A unified model of mammalian BCL-2 protein family interactions at the mitochondria

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX?and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.     

Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.     

Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.     

Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.     

Structural and Biochemical Bases for the Inhibition of Autophagy and Apoptosis by Viral BCL-2 of Murine ��-Herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine γ-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.     

A stapled BID BH3 helix directly binds and activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.     

BCL-2 selectively interacts with the BID-induced open conformer of BAK,inhibiting BAK auto-oligomerization

Caspase-8 cleaves BID to tBID, which targets mitochondria and induces oligomerization of BAX and BAK within the outer membrane, resulting in release of cytochrome c from the organelle. Here, we have initiated these steps in isolated mitochondria derived from control and BCL-2-overexpressing cells using synthetic BH3 peptides and subsequently analyzed the BCL members by chemical cross-linking. The results show that the BH3 domain of BID interacts with and induces an "open" conformation of BAK, exposing the BAK N terminus. This open (activated) conformer of BAK potently induces oligomerization of non-activated ("closed") conformers, causing a cascade of BAK auto-oligomerization. Induction of the open conformation of BAK occurs even in the presence of excess BCL-2, but BCL-2 selectively interacts with this open conformer and blocks BAK oligomerization and cytochrome c release, dependent on the ratio of BID BH3 and BCL-2. This mechanism of inhibition by BCL-2 also occurs in intact cells stimulated with Fas or expressing tBID. Although BID BH3 interacts with both BCL-2 and BAK, the results indicate that when BCL-2 is in excess it can sequester the BID BH3-induced activated conformer of BAK, effectively blocking downstream events. This model suggests that the primary mechanism for BCL-2 blockade targets activated BAK rather than sequestering tBID.     

A membrane-targeted BID BCL-2 homology 3 peptide is sufficient for high potency activation of BAX in vitro

The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni(2+)-nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic activity.     

Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles

A peptide corresponding to the BH3 region of the proapoptotic protein, BID, could be bound in the cleft of the antiapoptotic protein, BCL-w. This binding induced major conformational rearrangements in both the peptide and protein components of the complex and led to the displacement and unfolding of the BCL-w C-terminal alpha-helix. The structure of BCL-w with a bound BID-BH3 peptide was determined using NMR spectroscopy and molecular docking. These studies confirmed that a region of 16 residues of the BID-BH3 peptide is responsible for its strong binding to BCL-w and BCL-x(L). The interactions of BCL-w and the BID-BH3 peptide complex with dodecylphosphocholine micelles were characterized and showed that the conformational change of BCL-w upon lipid binding occurred at the same time as the release and unfolding of the BH3 peptide.     

Solution structure of human BCL-w: modulation of ligand binding by the C-terminal helix

The structure of human BCL-w, an anti-apoptotic member of the BCL-2 family, was determined by triple-resonance NMR spectroscopy and molecular modeling. Introduction of a single amino acid substitution (P117V) significantly improved the quality of the NMR spectra obtained. The cytosolic domain of BCL-w consists of 8 alpha-helices, which adopt a fold similar to that of BCL-xL, BCL-2, and BAX proteins. Pairwise root meant square deviation values were less than 3 A for backbone atoms of structurally equivalent regions. Interestingly, the C-terminal helix alpha8 of BCL-w folds into the BH3-binding hydrophobic cleft of the protein, in a fashion similar to the C-terminal transmembrane helix of BAX. A peptide corresponding to the BH3 region of the pro-apoptotic protein, BID, could displace helix alpha8 from the BCL-w cleft, resulting in helix unfolding. Deletion of helix alpha8 increased binding affinities of BCL-w for BAK and BID BH3-peptides, indicating that this helix competes for peptide binding to the hydrophobic cleft. These results suggest that although the cytosolic domain of BCL-w exhibits an overall structure similar to that of BCL-xL and BCL-2, the unique organization of its C-terminal helix may modulate BCL-w interactions with pro-apoptotic binding partners.     

Solution structure of BID, an intracellular amplifier of apoptotic signaling

We report the solution structure of BID, an intracellular cross-talk agent that can amplify FAS/TNF apoptotic signal through the mitochondria death pathway after Caspase 8 cleavage. BID contains eight alpha helices where two central hydrophobic helices are surrounded by six amphipathic ones. The fold resembles poreforming bacterial toxins and shows similarity to BCL-XL although sequence homology to BCL-XL is limited to the 16-residue BH3 domain. Furthermore, we modeled a complex of BCL-XL and BID by aligning the BID and BAK BH3 motifs in the known BCL-XL-BAK BH3 complex. Additionally, we show that the overall structure of BID is preserved after cleavage by Caspase 8. We propose that BID has both BH3 domain-dependent and -independent modes of action in inducing mitochondrial damage.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis¹. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)¹. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues².In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)^3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation^7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members^7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization¹⁰. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)¹⁰. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)¹¹. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.     

BIM and tBID are not mechanistically equivalent when assisting BAX to permeabilize bilayer membranes

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-X(L), BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency.     

Heterodimerization of BAK and MCL-1 activated by detergent micelles

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.     

After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization

Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1–3 (BH1–3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM.     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

Mechanisms of Action of Bcl-2 Family Proteins

The Bcl-2 family of proteins controls a critical step in commitment to apoptosis by regulating permeabilization of the mitochondrial outer membrane (MOM). The family is divided into three classes: multiregion proapoptotic proteins that directly permeabilize the MOM; BH3 proteins that directly or indirectly activate the pore-forming class members; and the antiapoptotic proteins that inhibit this process at several steps. Different experimental approaches have led to several models, each proposed to explain the interactions between Bcl-2 family proteins. The discovery that many of these interactions occur at or in membranes as well as in the cytoplasm, and are governed by the concentrations and relative binding affinities of the proteins, provides a new basis for rationalizing these models. Furthermore, these dynamic interactions cause conformational changes in the Bcl-2 proteins that modulate their apoptotic function, providing additional potential modes of regulation.Apoptosis was formally described and named in 1972 as a unique morphological response to many different kinds of cell stress that was distinct from necrosis. However, despite the novelty and utility of the concept, little experimental work was performed during the following 20 years because no tools existed to manipulate the process. In the early 1990s, two seminal observations changed the landscape. First, as the complete developmental sequence of the nematode Caenorhabditis elegans was painstakingly elucidated at the single-cell level, it was noted that a fixed, predictable number of “intermediate” cells were destined to die, and that this process was positively and negatively regulated by specific genes. Second, a novel gene called B-cell CLL/lymphoma 2 (Bcl-2; encoded by BCL2) that was discovered as a partner in a reciprocal chromosomal translocation in a human tumor turned out to function not as a classic oncogene by driving cell division, but rather by preventing apoptosis. When it was discovered that the mammalian BCL2 could substitute for CED-9, the C. elegans gene that inhibits cell death, the generality of the process was recognized, and the scientific literature exploded with now well over 10⁵ publications on apoptosis. However, it is ironic to note that after a further 20 years of intensive investigation, it is clear that the mechanism of action of Bcl-2 is quite distinct from Ced-9, which sequesters the activator of the caspase protease that is the ultimate effector of apoptosis. In contrast, Bcl-2 works primarily by binding to other related proteins that regulate permeabilization of the mitochondrial outer membrane (MOM).This review examines how apoptosis is regulated by the members of the (now very large) Bcl-2 family, composed of three groups related by structure and function (illustrated in Fig. 1): (1) the BH3 proteins that sense cellular stress and activate (either directly or indirectly); (2) the executioner proteins Bax or Bak that oligomerize in and permeabilize the MOM, thereby releasing components of the intermembrane space that activate the final, effector caspases of apoptosis; and (3) the antiapoptotic members like Bcl-2 that impede the overall process by inhibiting both the BH3 and the executioner proteins. To understand the consequence of the interactions among the three subgroups, several models have been proposed (“direct activation,” “displacement,” “embedded together,” and “unified” models; illustrated in Fig. 2) that are briefly described here before a more detailed discussion of the Bcl-2 families.Open in a separate windowFigure 1.Schematic overview of the Bcl-2 family of proteins. The family is divided into two subgroups containing proteins that either inhibit apoptosis or promote apoptosis. The proapoptotic proteins are further subdivided functionally into those that oligomerize and permeabilize the MOM, such as Bax and Bak, or those that promote apoptosis through either activating Bax or Bak or inhibiting the antiapoptotic proteins, such as tBid, Bim, Bad, and Noxa. Proteins are included in the Bcl-2 family based on sequence homology to the founding member, Bcl-2, in one of the four Bcl-2 homology (BH) regions. All the antiapoptotic proteins, as well as Bax, Bak, and Bid, have multiple BH regions, are evolutionarily related, and share a three-dimensional (3D) structural fold. The BH3 proteins contain only the BH3 region, are evolutionarily distant from the multiregion proteins, and are intrinsically unstructured. Most members of the Bcl-2 family proteins contain a membrane-binding region (MBR) on their carboxyl termini in the form of a tail anchor, mitochondrial-targeting sequence, or as a hydrophobic amino acid sequence that facilitates binding and localization of these proteins to the MOM or to the endoplasmic reticulum (ER) membrane.Open in a separate windowFigure 2.Schematics of the core mechanisms proposed by various models for the regulation of MOMP by Bcl-2 proteins. (↑) Activation; (⊥) inhibition; (⊥↑) mutual recruitment/sequestration. Paired forward and reverse symbols indicate the model makes explicit reference to equilibria. (A) The direct activation model divides the different BH3 proteins by qualitative differences in function. The BH3 proteins with high affinity for binding and activating Bax and Bak are termed as “activators,” whereas those that only bind the antiapoptotic proteins are termed “sensitizers.” The activator BH3 proteins directly interact with and activate Bax and Bak to promote MOMP. The antiapoptotic proteins inhibit MOMP by specifically sequestering the BH3 activators. The BH3 sensitizer proteins can compete for binding with the antiapoptotic proteins, thus releasing the BH3 activator proteins to avidly promote MOMP through activation and oligomerization of Bax and Bak. (B) The displacement model categorizes the BH3 proteins solely based on their affinities of binding for the antiapoptotic proteins (hence, does not recognize them as activators). In this model, Bax and Bak are constitutively active and oligomerize and induce MOMP unless held in check by the antiapoptotic proteins. Therefore, for a cell to undergo apoptosis, the correct combination of BH3 proteins must compete for binding for the different antiapoptotic proteins to liberate Bax and Bak and for MOMP to ensue. (C) The embedded together model introduces an active role for the membrane and combines the major aspects of the previous models. The interactions between members of the Bcl-2 family are governed by equlibria and therefore are contingent on the relative protein concentrations as well as their binding affinities. The latter are determined by posttranslational modifications, fraction of protein bound to the membrane, and cellular physiology. At membranes, the activator BH3 proteins directly activate Bax and Bak, which then oligomerize, inducing MOMP. Both activator and sensitizer BH3 proteins can recruit and sequester antiapoptotic proteins in the membrane. The antiapoptotic proteins inhibit apoptosis by sequestering the BH3 proteins and Bax and Bak in the membrane or by preventing their binding to membranes. At different intracellular membranes, the local concentrations of specific subsets of Bcl-2 family members alter the binding of Bcl-2 proteins to the membrane and the binding equilibria between family members. As a result, Bcl-2 family proteins have distinct but overlapping functions at different cellular locations. (D) The unified model builds on the embedded together model by proposing that the antiapoptotic proteins sequester the activator BH3 proteins (mode 1) and sequester Bax and Bak (mode 2). It differs in that in the unified model, inhibition of apoptosis through mode 1 is less efficient (smaller arrow in panel D) and therefore easier to overcome by sensitizer BH3 proteins. In addition, the unified model extends the role of Bcl-2 family proteins and the regulation of MOMP to mitochondria dynamics (not shown).