Pharmacological manipulation of Bcl-2 family members to control cell death (original) (raw)
Bcl-2 was initially cloned from the breakpoint of the t(14;18) chromosomal translocation found in the vast majority of patients with follicular lymphoma, an indolent B cell non-Hodgkin lymphoma (1–3). Expression of Bcl-2 blocked cell death following numerous cell insults (4, 5). As a test of its oncogenic function, a minigene bearing the _bcl-2_–immunoglobulin gene fusion, after a period of follicular hyperplasia (4, 5), induced lymphoma in transgenic mice (6). While previously characterized oncogenes shared the ability to increase cellular proliferation, Bcl-2 established a new class of oncogenes: inhibitors of programmed cell death.
Since the cloning of Bcl-2, an entire family of proteins related by sequence homology and participation in the control of apoptosis has been identified. Certain proteins share Bcl-2’s ability to oppose programmed cell death: Bcl-xL (7), Bcl-w (8), Mcl-1 (9), and Bfl-1 (A1) (10). These proteins share sequence homology in 4 α-helical Bcl-2 homology (BH) regions, BH1–BH4. Bax (11) and Bak (12), which promote cell death, share only the BH1–BH3 domains. Later, a third class of protein was discovered (13). These include Bid, Bad, Bik, Puma, Noxa, Bmf, and Hrk, which are called “BH3-only” proteins and demonstrate homology only in the BH3 region (14, 15). Like Bax and Bak, the proapoptotic BH3-only proteins require an intact BH3 domain to promote apoptosis (14, 16).
Bcl-2 proteins control mitochondrial permeabilization. Complex interactions among Bcl-2 family members govern mitochondrial outer membrane permeabilization (MOMP), the final common endpoint for execution of death signals by the Bcl-2 family (17) (Figure 1). Data show that activation of either Bax or Bak is required for MOMP (18–20), suggesting that Bax and Bak are the effectors in the Bcl-2 family most proximal to MOMP. In healthy cells, inactive Bax monomers reside either in the cytosol or in loose association with the mitochondrial outer membrane (21), while monomeric Bak is inserted in the outer membrane. Activation of Bax and/or Bak is accompanied by an allosteric change detectable by conformation-specific antibodies (22–24). Following activation, Bax inserts into the membrane, Bax and Bak homo-oligomerize, and then MOMP occurs (19, 25–27). Permeabilization releases proapoptotic factors, including cytochrome c (28), omi/htra2 (29), Smac/DIABLO (30, 31), endonuclease G (29), and AIF (32), to the cytosol. Released cytochrome c participates in a holoenzyme complex with Apaf-1 and caspase-9 that activates effector caspases by cleavage, resulting in widespread proteolysis. An extrinsic pathway of caspase activation initiated by cell surface death receptor signaling, which operates independently of the mitochondrion and Bcl-2 family members, exists but is beyond the scope of this discussion (33). While oligomers of Bax can form pores in artificial membranes that permit the passage of cytochrome c (34) or high–molecular weight dextran (35, 36), it is not clear whether activated Bax and/or Bak independently form pores in vivo, or whether Bax and/or Bak cooperate with other factors (17).
A model of Bcl-2 family member control over programmed cell death. In response to myriad death, damage, or derangement signals, BH3-only family members are activated (i). Activator BH3-only proteins interact with multidomain proapoptotic Bax and/or Bak (Bax/Bak), inducing their oligomerization (ii) and thus resulting in MOMP, release of cytochrome c, apoptosome formation, and caspase activation (iii). Bcl-2 and other multidomain antiapoptotic proteins interrupt the death signal by binding and sequestering activator BH3-only family members, and perhaps also Bax/Bak (iv). Bcl-2 antiapoptotic function may be antagonized by the competitive displacement of activator BH3-only molecules by sensitizer BH3-only proteins (v).
Activation of Bax and/or Bak. The mechanism of Bax and/or Bak activation has been controversial. Recent evidence supports activation of Bax and/or Bak via interaction with select BH3-only proteins. Bid protein and BH3 domains from Bid and Bim, but not other BH3-only proteins, induce MOMP in a Bax and/or Bak–dependent fashion and induce Bak and Bax oligomerization (19, 37, 38). Induction of these apoptotic changes requires an intact BH3 domain. The ability of Bid, Bims, or their BH3 domains to stimulate Bax oligomerization and pore formation required no other proteins in a defined synthetic liposomal system; this supports a direct interaction (35, 36). It has been hypothesized that Bid performs primarily as an inhibitor of Bcl-2. However, a Bid mutant that lacks the ability to interact with Bcl-2 but maintains interaction with Bax is still potently proapoptotic, which suggests that interaction with Bax and/or Bak is important in Bid’s function (14).
Complexes of Bid or Bim with Bax or Bak have been difficult, but not impossible, to isolate (14, 39–41). Interactions between BH3 domains and Bax and/or Bak may be transient, with the BH3 domain leaving after allosteric activation of Bax and/or Bak in a “hit and run” model. While the BH3 domains of Bid and Bim are necessary for interaction with Bax and Bak, their most efficient presentation to Bax and Bak may require conformational changes, posttranslational modifications, and/or certain isoforms of the entire protein (39, 40, 42–45). In addition, proteins outside of the Bcl-2 family bind and modulate function of Bax (46–48) and Bak (49).
Bcl-2 blocks Bax and/or Bak–dependent MOMP. Bcl-2 and the related antiapoptotic proteins Bcl-xL, Mcl-1, Bcl-w, and Bfl-1 inhibit MOMP by binding pro-death family members. Like the proapoptotic multidomain proteins Bax and Bak, the antiapoptotic proteins possess a hydrophobic pocket made from the α-helices BH1, BH2, and BH3, where the hydrophobic face of amphipathic α-helical BH3 domains from proapoptotic members binds (50–53). While earlier work focused on the ability of antiapoptotic proteins to bind Bax or Bak, a Bcl-xL mutant lacking Bax or Bak binding, but still binding BH3-only proteins, retained the majority of its antiapoptotic function. This result suggests that binding and sequestration of BH3-only family members prior to their interaction with Bax and Bak is also an important function of the antiapoptotic proteins (26, 54). It is also consistent with the finding that Bcl-2 inhibits apoptosis upstream of Bax and/or Bak conformational change, membrane insertion (in the case of Bax), and oligomerization (26, 37, 55, 56). By binding and sequestering activator BH3 domains like Bid and Bim, the antiapoptotic proteins inhibit Bax and/or Bak activation and subsequent MOMP.
Antagonism of Bcl-2 by sensitizer BH3-only proteins. While all BH3-only proteins are proapoptotic, it is likely that only a subset interacts with Bax and Bak. Using a series of BH3 peptides, BH3 domains have been divided into 2 classes: the activators (including Bid and Bim), which can induce Bax and/or Bak oligomerization and MOMP, and the sensitizers, which cannot (37). The ability of p53 to activate Bax suggests there may be other “cryptic” activator molecules outside the Bcl-2 family (57). The sensitizer BH3 domains interact with the antiapoptotic molecules and only indirectly induce Bax and/or Bak activation by competitive displacement of activator BH3 proteins from the Bcl-2–binding cleft (37). These so-called sensitizer BH3 domains are thus prototypes of selective inhibitors of the antiapoptotic proteins (see below) (37, 58). Binding of a given BH3 protein to antiapoptotic proteins is not necessarily promiscuous. For instance, while BH3 domains from Bid, Bim, and Puma interact with all of the antiapoptotic proteins tested, the remainder interact only with select antiapoptotic partners, suggesting that antiapoptotic proteins have biophysically distinct binding pockets (36, 37, 59, 60). In theory, therefore, individual antiapoptotic family members can be selectively targeted by small molecules that mimic sensitizer BH3 domain behavior.
BH3-only proteins’ response to death stimuli. There remains the question of how BH3-only molecules are triggered to respond to death stimuli (61). In some cases, activation of BH3-only molecules is transcriptional. Noxa and Puma are p53-inducible genes that are transcriptionally induced in response to numerous DNA-damaging agents (62–66). While Bim can be transcriptionally regulated (67, 68), its regulation may also depend on cytoskeletal interaction (69) and phosphorylation, which may affect interaction with Bax (40) as well as ubiquitination and proteosomal degradation (70–72). In response to death signals that activate the extrinsic apoptotic pathway, Bid is first cleaved by caspase-8 (42, 43), and then the new amino terminus is myristoylated to facilitate targeting to the mitochondria (44). Bad is controlled, at least in part, by phosphorylation that mediates its sequestration by cytoplasmic 14-3-3 protein (73). Interaction of Bad with members of the glycolytic pathway suggests a role for Bad in glucose homeostasis (74). It appears that BH3-only family members serve individual but overlapping roles in sensing different types of cellular derangement and communicating these to the core death pathway.
To summarize, conduction of a death signal by the Bcl-2 family members begins with activation of the BH3-only proteins, which act as sentinels of myriad damage signals. Activator BH3 domains then trigger allosteric activation of Bax and/or Bak, which oligomerize at the mitochondria, inducing MOMP. Proapoptotic factors are released, including cytochrome c, which forms a holoenzyme complex with Apaf-1 and caspase-9, termed the apoptosome. This complex then catalytically cleaves effector caspases like caspase-3, resulting in widespread proteolysis and commitment to cell death. This pathway can be interrupted by Bcl-2 and related antiapoptotic proteins that bind and sequester the activator BH3 molecules. Sensitizer BH3-only molecules can further assist the progression of a death signal by competitive displacement of activator BH3 signals from the Bcl-2 pocket (Figure 1).
Alternative models of the control of apoptosis by Bcl-2 family members exist. In one, Bcl-2 regulates caspases, directly or via an as-yet undiscovered mammalian adaptor (13, 61, 75). In another, Bcl-2 tonically inhibits oligomerization of a Bax and/or Bak that is ready to induce MOMP without BH3 interaction (76). In a variation of this model, combinations of particular Bcl-2–like proteins must be neutralized by BH3 ligands to allow Bax and/or Bak activation (60). In another, Bax and/or Bak inhibit a dominantly acting Bcl-2 to induce death (13). However, recent results that emphasize the centrality of Bax and Bak (18, 20) and their activation by activator BH3 domains (19, 26, 35–37) to MOMP may call into question the consistency of the above alternatives with mounting experimental data.