C. Elegans and the Evolutionary Conservation of Apoptotic Regulators
The striking morphologic similarity between disparate cell types undergoing apoptosis suggests that the underlying molecular processes may be similar.22 Many of the features of this central apoptotic control system have been defined, and the outline of this pathway provides insight into potential mechanisms for viral and cellular oncogenic transformation.
Many of the critical factors involved in the control of apoptosis were first defined in the nematode Caenorhabditis elegans. Every cell division and cell death event in the normal pathway of C. elegans development is known, and this developmental pathway follows a determined, predictable program.23 All 131 programmed cell death events in the developing worm depend on the normal function of three proteins: CED-3, CED-4, and CED-9. Loss of CED-3 or CED-4 function or a gain-of-function mutation of the CED-9 protein will lead to complete abrogation of cell death in C. elegans development.25,25 Loss of CED-9 function leads to increased apoptotic cell death.25 CED-9 appears to function by suppressing inappropriate activity of CED-3 and CED-4.26 CED-4 interacts with CED-3 to facilitate its activation to a protease capable of generating an apoptotic phenotype.27-31
Strong evolutionary pressures would be expected to preserve the mechanisms involved in a process as fundamental to the organism as the control of cell death. Many of the proteins central to apoptotic control indeed have been highly conserved, from the roundworm to the human (Fig. 24-4). CED-9 is highly homologous to mammalian Bcl-2.32,33 Bcl-2 expression in C. elegans mimics a CED-9 gain of function and can partially revert a CED-9 loss-of-function mutant. A mammalian homologue of CED-4, Apaf-1, recently was identified and appears to function as a signal transducer between Bcl-2 family members and CED-3-related molecules.34 CED-3 was found by database searching to be related to the mammalian interleukin-1β converting enzyme, now known as caspase 1. 35 As discussed below, caspases have been found to play a central role in the effector phase of apoptosis.
Critical components of apoptotic regulation have been evolutionarily conserved.
Bcl-2 overexpression is capable of inhibiting cell death in response to many disparate apoptotic signals, suggesting that it acts at the convergence of many apoptotic pathways. Bcl-2 has been found to be one of a family of related proteins, several members of which appear to play important positive and negative roles in the control of apoptosis (Table 24-1). Antiapoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 have been found to be overexpressed in several tumor types. Members of the Bcl-2 family have been found to form both homo- and heterodimers, and the relative balance of antiapoptotic and proapoptotic members in these complexes may be a critical determinant of apoptotic sensitivity.43
Table 24-1: Mammalian Bcl-2-Related Proteins |Favorite Table|Download (.pdf) Table 24-1: Mammalian Bcl-2-Related Proteins
|Antiapoptotic ||Proapoptotic ||BH-3 Proteins (Inhibit Antiapoptotic Function) |
|Bcl-2 ||Bcl-xS ||Bad |
|Bcl-xL ||Bax ||Bid |
|Mcl-1 ||Bak ||Bik |
|Bcl-w ||Mtd/Bok ||Hrk |
|A-1 || ||Bim |
| || ||Blk |
Bcl-2 family members can be divided into three categories: antiapoptotic factors such as Bcl-2 that inhibit cell death, proapoptotic factors that when overexpressed trigger cell death, and factors that while not intrinsically proapoptotic nevertheless can bind to and inhibit the function of antiapoptotic factors. Members of the third category typically share only a limited homology with the other family members, in a domain known as BH-3.44
The mechanism of action of Bcl-2 family members has not been determined, although several important insights have been established. The structure of Bcl-xL has been determined by x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.45 The molecular structure of Bcl-xL surprisingly was found to be similar to that of members of the colicin family of bacterial proteins. Colicins are proteins secreted by bacteria that form pores in the surface membranes of other bacterial strains, causing cell death.46 The relevance of this unexpected structural link between families of proteins involved in mammalian and bacterial cell death is currently unclear.
Bcl-2 appears to be localized to the nuclear endoplasmic reticular and outer mitochondrial membranes.47-49 Commitment to apoptotic cell death has been associated with loss of mitochondrial membrane potential, and this membrane potential gradient can be maintained by Bcl-2 overexpression.50-54 Many of the characteristic nuclear changes of apoptosis can be induced by the addition of dATP and cytochrome c to cytoplasmic extracts in vitro.55 Since cytochrome c normally is tightly sequestered within mitochondria, Bcl-2 family members may function by directly controlling mitochondrial membrane permeability.56 Indeed, recent observations indicate that Bcl-2 and Bcl-xL inhibit, whereas the proapoptotic family member Bax promotes, cytochrome c release from the mitochondrial intermembrane space.56-59
Antiapoptotic family members also may function by complexing with and inactivating factors that would otherwise trigger caspase activation. As described earlier, in C. elegans CED-9 has been found to interact directly with CED-4; this interaction in turn prevents CED-4 from activating the cysteine protease CED-3.27,28 Bcl-xL similarly can interact with the mammalian CED-4 homologue Apaf-1, and this interaction may play a role in the inhibition of Apaf-1 activation of downstream caspases.34,62,63
Another group of proteins that has been implicated strongly in the central apoptotic pathway are the caspases (cysteine proteases with aspartic acid specificity).64,65 Unlike the Bcl-2 family, which appears to modulate apoptotic threshold without participating directly involved in cellular autodigestion, caspase activity has been associated closely with the apoptotic morphology of the dying cell. The family has at least 13 members, which can be subgrouped on the basis of similarity and target specificity65-69 (Table 24-2). Overexpression of most caspases has been shown to trigger apoptosis in cell lines, although not all appear to be involved in physiological apoptosis. A central role for this family of proteases in the process of apoptosis in mammalian cells has been suggested by studies showing that specific inhibitors of caspases can prevent cell death, or at least an apoptotic morphology, in response to many of the known triggers of programmed cell death.70,71 These inhibitors include viral products such as p35 and crmA (see below) as well as synthetic oligopeptides that occupy and block the protease activity site.
Table 24-2: Mammalian Caspase Subfamilies Grouped by Substrate Specificity |Favorite Table|Download (.pdf) Table 24-2: Mammalian Caspase Subfamilies Grouped by Substrate Specificity
|Group 1 ||Group 2 ||Group 3 |
|Caspase 1 (ICE) ||Caspase 2 (ICH-1/NEDD-2) ||Caspase 6 (Mch2) |
|Caspase 4 (ICE rel II/ICH-2/TX) ||Caspase 3 (CPP32/Yama/Apopain) ||Caspase 8 (FLICE/MACH/Mch5) |
|Caspase 5 (ICE rel III/TY) ||Caspase 7 (ICE-LAP3/Mch3/CMH-1) ||Caspase 9 (ICE-LAP6/Mch6) |
|Caspase 11 (ICH-3) || ||Caspase 10 (FLICE2/Mch4) |
|Caspase 12 || || |
|Caspase 13 (ERICE) || || |
Regulation of caspase activity may occur on several levels. All the caspases are synthesized as larger inactive proenzymes that must undergo proteolytic processing to the active enzymatic forms.72 The cleavage sites in these proenzymes are consistent with processing by caspases themselves. High local concentrations of some caspases may be sufficient to permit autocatalysis and activation.73 The processing sites of some family members appear more likely to be target sites for other caspases, suggesting a sequential cascade of protease activation. Initial activation of a caspase may generate a rapidly and irreversibly amplified signal by initiating autocatalysis as well as triggering activation of downstream proteases. An additional layer of regulation may derive from alternate mRNA splicing. At least four of the caspase genes encode truncated forms as well as full-length proteases.74-77 These truncated proteins may down-regulate protease activity by directly inhibiting the active proteases or by binding and stabilizing the proenzyme forms.
Among the caspase family, caspase 9 has been most clearly implicated in the central pathway of apoptotic induction (Fig. 24-5). Caspase 9, along with cytochrome c and Apaf-1, was identified as a factor required for induction of apoptotic events in a cell-free system.55,61 Apaf-1 interacts with and activates caspase 9, which can then process other caspases, including caspase 3.61 Caspase 3, in turn, has been found to be integrally involved in the generation of apoptotic nuclear morphology (condensation and DNA degradation).78
A hypothetical model of the central apoptotic pathway. Initiation of apoptosis is held in check by survival signals received by cell surface receptors. Removal of the cell from its in vivo context or blockade of these survival signals allows induction of the apoptotic pathway, resulting in loss of mitochondrial outer membrane integrity and cytochrome c release. Cytosolic cytochrome c interacts with Apaf-1, which in the presence of ATP leads to caspase 9 processing and activation. This initiates a cascade of caspase activation, and ultimately in the characteristic morphologic changes of apoptosis. Bcl-2 and Bcl-xL reside in the mitochondrial outer membrane and can inhibit cytochrome c release. Other important apoptotic initiators include cell surface receptor-mediated cell death signals, DNA damage, cell cycle dysregulation, and metabolic alterations.
Many potential downstream targets with caspase cleavage sites have been identified and together may elucidate some of the mechanisms underlying apoptotic physiology. Among the defined caspase substrates are proteins involved in nuclear and cytoplasmic structure (e.g., nuclear lamins, actin), signal transduction (e.g., c-Abl, Raf-1, NF-κB p65 and p50), cell cycle control (e.g., MDM-2, Rb), genomic repair and integrity (e.g., poly-ADP ribose polymerase (PARP), DNA-dependent protein kinase), and apoptotic regulation (Bcl-xL, Bcl-2).79-93 Nevertheless, clear definition of the roles and relative importance of the various caspases and their downstream targets has been difficult. For example, PARP can be processed by caspase 3, 8, or 9 and has been used as a marker for the nuclear changes associated with apoptosis.77,92,93 However, PARP cleavage is neither necessary nor sufficient for apoptosis.94,95