form a continuous 12-stranded β-sheet in the active enzyme. Several α-helices and short β-strands are located on either side of the central β-sheet, which gives rise to a globular fold. The active sites, formed by four protruding loops (L1–L4), are located at two opposite ends of the β-sheet. Loop L1 and a portion of L2, which contains the catalytic Cys residue, are a part of the p20 subunit, whereas L3 and L4 come from the p10 subunit (Figures 1a and b). During caspase activation, the L2 loop is cleaved between p20 and p10 subunits into two segments. The C-terminal segment of L2 loop will stabilize the active site of the neighboring monomer (Figure 1b). Among the four loops, L1 and L3 have a relatively conserved length and composition among all caspases, whereas L2 and L4 display a greater degree of variation (Figure 1c).
These four active-site loops determine the sequence specificity of caspase substrates. The binding pockets for the P4–P3–P2–P1 positions in the substrate are known as the S4–S3–S2–S1 subsites, respectively. The S1 and S3 subsites are nearly identical among all caspases, whereas the location of the S2 and S4 subsites is conserved. The S1 subsite is constructed by three invariant residues, an Arg residue from L1, a Gln residue at the beginning of L2 and an Arg residue at the end of L3 loop (Riedl and Shi, 2004). This deep, highly basic pocket is ideally shaped to accommodate Asp at the P1 position. The aromatic S2 subsite of caspase-3 and caspase-7 preferentially accommodates small aliphatic residues (Ala, Val). By contrast, the S2 subsite is larger in initiator caspses and therefore tolerates well residues with bulkier side chains (Fuentes-Prior and Salvesen, 2004). The Arg residue on L3 loop, which contributes to the S1 subsite, is also engaged in main-chain–main-chain hydrogen bonds with the P3 residue, preferentially Glu, at the S3 subsite.
The S4 subsite provides major specificity-conferring elements to different caspases (Degterev et al., 2003; Schweizer et al., 2003). Caspase-1 has the widest S4 subsite, which favors large hydrophobic residues. In contrast, caspase-2, -3 and -7 possess the narrowest pocket and prefer an Asp residue in the P4 position. The S4 subsites of caspase-8 and -9 occupy intermediate positions and show preference for small hydrophobic Val or Leu residue. However, caspase-8 tolerates equally well both small hydrophobic (Ile) and acidic (Asp) residues at the P4 position.
Caspase-2 is exceptional in its requirement for an additional occupied S5 subsite for efficient substrate cleavage (Schweizer et al., 2003). In contrast to caspase-3 and -7, the presence of a small hydrophobic P5 residue conferred a 35-fold increase in catalytic efficiency, which may partly reflect a better burial of the P4 Asp residue. A systematic study using fluorescent peptidyl substrates indicated a clear degree of selectivity of the residues at P1′ position (Stennicke et al., 2000). Small residues (Gly, Ala and Ser) are preferred at position P1′ and large aromatic side chains (Phe and Tyr) are also well tolerated, whereas charged residues or proline are prohibited.
Cellular pathways and complexes controlling caspase activation
Death-inducing signaling complex and the death receptor pathway
The death receptors are a family of type I transmembrane proteins characterized by the presence of multiple cysteine-rich repeats in the extracellular domain and the protein–protein interaction module known as the death domain (DD) in the cytoplasmic tails (Figure 2). The death receptors function as cell surface sensors to detect extracellular danger signals by ligand binding. The members of death receptor family include tumor necrosis factor receptor 1 (TNFR1; also known as DR1, CD120a, p55 and p60), Fas (also known as DR2, APO-1 and CD95), DR3 (also known as APO-3, LARD, TRAMP and WSL1), TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as DR4 and APO-2), TRAILR2 (also known as DR5, KILLER and TRICK2), DR6, ectodysplasin A receptor (EDAR) and nerve growth factor receptor (Lavrik et al., 2005).
Figure 2
Caspase activation pathways. Schematic representations of the extrinsic (death receptor-mediated) and the intrinsic (internally initiated by damage, stress, and so on) pathways of caspase activation. Death receptor signaling may involve direct caspase-8-mediated caspase-3 activation or a Bid-cleavage-dependent mitochondrial amplification step. In the intrinsic pathway, diverse stimuli activate BH3-only proteins, which promote the assembly of BAK–BAX oligomers and the mitochondria outer membrane permeability change. Cytochrome c releases into cytosol and seeds apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of future caspase activation events.
Upon interacting with their respective ligands, the death receptors undergo multimerization to form the intracellular death-inducing signaling complexes (DISCs) that may include multiple adapter molecules (Figure 3). For Fas and TRAIL receptors, FADD is recruited to the DISC through its C-terminal DD, which in turn interacts through its N-terminal death effector domain with the death effector domain of caspase-8. The recruitment and oligomerization of caspase-8 in the DISC result in its autocatalytic activation and is critical for the initiation of cell death (Figure 3a) (Juo et al., 1998; Varfolomeev et al., 1998).
Figure 3
Death-inducing signaling complex (DISC). (
a) DISC of Fas and TRAIL receptors. Death domain (DD) of Fas recruits the adapter protein FADD. FADD in turn, via its death effector domain (DED), recruits and activates caspase-8.
(b) DISC of tumor necrosis factor (TNF) receptor 1. After the binding of TNF to TNFR1, rapid recruitment of TRADD, RIP1 and TRAF2 occurs (complex I). Subsequently, TNFR1, TRADD and RIP1 are modified and dissociate from TNFR1. The liberated DD of TRADD (and/or RIP1) binds to FADD, resulting in caspase-8 recruitment (complex II) and apoptosis. Complex I activates nuclear factor-κB (NF-κB) and promotes FLIP expression, which inhibits caspase-8 and antagonizes apoptosis.
In certain cell types, known as type I cells, the activation of caspase-8 by the DISC may be sufficient to induce the activation of downstream caspases, such as caspase-3 and caspase-7, to complete the execution of apoptosis (Scaffidi et al., 1998). On the other hand, in type II cells, where apoptotic signaling initiated by an insufficient amount of active caspase-8, death receptor apoptotic signaling must be amplified by a mitochondrial amplification step (Figure 2). In this case, caspase-8 cleaves cytosolic BH3-only proapoptotic Bcl-2 family member Bid, and the cleaved Bid (tBid) translocates to the mitochondria to activate the downstream components of the mitochondria apoptotic pathway (Li et al., 1998; Luo et al., 1998).
The activation of death receptors does not necessarily lead to cell death. The interaction of TNFα with TNFR1 leads to the formation of two distinct signaling complexes (Micheau and Tschopp, 2003). The rapidly formed plasma membrane-bound complex I is composed of TNFR1, TRADD, RIP, TRAF2 and c-IAP1 and triggers a nuclear factor-κB (NF-κB) response that regulates inflammation and promotes cell survival by inducing the expression of anti-apoptotic proteins such as FLIPL. Subsequently, TRADD, RIP and TRAF2 dissociate from TNFR1 and form a second complex (complex II) with FADD and caspase-8/10 in the cytoplasm. The formation of complex II initiates a pro-apoptotic signaling (Figure 3b). The ultimate fate of life or death depends on the balance of the complex I and complex II through a process not yet fully understood.
Apoptosome and mitochondria pathway
The intrinsic apoptotic pathway (mitochondria pathway) is triggered by stimuli such as DNA damage and cytotoxic drugs. Diverse damages or stress signals activate one or more members of the BH3-only protein family. The BH3-only protein activation above certain crucial threshold overcomes the inhibitory effect of the anti-apoptotic Bcl-2 family and promotes the assembly of Bak–Bax oligomers within the mitochondrial outer membranes, which leads to the release of cytochrome c (Chipuk and Green, 2008). Cytosolic cytochrome c in turn promotes the assembly of apoptosome (Figure 2).
Apoptosome is a multimeric protein complex involving Apaf-1, cytochrome c and the cofactor dATP/ATP (Liu et al., 1996; Li et al., 1997; Zou et al., 1997). Apaf-1, the central component of the apoptosome, contains an N-terminal CARD, an expanded nucleotide-binding domain and a C-terminal WD40 domain. The CARD is responsible for the interaction with the prodomain (CARD) of caspase-9, which is essential to the recruitment and the activation of caspase-9 (Qin et al., 1999). The CARD and the nucleotide-binding domains of Apaf-1 are responsible for the oligomerization in the presence of cytochrome c and dATP, whereas the WD40 domain is believed to interact with cytochrome c. In the absence of apoptotic stimuli, Apaf-1 exists in monomeric form. In the presence of cytochrome c and dATP, Apaf-1 oligomerizes to form a wheel-shaped signaling platform, apoptosome, which contains seven Apaf-1 molecules, each bound to one molecule of cytochrome c and one caspase-9 (Acehan et al., 2002; Yu et al., 2005). Caspase-9, which is activated through an apoptosome-induced conformational change, further processes the downstream caspases, such as caspase-3 and caspase-7, to carry out the execution of apoptosis (Slee et al., 1999) (Figure 2).
PIDDosome and caspase-2 activation
Caspase-2 is a long prodomain initiator caspase involved in stress-induced apoptosis (Lassus et al., 2002). Caspase-2-deficient germ cells and oocytes are resistant to cell death after treatment with chemotherapeutic drugs (Bergeron et al., 1998). The activation of caspase-2 promotes mitochondrial membrane permeabilization. Although caspase-2 may promote mitochondrial damage by cleaving and activating Bid, it can also directly induce mitochondrial cytochrome c release through a mechanism not yet understood (Guo et al., 2002; Robertson et al., 2002).
A large protein complex, named PIDDosome, that contains the p53-induced protein with a death domain (PIDD) and an adapter protein RAIDD, has been shown to mediate the activation of caspase-2 in response to genotoxic stimuli (Tinel and Tschopp, 2004). RAIDD contains one CARD and one DD, which associates with the CARD of caspase-2 and the DD of PIDD, respectively (Figure 4). PIDD was initially identified as a prime p53 target gene in an erythroleukemia cell line that undergoes G1 cell cycle arrest and subsequently apoptosis after p53 expression (Lin et al., 2000b). Increased amount of PIDD expression results in spontaneous activation of caspase-2 and sensitization to apoptosis by genotoxic stimuli (Tinel and Tschopp, 2004). RAIDD−/− mice have normal development. The complete resistance of RAIDD−/− MEFs to PIDD-induced apoptosis and caspase-2 activation represents the only measurable phenotype in cells that lack RAIDD, which highlights the essential role for RAIDD in PIDD-induced apoptosis (Berube et al., 2005). In contrast to RAIDD−/− MEFs, caspase-2−/− MEFs are only partially resistant to PIDD-mediated apoptosis and growth suppression, suggesting that multiple effectors, in addition to caspase-2, might be present downstream of PIDD signaling pathway (Berube et al., 2005).
Figure 4
Caspase-activating complexes: PIDDosome and inflammasome. CARD, caspase-recruitment domain; DD, death domain; FIIND, F-interacting domain; LRR, leucine-rich repeats; NACHT, NAIP, CIITA, HET-E, TP1 domain; NAD, NACHT-associated domain; PYD, pyrin domain; ZU5, ZU-5 domain.
Further characterization of PIDD indicated that PIDD is also able to form a complex with the kinase RIP1 and a component of NF-κB-activating kinase complex, NEMO, leading to sumoylation and ubiquitination of NEMO and NF-κB activation, in response to DNA damage (Janssens et al., 2005). Three isoforms of PIDD differentially activate NF-κB and caspase-2 in response to genotoxic stress (Cuenin et al., 2008). Thus, PIDD may act as a molecule switch controlling the balance between cell survival and cell death in response to DNA damage.
Inflammasome and pro-inflammatory caspase activation
Pro-inflammatory caspases has a dual function in regulating both cytokine processing and apoptosis. Murine pro-inflammatory caspases are clustered on chromosome 9A1 in the following order from the telomere: caspase-1, caspase-11 and caspase-12. The organization of the syntenic region in humans (chromosome 11q22) is similar, except that the caspase-5 and caspase-4 genes replace caspase-11 gene (Martinon and Tschopp, 2004). Mouse genetic knockout studies have clearly shown that caspsae-11 mediates the activation of caspase-1, which in turn processes cytokines IL-1β and IL-18 (Li et al., 1995; Ghayur et al., 1997; Wang et al., 1998). Lipopolysaccharide (LPS) induces caspase-5 expression in human cells and caspase-11 expression in murine cells (Wang et al., 1996; Lin et al., 2000a). Caspase-5 together with caspase-1 was found to be the components of the NALP1 inflammasome, a complex involved in the activation of caspase-1 (Martinon et al., 2002) (see below).
A family of intracellular receptors structurally related to Apaf-1 was described in vertebrates recently. These proteins named NOD-like receptors (NLRs) are intracellular sensors of pathogens and other stresses (Martinon and Tschopp, 2005). NLRs include NODs and three subfamilies of proteins involved in the formation of caspase-1-activating inflammasome complexes, namely NALPs, IPAF and NAIPs (Figure 4).
IPAF was identified in a genomic screen for genes having sequence similarity of the CARD of caspase-1 (Poyet et al., 2001). IPAF associates directly and specifically with the CARD domain of caspase-1 through the CARD–CARD interaction. The NACHT domain of IPAF induces oligomerization and promotes proximity of the caspases, whereas the C-terminal leucine-rich repeat (LRR) is probably involved in ligand sensing (Figure 4).
NAIP shares with IPAF the highest sequence similarity of the NACHT and LRR domains. Instead of a CARD, NAIP harbors three N-terminal baculovirus inhibitor-of-apoptosis repeats (BIR). NAIP was proposed to interact with IPAF, suggesting that it may be part of the same caspase-1-activating complex (Zamboni et al., 2006; Vinzing et al., 2008).
NALP1, NALP2 and NALP3 were shown to be the central scaffold of caspase-1-activating complexes known as inflammasomes. These proteins harbor an NACHT and an LRR similar to IPAF and NAIP but are characterized by an N-terminal pyrin domain (PYD). The PYD of NALPs interacts and recruits the adapter ASC via the PYD–PYD interaction. ASC (apoptosis associated speck-like protein containing a CARD) contains an N-terminal PYD and a C-terminal CARD, and is an essential component for inflammasome formation. The CARD domain within ASC binds and recruits caspase-1 to the inflammasome. The inflammasome may also recruit other caspases, such as caspase-5 via the C-terminal CARD of NALP1 or a second caspase-1 via the C-terminal CARD of CARDINAL, another component of the NALP2/3 inflammasome (Figure 4).
The natural stimuli that lead to inflammasome assembly are agents inducing potassium efflux and bacteria toxins (Martinon and Tschopp, 2007). The differential requirement of IPAF, ASC and NALP3 in sensing different stimuli to activate caspase-1 has been shown with genetic knockout studies (Mariathasan et al., 2004, 2006). With 14 NALPs, plus IPAF and NAIP, the repertoire of caspase-1-activating molecular machines is potentially very complex.
Molecular basis of caspase activation
Effector caspases: cleavage leads to activation
On the basis of biochemical and structural characterization, effector caspases are known to exist as a homodimer, both before and after the activation cleavage at the intersubunit linkers into p20 and p10 subunits (Figure 1). Crystal structure analysis on caspase-7 reveals that cleavage of the intersubunit linkers allows for the rearrangement of crucial chains that are in disarray and block the formation of the active sites in the uncleaved caspases. Cleavage is the only requirement for providing the correct placement of the catalytic residues and the proper alignment of the substrate-binding pockets (Chai et al., 2001; Riedl et al., 2001).
Initiator caspases: proximity-induced dimerization vs induced conformation model
The precise details of initiator caspase activation remain inconclusive. Initiator caspases, such as caspase-9 and caspase-8, exist in an inactive monomeric form in the absence of an activation signal. Both are activated by their respective activation platforms, apoptosome and DISC, as we discussed above. The induced proximity model, first proposed in 1998, states that the initiator caspases autoprocess themselves when brought into close proximity of each other (Salvesen and Dixit, 1999). Later on, this model has been refined as the ‘proximity-induced dimerization’ by Salvesen and colleagues because biochemical studies of caspase-9 and caspase-8 show that dimerization of the initiator caspases drives their activation. Internal proteolysis does not activate these apical caspases but is a secondary event, resulting in partial stabilization of activated dimers (Boatright et al., 2003).
The fact that caspase-9 exhibits a much higher level of catalytic activity in the apoptosome suggests that a conformational change may occur in the active site of the apoptosome-bound caspase-9. The ‘induced conformation model’ proposes that the apoptosome may directly activate monomeric caspase-9 through modification of its active site conformation; otherwise, the apoptosome may assemble the dimeric caspase-9 into a higher order complex that results in the modification of the active site conformation for enhanced activity (Shi, 2004).
Additional regulatory mechanisms for caspases
Gene expression
Although apoptosis in most cases does not require new caspase synthesis, regulation of caspase transcription does have an important function in certain circumstances. For example, the basal expression level of caspase-11 in healthy mice and resting cells is very low, but treatment with LPS or other pathological stimuli such as ischemia in vivo leads to a dramatic transcriptional upregulation of caspase-11 expression (Wang et al., 1996; Kang et al., 2000). The putative human caspase-11 homolog, caspase-5, is also transcriptionally upregulated by LPS (Lin et al., 2000a). Caspase-14 is transcriptionally induced during terminal differentiation of keratinocytes (Eckhart et al., 2000).
Deregulation of E2F by adenovirus E1A, loss of Rb or enforced E2F-1 expression results in the accumulation of zymogen forms of multiple caspases through a direct transcriptional mechanism, which may potentiate p53-mediated apoptotic signals (Nahle et al., 2002). These results may explain the increased sensitivity of oncogene-expressing cells to multiple apoptotic stimuli, which is exploited by anti-cancer therapy.
Phosphorylation
Protein phosphorylation is the most widely used cellular regulatory mechanism. Caspase-9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis. In response to growth factor stimulation, Akt and ERK (extracellular signal regulated kinase)/MAPK (mitogen-activated protein kinase) have been shown in separate studies to phosphorylate caspase-9 on Ser196 and Thr125, respectively, and these phosphorylation events lead to inhibition of caspase-9 activity and apoptosis (Cardone et al., 1998; Allan et al., 2003). Later studies also show that phosphorylation and inhibition of caspase-9 by PKCzeta on Ser144 restrain the intrinsic apoptotic pathway during hyperosmotic stress (Brady et al., 2005). In mitosis, caspase-9 is phosphorylated at Thr125 by CDK1/cyclinB1 to restrain apoptosis (Allan and Clarke, 2007).
Metabolic regulation of caspase activation
An increasing number of studies show that the signaling pathways linking cell survival and metabolism influence the activation of caspase-dependent cell death (Hammerman et al., 2004). For example, cell survival mediated by the oncogene, Akt, is dependent on glucose metabolism to inhibit Bax activation and cytochrome c release (Gottlob et al., 2001; Rathmell et al., 2003). Glucose metabolism can regulate apoptosis by directly controlling the activation of caspases. In Xenopus oocyte system, the addition of glucose-6-phosphate delays the activation of caspases. It was discovered that the pentose phosphate pathway generation of NADPH (nicotinamide adenine dinucleotide phosphate) is important for the inhibition of caspase-2 activation through inhibitory phosphorylation of caspase-2 at Ser135 by calcium/calmodulin-dependent protein kinase II (CaMKII) (Nutt et al., 2005).
In a genome-wide screen for genes required for apoptosis in Drosophila cells, multiple genes directly involved in cellular metabolism, such as genes encoding citrate synthase (CS) and 3-ketoacyl-acyl carrier protein synthase, were found to protect against apoptosis induced by the removal of DIAP1, which is a direct inhibitor of Drosophila caspases (Yi et al., 2007). Additional studies will be needed to identify the exact metabolic changes mediated by the downregulation of these ‘housekeeping’ genes that protect cells against apoptosis.
Apoptotic and non-apoptotic functions of caspases
Our knowledge of the physiological functions of individual caspases is largely gained from genetic knockout mouse studies. The apoptotic and non-apoptotic phenotypes of the available caspase knockout mice are summarized in Table 1. Increasing evidence suggests that caspases have important functions to regulate cell proliferation, differentiation and migration in addition to regulate apoptosis.
Table 1 The apoptotic and non-apoptotic phenotypes of caspase knockout mice
Caspases in cell proliferation
Several studies demonstrate an essential role for caspase-8 in the proliferation of immune cells (Chun et al., 2002; Salmena et al., 2003; Beisner et al., 2005; Su et al., 2005). Human individuals with homozygous caspase-8 reduction-of-function mutations not only manifest defective lymphocyte apoptosis, characteristic of ALPS (autoimmune lymphoproliferative syndrome) found in patients with defects in Fas signaling, but also display impaired proliferation of T, B and natural killer cells, which leads to immunodeficiency (Chun et al., 2002). Similarly, peripheral murine T cells with conditional deletion of caspase-8 are unable to proliferate after TcR activation (Salmena et al., 2003). In human B cells harboring homozygous caspase-8 mutations, both NF-κB activation and proliferation in response to B-cell receptor or LPS simulation are abrogated (Su et al., 2005). Similarly in mice, LPS or dsRNA-induced proliferation of caspase-8-deficient B cells is impaired, although TLR-4-induced activation of NF-κB activation is not impeded in these cells (Beisner et al., 2005). The molecular mechanism that is responsible for caspase-8-regulated proliferation of immune cells is still not clear.
Caspases in cell differentiation
Caspases are involved in the terminal differentiation of a variety of cell types, including enucleation processes such as lens cell differentiation, erythrocyte and platelet formation and the terminal differentiation of keratinocytes (Dahm, 1999; Shcherbina and Remold-O'Donnell, 1999; Lippens et al., 2000; Zermati et al., 2001). In addition, caspase-8 has been shown to be required for myelomonocytic lineage differentiation into macrophages (Kang et al., 2004). Transient activation of caspase-3 mediates differentiation of long-lived cell types, such as skeletal muscle, osteoblasts and neurons (Fernando et al., 2002; Miura et al., 2004; Fernando and Megeney, 2007). Very recently, two elegant studies reported that caspase-3 is required for the differentiation of embryonic stem cells and hematopoietic stem cells, respectively (Fujita et al., 2008; Janzen et al., 2008). It is not clear, however, whether caspase-3 promotes stem cell differentiation indirectly by limiting self-renewal or by directly engaging in differentiation program or both. How caspase activity is restrained and guided to influence differentiation without inducing cell death awaits future studies.
Caspases in cell migration
Regulation of cell migration is another emerging non-apoptotic function of caspases. Cell migration has been shown to be severely hampered in caspase-8-deficient MEFs. Caspase-8 activates calpains through a mechanism not yet clear and promotes subsequent cytoskeletal remodeling downstream of calpains (Helfer et al., 2006).
A role of pro-inflammatory caspase in regulating cell migration was illustrated by the studies on caspase-11-mutant mice. Caspase-11-deficient leukocytes were found to show reduced motility. Interestingly, caspase-11 was shown to promote actin depolymerization and cell migration through interaction with Aip1 (Li et al., 2007). Thus, consistent with a pro-inflammatory role of caspase-11, the upregulation of caspase-11 expression in response to inflammatory signals may also coordinate the recruitment of inflammatory cells through an intracellular mechanism.
Caspases and apoptosis in the pathogenesis of diseases
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Owing to its role in the elimination of virally infected and damaged cells, apoptosis has a central role in the prevention of diseases. Aberrant activation or lack of activation of caspases leads to a host of pathologies, including cancer, autoimmune diseases, sepsis, immunodeficiency and neurodegenerative disorders.
Disorders associated with excess apoptosis and/or caspase activation
Evidence of caspase activation has been demonstrated in multiple neurodegenerative diseases, for example, Alzheimer's disease, Parkinson's disease and Huntington's disease and amyotrophic lateral sclerosis (ALS) from studies of post-mortem human brain samples and animal models (Vila and Przedborski, 2003). Caspase inhibition has been shown to delay cell death in different experimental models of neurodegeneration. However, apoptosis is not the sole mediator of cell demise in these disorders but a key component within a coalition of deleterious events such as inflammation, mitochondria dysfunction, oxidative stress or protein misfolding and aggregation. Thus, apoptosis may be only one of the downstream events. Inhibition of caspases should be combined with other treatments directed toward the upstream events to achieve optimal therapeutic neuroprotection.
Sepsis represents an initial overly exuberant inflammatory response, where unbridled cytokine-mediated host defense mechanism induces significant cell and organ injury, and subsequently a substantial impaired immune response due to extensive death of lymphocytes and dendritic cells (Hotchkiss and Nicholson, 2006). Caspase-1 mediates its response in sepsis by processing IL-1β and IL-18. However, IL-1-specific therapy failed to show a significant clinical benefit in septic patients (Zeni et al., 1997). Caspase-2, -3, -6 and -9 have also been implicated in lymphocyte apoptosis (Tinsley et al., 2000), and prevention of lymphocyte apoptosis by caspase inhibitors and siRNAs improved survival in sepsis models (Hotchkiss et al., 1999, 2000; Wesche-Soldato et al., 2005).
Mutations in the components of the inflammasome affect caspase-1 activation and inflammatory response by altering IL production. Missense mutations in the NACHT domain of NALP3 are involved in several autosomal-dominant autoinflammatory syndromes (Martinon and Tschopp, 2007). These mutations in NALP3 confer a gain-of-function mutation and lead to an overactivation of caspase-1 in monocytes, resulting in an aberrant maturation of IL-1β. Treatment of those patients with a natural decoy IL-1 molecule (IL1Ra) rapidly and dramatically decreases disease manifestations (Hawkins et al., 2003; Hoffman et al., 2004). Caspase-1 inhibitors are under clinical trials for the treatment of familial cold urticaria, rheumatoid arthritis and other autoinflammatory diseases (Howley and Fearnhead, 2008).
Disorders associated with inhibition of apoptosis and/or caspase activation
Apoptosis is essential for the removal of potentially autoreactive lymphocytes during development and that of excess effector cells after the completion of an immune response. Failure to remove autoimmune cells can result in autoimmune diseases. Loss-of-function mutation in Fas, as in the mouse lpr mutation, is associated with systemic autoimmunity and lymphoproliferation (Watanabe-Fukunaga et al., 1992). A similar mouse syndrome is caused by the recessive gld mutation in the Fas ligand Fasl gene (Lynch et al., 1994). In humans, the ALPS, which exhibits many similarities to lpr-associated disease in mice, is also associated with dominant mutations in Fas gene (Straus et al., 1999). Missense mutations in human caspse-10, which decrease enzymatic activity, have been found to be genetically associated with ALPS, similar to the abnormal homeostasis of lymphocytes and dendritic cells caused by defective FasL/Fas (Wang et al., 1999).
‘Evading apoptotsis’ has been recognized as one of the six hallmarks of cancers (Hanahan and Weinberg, 2000). Functional inactivation of p53, found in more than 50% of human cancers, results in the removal of a key component of the DNA damage sensor that can induce the apoptosis. The activation of PI3K–Akt survival pathway is responsible for suppressing apoptosis in a substantial fraction of human tumors. In addition, inhibitory proteins of caspases, like IAPs and FLIP, are frequently overexpressed in cancers and may contribute to tumor chemoresistance. Different strategies are being developed either to enhance the death signaling input or to relieve the inhibition on apoptotic signaling as new anti-cancer therapies (Meng et al., 2006). These strategies include enhancing death receptor ligand activity, chemical inhibition of Bcl-2/Bcl-xL, activating apoptosis by IAP antagonists. Such exciting developments indicate that the 15-years-old ‘apoptosis’ tree is finally beginning to bear fruits, which may bring in new breakthroughs for the treatment of human diseases.
Conclusions
Evolution has modified a primitive, dedicated programmed cell death mechanism, such as the one found in C. elegans, into multiple, more flexible and adaptive cell death pathways in mammals, which are highly responsive to a much broader variety of both internal and external stimuli. The roles of caspases have been significantly expanded from regulating cell death to multiple other physiological processes. Mammals may have evolved alternative strategies to delete unwanted cells when caspases or caspase functions are abrogated, which may occur frequently as a result of oncogenic mutations or viral infection.
Although a critical role of caspases in regulating apoptosis has been well accepted, many important questions remain unresolved. Here are a few examples. First, all of the caspase knockout mice that have been generated so far are constitutive mutants. Although these mutant mice have been very helpful in deciphering the functional roles of caspases in regulating apoptosis, most of the apoptotic defects from such constitutive caspase mutant mice were relatively mild. To rule out the roles of compensatory expression of alternative caspases that may have masked the true phenotypes, we need to obtain conditional caspase knockout mice. Second, increasing evidence suggests that cellular metabolic activity may have an important function in regulating cellular sensitivity to apoptosis by controlling caspase activation. It is important for us to understand how intermediate metabolites might regulate caspase activation. Third, as caspases have been found to be involved in regulating multiple cellular processes that do not lead to cell death, we need to identify the mechanisms that control the activation and inactivation of caspases and the caspase substrates under non-apoptotic conditions. Fourth, although the roles of caspases in human diseases have been amply demonstrated, it is unfortunate that small molecule inhibitors of caspases cannot be made easily available to the diseased tissue areas in most cases. Thus, identification of molecular targets, which can be targeted by small molecule inhibitors to inhibit caspases in the diseases areas, is directly relevant for the treatment of multiple human diseases such as acute neurological injuries. We are optimistic that careful biochemical and molecular analyses coupled with genetic studies in various model systems will clarify these unresolved issues and provide fundamental information and guidance for the development of better therapeutics to treat human diseases.
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