Molecular Cell
`
`Review
`
`The BCL-2 Family Reunion
`
`Jerry E. Chipuk,1,2 Tudor Moldoveanu,1 Fabien Llambi,1 Melissa J. Parsons,1 and Douglas R. Green1,*
`1Department of Immunology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
`2Present address: Department of Oncological Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place 15-20E,
`New York, NY 10029, USA
`*Correspondence: douglas.green@stjude.org
`DOI 10.1016/j.molcel.2010.01.025
`
`B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally
`characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis.
`Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will
`discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting
`the complex integration and regulation of the BCL-2 family in cell fate decisions.
`
`A Death in the Family
`Like all living things, cells die. In animals, the predominant mode
`of cell death during development and tissue homeostasis is
`apoptosis. During this process, the caspase proteases effec-
`tively package and label
`(e.g., by inducing cellular blebbing
`and shrinkage, DNA fragmentation, and plasma membrane
`changes) dying cells for rapid clearance.
`In vertebrates, the BCL-2 family regulates the mitochondrial
`pathway of apoptosis by complex interactions that dictate the
`integrity of the outer mitochondrial membrane (OMM) (Green
`and Evan, 2002). This pathway is initiated by mitochondrial outer
`membrane permeabilization (MOMP), which allows soluble
`proteins (e.g., cytochrome c) in the mitochondrial intermembrane
`space (IMS) to diffuse into the cytosol. Cytochrome c engages
`apoptotic protease activating factor-1 (APAF-1) to oligomerize
`into a caspase activation platform termed the apoptosome.
`This binds and promotes the activation of initiator caspase-9,
`which then activates executioner caspases-3 and -7. The cas-
`pases cleave cellular substrates to elicit the apoptotic phenotype
`(Figure 1A) (Riedl and Salvesen, 2007). Temporally, MOMP inde-
`terminately occurs following proapoptotic stress, but studies
`suggest that this timing is dependent on the concentrations of
`diverse cellular proteins (Spencer et al., 2009). After MOMP, cas-
`pase activation and apoptosis ensue often within minutes.
`Here we discuss the BCL-2 family and its regulation of mitochon-
`drial integrity, apoptosis, and other cellular processes including
`mitochondrial dynamics, endoplasmic reticulum (ER) calcium
`stores, and autophagy. Although the BCL-2 family is conserved
`among animals, its role in controlling the integrity of the OMM is
`only described for the vertebrates, and limited data exist for func-
`tions in invertebrates. In C. elegans, the BCL-2 protein cell death
`abnormality-9 (CED-9) does not control MOMP, but instead inhibits
`apoptosis by sequestering the APAF-1 homolog, CED-4 (reviewed
`in Lettre and Hengartner, 2006). Similarly, the Drosophila BCL-2
`homologs do not appear to control cell death, and their functions
`remain obscure (reviewed in Mollereau, 2009). For these reasons,
`our discussion focuses on vertebrate BCL-2 family function.
`
`A Family Portrait
`BCL-2 and its relatives are functionally classified as either antia-
`poptotic or proapoptotic (Figure 1B). Most cells express a variety
`
`of antiapoptotic and proapoptotic BCL-2 proteins, and the
`regulation of their interactions dictates survival or commitment
`to apoptosis (Figure 1C).
`Antiapoptotic BCL-2 proteins contain four BCL-2 homology
`domains (BH1–4) and are generally integrated within the OMM,
`but may also be in the cytosol or ER membrane (Figure 2A).
`BCL-2-related gene A1 (A1), BCL-2, BCL-2-related gene, long
`isoform (BCL-xL), BCL-w, and myeloid cell leukemia 1 (MCL-1)
`are the major members of the antiapoptotic BCL-2 repertoire
`and preserve OMM integrity by directly inhibiting the proapop-
`totic BCL-2 proteins.
`The proapoptotic BCL-2 members are divided into the effector
`proteins and the BH3-only proteins. The effector proteins BCL-2
`antagonist killer 1 (BAK) and BCL-2-associated x protein (BAX)
`were originally described to contain only BH1-3; however, struc-
`ture-based alignment of globular BCL-2 family proteins revealed
`a conserved BH4 motif (Figure 2A) (Kvansakul et al., 2008). Upon
`activation, BAK and BAX homo-oligomerize into proteolipid
`pores within the OMM to promote MOMP (Figure 2B). There is
`a potential third effector molecule, BCL-2-related ovarian killer
`(BOK), but no biochemical evidence supports a function akin
`to BAK or BAX.
`The BH3-only proteins function in distinct cellular stress
`scenarios and are subdivided based on their ability to interact
`with the antiapoptotic BCL-2 repertoire or both the antiapoptotic
`proteins and the effectors (Figures 1C, 3A–3C). BH3-only
`proteins that only bind to the antiapoptotic repertoire are referred
`to as ‘‘sensitizer’’ and/or ‘‘derepressor’’ BH3-only proteins; e.g.,
`BAD (BCL-2 antagonist of cell death) and Noxa. BID (BCL-2-
`interacting domain death agonist) and BIM (BCL-2-interacting
`mediator of cell death) interact with the antiapoptotic repertoire
`as well as the effectors, and can directly induce BAK and BAX
`oligomerization and MOMP. These BH3-only proteins are
`referred to as ‘‘direct activators’’ (Figure 3B). The interactions
`between the antiapoptotic repertoire, direct activator/sensi-
`tizer/derepressor BH3-only proteins, and effectors determine
`MOMP and apoptosis (Figures 1C, 3A–3C).
`BAK and BAX Activation
`Central to the initiation of apoptosis is BAK/BAX activation at the
`OMM. While there are competing models explaining the control
`of BAK/BAX activation (Figures 3A–3C), the contribution of
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`Figure 1. The Mitochondrial Pathway of
`Apoptosis and the BCL-2 Family
`(A) Cellular stress causes transcriptional and post-
`transcriptional regulation of the BCL-2 family to
`promote MOMP. MOMP is induced by interactions
`between the BH3-only and effector proteins and
`leads to cytochrome c release, APAF-1 recruit-
`ment, and caspase activation. At the time of
`MOMP (middle), the intact mitochondrial network
`(green, left) undergoes fragmentation (gray, right),
`and soon after the cell is disassembled. The mito-
`chondria in the middle are enlarged from the white
`box.
`(B) The BCL-2 family is divided into antiapoptotic,
`effector, and direct activator/sensitizer/derepres-
`sor BH3-only proteins.
`(C) The antiapoptotic BCL-2 protein binding
`profiles for the BH3-only proteins.
`
`The series of events leading to BID-
`mediated activation of BAX has been
`elucidated in vitro (Lovell et al., 2008).
`BAX is soluble and undergoes activation
`in the presence of a direct activator and
`suitable membrane (e.g., the OMM or
`LUV); this results in oligomerized BAX
`and membrane permeabilization. The first step for BID-induced
`BAX activation is the association of BID with a membrane, fol-
`lowed by BAX recruitment,
`insertion, and oligomerization
`(Figure 2B) (Leber et al., 2007). Binding between BID and BAX
`has been difficult to study, but FRET analysis revealed the inter-
`action in vitro (Lovell et al., 2008).
`The interaction between BIM and BAX was demonstrated by
`NMR (Gavathiotis et al., 2008). Several other proteins are
`described to directly activate BAX, and it will be interesting to
`determine if they utilize a similar mechanism. BAK and BAX
`activation can also be triggered by nonprotein factors: e.g.,
`mild heat, detergents, and high pH (Hsu and Youle, 1997; Khaled
`et al., 2001; Pagliari et al., 2005). Whether or not these mecha-
`nisms physiologically occur remains to be proven. However,
`observations that BAK/BAX-dependent apoptosis proceeds in
`the absence of BID and BIM argues that either direct activation
`
`Figure 2. BCL-2 Family Composition and
`Membrane Permeabilization
`(A) The BCL-2 proteins are comprised of BCL-2
`homology (BH) domains. A representation of an
`antiapoptotic (BCL-2), effector (BAX), and BH3-
`only (BID) protein is shown with the BH1-4 desig-
`nated underneath the corresponding a helices.
`(B) Proposed model of BAX activation. Soluble
`BAX interacts with a direct activator and the
`OMM to promote stable N-terminal exposure,
`and BAX a5, a6, and a9 insert within the OMM.
`
`BH3-only proteins in this process is undisputed (Chipuk and
`Green, 2008). At least two of the BH3-only proteins, BID and
`BIM, are capable of directly inducing effector function. The
`active form of BID (discussed below) promotes BAK and BAX
`oligomerization, MOMP, and cytochrome c release (Kuwana
`et al., 2002; Wei et al., 2000). Similar evidence exists for BIM-
`mediated BAK/BAX activation; and while PUMA was also
`suggested to promote BAK/BAX activation, this effect may not
`be direct (Chipuk et al., 2008; Kim et al., 2006; Kuwana et al.,
`2005; Letai et al., 2002). The BH3 domains of BH3-only proteins
`can be synthesized and represent the minimal unit of BH3-only
`protein function (referred to as BH3 peptides). BID and BIM
`BH3 peptides induce BAK and BAX oligomerization and pore-
`forming activity with isolated mitochondria or large unilamellar
`vesicles (LUVs, lipid vesicles that mimic the OMM) (Kuwana
`et al., 2002, 2005; Letai et al., 2002).
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`by proteins is unnecessary, or alternative mechanisms for
`activation exist (Willis et al., 2007).
`Sensitization and Derepression
`Other BH3-only proteins, such as BAD, BCL-2-interacting killer
`(BIK), Harakiri (HRK), Noxa, and p53-upregulated modulator of
`apoptosis (PUMA) function predominantly by binding to the anti-
`apoptotic repertoire and not by directly activating BAK or BAX
`(Chen et al., 2005; Chipuk et al., 2008; Kuwana et al., 2005; Letai
`et al., 2002). The terms ‘‘sensitizer’’ and ‘‘derepressor’’ are used
`to indicate the consequences of binding between a BH3-only
`protein and an antiapoptotic BCL-2 protein. Each sensitizer/der-
`epressor BH3-only protein has a unique binding profile for the
`antiapoptotic repertoire, which was primarily determined by
`using BH3 peptides (Figure 1C). These BH3-only proteins estab-
`lish two distinct mechanisms that promote BAK/BAX activation:
`sensitization and derepression (Figures 3A and 3B) (Chipuk et al.,
`2008; Kuwana et al., 2005; Letai et al., 2002).
`Sensitization lowers the threshold for BAK and BAX activation
`and MOMP but does not cause apoptosis itself (Figure 3A). In
`this scenario, an antiapoptotic protein is in complex with a sensi-
`tizer BH3-only protein, which prevents the inhibition of subse-
`quent direct activators. For example, if BCL-2 is associated
`with PUMA, any future induction of BIM is not inhibited and
`MOMP proceeds. In the absence of PUMA, BIM would be
`sequestered and the cell may survive.
`For derepression, a direct activator is bound by an antiapop-
`totic BCL-2 protein, and a subsequent BH3-only protein releases
`the direct activator to promote MOMP (Figure 3B). For example,
`reparable cellular stress can induce BIM function, but this
`activity is inhibited by the antiapoptotic repertoire and the cell
`survives. If a derepressor BH3-only protein is induced while
`the direct activator is sequestered, the latter can be released,
`
`Figure 3. BH3-Only Protein Function
`(A) Sensitizer BH3-only protein function. A sensi-
`tizer BH3-only protein inhibits the antiapoptotic
`BCL-2 repertoire. Following minimal cellular
`stress, a direct activator is induced but cannot
`be inhibited and MOMP proceeds.
`(B) Derepressor BH3-only protein function. A
`direct activator is sequestered by an antiapoptotic
`BCL-2 protein. Following cellular stress, a dere-
`pressor BH3-only protein is
`induced and
`competes with the direct activator for binding to
`the antiapoptotic repertoire. When the direct acti-
`vator is released, MOMP proceeds.
`(C) The neutralization model of BCL-2 family func-
`tion. In this model, BAK/BAX are always compe-
`tent to promote MOMP but are actively inhibited
`by the antiapoptotic BCL-2 repertoire to promote
`survival. Following cellular stress, BH3-only pro-
`teins are induced, bind the antiapoptotic proteins,
`and displace effectors to promote MOMP.
`
`allowing for MOMP. Studies using FRET
`demonstrated that derepression and
`consequential direct activation occur via
`protein$protein interactions that are not
`readily detected in the absence of
`membranes (Lovell et al., 2008). For
`example, activated BID was bound by
`BCL-xL, and this interaction was disrupted by BAD. BID then inter-
`acted directly with BAX, followed by BAX oligomerization and LUV
`permeabilization. These studies highlight the rapidity of the inter-
`actions once the conditions for derepression and MOMP are satis-
`fied. Derepression also represents a means of pharmacological
`regulation in certain tumors, such as chronic lymphocytic leukemia
`(CLL), which undergo MOMP when treated with a derepressor
`BH3 mimetic (Certo et al., 2006; Del Gaizo Moore et al., 2007).
`An alternative hypothesis to the direct activator requirement is
`the neutralization model (Figure 3C). In this case, MOMP can
`proceed following inhibition of the antiapoptotic BCL-2 reper-
`toire independently of direct activator$effector
`interactions
`(Uren et al., 2007; Willis et al., 2007). This model assumes that
`cells harbor activated forms of BAK and BAX that are seques-
`tered by the antiapoptotic BCL-2 repertoire. BH3-only proteins
`then compete for the antiapoptotic proteins and apoptosis
`ensues. We recognize that inhibition of the antiapoptotic reper-
`toire contributes to MOMP, but contend it most efficiently occurs
`following the combined efforts of direct activator and sensitizer/
`derepressor BH3-only proteins, as recently suggested by
`elegant experiments in vivo (Merino et al., 2009).
`
`Communicating with the BCL-2 Family
`The BH3-only proteins are the major sentinels for cellular stress,
`and diverse signaling pathways converge upon these proteins.
`BH3-only proteins share little homology, and this may explain
`the variety of mechanisms capable of regulating their function.
`The multidomain BCL-2 proteins can also be regulated during
`apoptotic signaling by changes in stability or cooperation with
`other family members. Here we highlight a few of the pathways
`that regulate the BCL-2 family (see Table S1, available online,
`for a list of regulators and references).
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`Family Form: Structural Considerations
`of the BCL-2 Family
`The BCL-2 Core
`The BCL-2 family is structurally categorized into folded globular
`and intrinsically unstructured proteins (IUPs) (see Table S2 for
`a listing of structures and references). Among the globular
`proteins, all of
`the multidomain antiapoptotic and effector
`BCL-2 proteins share a conserved ‘‘BCL-2 core.’’ This is also
`preserved in BID, even though it has the least structural
`homology to the folded members. The remaining BH3-only
`proteins are IUPs and likely fold upon binding to a globular
`BCL-2 protein. MCL-1 is structurally distinct, as it contains an
`N-terminal unstructured domain followed by the BCL-2 core.
`The BCL-2 core was revealed by the X-ray and NMR struc-
`tures of BCL-xL and represents a 20 kDa globular domain
`comprised of seven or eight amphipathic alpha (a) helices, ar-
`ranged around a central buried helix, a5 (Figure 4A). The BH1
`(portions of a4–a5) and BH2 (a7–a8) regions on one side, and
`the BH3 region (a2) and a3 on the other side, coalesce to delimit
`a hydrophobic groove (referred to as the BCL-2 family BH3 and
`C-terminus-binding groove, BC groove) at the ‘‘front’’ of the
`BCL-2 core. In BCL-w, and probably BCL-xL (as well as BAX,
`see below) the C-terminal a helix binds to the BC groove. The
`BH4 region, which is structurally defined by the conserved a1
`positioned alongside a6, stabilizes the BH1-BH3 regions. All
`together, the BC groove and a1/a6 structural components
`comprise the BCL-2 core. The major differences between antia-
`poptotic and effector proteins are likely distinguished via the
`structural features of the individual BCL-2 cores, which reveals
`how the front pocket geometry, amino acid composition, and
`degree of obstruction by the C-terminal transmembrane (TM)
`tail modulate interactions with the BH3-only proteins.
`Conformational Changes during BAK and BAX
`Activation
`In unstimulated cells, BAX is cytosolic and the BC groove
`accommodates the C-terminal TM region as an amphipathic
`a helix (Figure 2B). To initiate MOMP, BAX undergoes a cyto-
`solic-to-mitochondrial
`redistribution,
`initially implicating the
`exposure of TM region with OMM targeting. In contrast, BAK is
`constitutively targeted to the OMM,
`likely explained by an
`occluded BC groove, which impedes binding of the TM tail.
`Constitutive targeting of BAK or induced redistribution of BAX
`to the OMM does not imply that effectors are active once the TM
`regions are embedded in the OMM. Instead, direct activation of
`BAK and BAX induces numerous conformational changes: (1) a1
`exposure,
`(2) transient BH3 exposure,
`(3) protection of the
`membrane-embedded BCL-2 core, and (4) increased proximity
`of embedded monomers. Furthermore, analysis of BCL-xL/
`BAX chimeras identified the regions of BAX required for
`membrane insertion and MOMP (George et al., 2007). Together,
`these activation-induced conformational changes correlate with
`MOMP. These studies also revealed that the a5 helix was both
`necessary and sufficient for BAX oligomerization and MOMP.
`Supporting the sequence of events postulated for direct acti-
`vation of the effectors, subtle conformational changes in BAX
`instigated by the BIM BH3 peptide were modeled using NMR
`(Figure 4H) (Gavathiotis et al., 2008). To improve the affinity of
`the BIM BH3 peptide for BAX, a BIM SAHB (‘‘stabilized a helix
`
`The proapoptotic function of the BID BH3 domain is revealed
`by a unique mechanism involving the proteolytic cleavage of the
`large unstructured loop joining the inhibitory N terminus and the
`BH3-containing C terminus. Cleavage can be achieved by
`a variety of proteases; e.g., caspase-8 (via death receptors),
`granzyme B (cytotoxic lymphocytes), and caspase-2 (via heat
`shock). The proapoptotic function of BID is also enhanced by
`N-myristoylation, which promotes OMM targeting and BAK acti-
`vation.
`BIM is regulated both at the transcriptional and posttransla-
`tional
`levels. BIM is expressed as three alternatively spliced
`isoforms (BIM-S, BIM-L, and BIM-EL), although the latter two
`are most often observed. Levels of bim mRNA are positively
`regulated by the forkhead transcription factor FOXO3A upon
`cytokine deprivation, and by C/EBPa and CHOP following ER
`stress. Translation of bim mRNA is negatively regulated by the
`miRNA cluster miRNA-17-92, and overexpression of miRNA-
`17-92 induces a bim-deficient phenotype (Xiao et al., 2008).
`BIM function is also controlled by a series of posttranslational
`modifications via ERK1/2 and bTrCP that differentially regulate
`the major BIM isoforms.
`BAD is involved in apoptotic and nonapoptotic processes, and
`these dual activities are regulated by posttranscriptional modifi-
`cations. Growth factors inhibit the proapoptotic function of BAD
`through phosphorylation by several kinases, such as Akt. Phos-
`phorylation results in cytoplasmic sequestration and inactivation
`of BAD by 14-3-3 proteins, which prevents interaction with antia-
`poptotic BCL-2 proteins. The BAD BH3 domain phosphorylation
`status also regulates a nonapoptotic function of BAD through the
`direct regulation of glucokinase and glucose-driven mitochon-
`drial respiration. Accordingly, mice deficient in bad or expressing
`a mutant with mutated phosphorylation residues display
`abnormal glucose homeostasis and deficient insulin responses,
`an effect that is reversed by BAD BH3 peptide treatment.
`BAK and BAX expression levels are generally sufficient to
`promote MOMP. Hence, posttranslational modification of BAK
`and BAX likely regulates interactions within the BCL-2 family.
`In line with this hypothesis, survival signaling through ERK1/2
`causes BAX phosphorylation and inhibition of its proapoptotic
`activity. Interestingly, the ubiquitously expressed b isoform of
`human BAX is capable of inducing MOMP and apoptosis, appar-
`ently in a BH3-only protein-independent manner. To thwart
`unwarranted MOMP, BAXb is constitutively degraded in the
`absence of proapoptotic stress, suggesting strict posttransla-
`tional control (Fu et al., 2009).
`Among the antiapoptotic proteins, the stability and function of
`MCL-1 have been extensively studied. After genotoxic stress,
`MCL-1 is ubiquitinylated by MULE, a HECT domain-containing
`E3 ligase, and rapidly degraded. MULE contains a BH3 domain
`that binds to MCL-1 similarly to the Noxa BH3, which can also
`promote MCL-1 degradation. MCL-1 stability is regulated by
`glycogen synthase kinase-3 (GSK-3), which phosphorylates
`MCL-1 to promote its degradation. The E3 ligase responsible
`for MCL-1 ubiquitination following GSK-3 phosphorylation is
`bTrCP, which can regulate BIM-EL stability. Also, removing ubiq-
`uitin groups conjugated to MCL-1 thwarts degradation and
`enhances cellular survival; this can be achieved by the deubiqui-
`tinase USP9X (Schwickart et al., 2010).
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`of BCL-2 domain’’ peptide) was used, in which the BH3 a helix is
`fixed by a chemical staple. The BAX$BIM SAHB complex was
`nevertheless unstable, precluding structural determination by
`conventional NMR. Instead, paramagnetic relaxation enhance-
`ment NMR oriented the BIM SAHB on the back face opposite
`the BC groove of BAX. A shallow hydrophobic groove in BAX
`devoid of pronounced ridges was defined by a1 and a6. This
`occurred by displacing the a1-a2 loop and rearranging the
`peripheral side chains (e.g., BAX Lys21 next to BIM Glu158) to
`accommodate the BIM SAHB;
`the newly identified groove
`appears similar to antiapoptotic BC grooves. Binding of the
`BIM SAHB also induced subtle conformational changes to the
`BAX BC groove, where a9 became loosely attached compared
`to the free BAX structure. In addition, the BID and PUMA BH3
`can promote remodeling of the BAX structure (Kim et al., 2009).
`Whether a similar direct activator-binding strategy mediates
`BAK activation is unknown. Modeling of the BIM SAHB on the
`back face identified fewer clashes in BAK compared to BAX
`(Figure 4I). However, NMR revealed a low-affinity complex
`between the BID BH3 peptide and BAK-DTM (which mimics
`the globular domain of membrane-targeted BAK) (Figure 4G).
`The BID BH3 binding site overlapped with the partially occluded
`
`Figure 4. Structural Highlights for the BCL-2
`Family
`(A) The ‘‘front’’ view of BCL-xL identifies the BCL-2
`core and the respective locations of the four BH
`regions. The PDB identifier is in parentheses.
`(B) Surface representation of free BCL-xL, empha-
`sizing amino acids participating in BH3 peptide
`binding at
`the ‘‘front’’
`face,
`identifies the BC
`groove. Representations are partially transparent,
`permitting identification, beneath the surface, of
`contact side chains positioned 4 A˚ from a peptide.
`With the exception of free BCL-xL and MCL-1 (C),
`both having surface coloring based on all atoms of
`peptide amino acids, surface coloring highlights
`strictly the atoms within 4 A˚ from a peptide. PDB
`residue numbering has been maintained. Labels
`of acidic and basic amino acids are colored red
`and blue, respectively.
`(C) Free MCL-1.
`(D) A1 bound to BIM BH3. The four conserved
`hydrophobic residues/sites and the conserved
`charged interactions are marked. Peptide amino
`acid labels are bold.
`(E) BCL-xL bound to BIM BH3.
`(F) MCL-1 bound to BIM BH3.
`(G) A model between BID BH3 (from the A1$BID
`BH3 complex) and BAK-DTM, overlapping sites
`of interaction (colored) identified by NMR spec-
`troscopy.
`(H) BIM SAHB modeling to the ‘‘back’’ face of free
`BAX.
`(I) BIM SAHB binding site on BAX.
`
`BC groove of BAK, and major conforma-
`tional changes were not detected. The
`BAK BC groove is also implicated in ho-
`modimerization, as illustrated by cross-
`linking studies at cysteine residues engi-
`neered within the BH3 and the BC
`groove (Dewson et al., 2008). These
`studies indicated that the formation of
`BAK dimers involves reciprocal BH3$BC groove interactions;
`and most recent studies revealed that high-order BAK oligomer
`formation requires a6$a6 interactions to promote MOMP (Dew-
`son et al., 2009).
`Structures of Antiapoptotic and BH3-Only Protein
`Complexes
`The antiapoptotic BCL-2 family members have been extensively
`studied at the molecular level by both NMR spectroscopy and
`X-ray crystallography. A comprehensive review of antiapoptotic
`BCL-2 family structures described free BCL-2, BCL-xL, BCL-w,
`and the complexes between BCL-xL and the BAK and BAD BH3
`peptides (Petros et al., 2004). The current portrait of antiapop-
`totic proteins now includes A1 and MCL-1 structures in complex
`with an extensive panel of BH3 peptides (Figures 4C–4F and 5A).
`These recent structural insights are significant to cancer drug
`discovery because MCL-1 and A1 are not targeted by the most
`promising BH3 mimetics (e.g., ABT-737 and its analogs), which
`only inhibit BCL-2, BCL-w, and BCL-xL (Oltersdorf et al., 2005).
`There are structural similarities and differences between the
`BCL-2 cores of A1, BCL-xL, and MCL-1 in the free and BH3
`peptide-bound states (Figures 4B–4F). Analyses of their BC
`grooves revealed a deep hydrophobic groove of variable width,
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`Figure 5. Binding of BH3-Only Peptides,
`Proteins, and ABT-737 to BC Grooves
`(A) Structure-based alignment of BH3 peptides
`from complexes with A1 (BAK, 2VOH; BID, 2VOI;
`BIM, 2VM6; PUMA, 2VOH; BMF, 2VOG), BCL-xL
`(2BZW,
`full-length BAD; 2P1L, BECLIN), and
`MCL-1 (Noxa A, 2ROD; Noxa B, 2NLA; see Table
`S2). BC grooves accommodate different types of
`BH3 amino acids: h, hydrophobic; m, mixed; s,
`small; c, charged. The degree of conservation,
`illustrated in black (100%), green (>75%), blue
`(>50%), and yellow (25%), identifies a conserved
`core defining BH3 peptide specificity. The five
`conserved hydrophobic residues/sites and the
`conserved charged interactions are marked.
`(B) Structure of BCL-xL illustrates overlapping
`sites of
`interaction with full-length BAD and
`ABT-737.
`
`full-length BAD in complex with BCL-xL,
`the antiapoptotic BCL-2 protein struc-
`tures remain to be solved in complex
`with their putative full-length BH3-only
`protein partners (Figure 5B).
`
`A Dysfunctional Family
`BCL-2 was identified in follicular B cell
`lymphoma as a translocation to the immu-
`noglobulin heavy-chain locus t(14:18)
`rendering the gene constitutively hyper-
`expressed. To determine if BCL-2 was
`capable of synergizing with oncogenes ex vivo, bcl-2 was retro-
`virally introduced into the bone marrow of wild-type mice or
`transgenic mice overexpressing the c-myc oncogene (Em-myc).
`BCL-2 cooperated with c-Myc to promote transformation of B
`cell precursors (Vaux et al., 1988). However, in the absence of
`such an additional oncogene, bcl-2 did not promote proliferation,
`although it did protect against apoptosis induced by cytokine
`withdrawal. In vivo, this was mirrored by a bcl-2 transgenic model
`(McDonnell et al., 1989). Further evidence for bcl-2 and c-myc
`synergy in a double transgenic (Em-bcl-2/c-myc) model con-
`firmed that bcl-2 contributes to lymphomagenesis by allowing
`a cell to survive despite c-myc expression (Strasser et al., 1990).
`Although developmentally normal, the adult bcl-2/ mouse
`displays thymus and spleen involution, polycystic kidney
`disease, and hypopigmented hair due to increased apoptosis.
`Genetic knockouts of the other antiapoptotic proteins revealed
`specific physiologic roles for the antiapoptotic BCL-2 proteins
`(see Table S3 for a list of animal models and references). For
`example, the bcl-x/ mouse dies at E13.5 due to massive
`apoptosis of hematopoietic and neuronal cells, while the
`bcl-w/ mouse elucidated an important role for BCL-w in sper-
`matogenesis. Loss of MCL-1 blocked embryo implantation and
`is early embryonic lethal. Although these proteins are classified
`into one category, each regulates a distinct pathway presumably
`due to different subsets of proapoptotic molecules. Tissue-
`specific knockouts for antiapoptotic BCL-2 proteins are
`currently providing insights where the complete knockouts pre-
`vented observation. For example, mcl-1 deletion in the adult
`mouse revealed a requirement
`in hematopoietic stem cell
`
`stretching across the entire length of the BCL-2 core, lined on
`either side by ridges composed of distinct combinations of polar
`and charged amino acids. Most likely, the groove and ridge
`combinations as captured in these structures define antiapop-
`totic protein selectivity for BH3 peptides. Compared to the
`MCL-1 BC groove, which shows a constant width throughout,
`the BCL-xL BC groove is partially constricted due to obstruc-
`tions on either side of the groove along its entire length. Large
`amino acid side chains from a4 (Glu129) and the BH2 region
`(Tyr195) delimit, respectively, the lower and upper ends of the
`compacted BCL-xL BC groove, and the conserved BH1
`Arg139 side-chain projects centrally within the groove. In addi-
`tion, a2 and a3 assemble to delimit the tighter groove from the
`opposite side.
`Upon BH3 peptide binding and regardless of the extent of
`obstruction in the free protein, A1, BCL-xL, and MCL-1 are
`induced to present a rearranged wide-open BC groove. In con-
`trast to the subtle changes in the preformed groove of MCL-1,
`binding of BH3 peptide to BCL-xL induces significant rearrange-
`ment of the BH3-containing ridge, including winding of additional
`turns in a2 and concomitant unwinding in a3, along with major
`side-chain rearrangements on both sides of the BC groove.
`Five main conserved hydrophobic pockets are induced con-
`tiguously along the BC groove to accommodate the hydropho-
`bic residues projected by a snuggly fitting amphipathic helix
`assumed by incoming BH3 peptides (Figure 5A). Additional
`interactions from the ridge residues to solvent-exposed, mainly
`hydrophilic residues of the BH3 peptide may significantly con-
`tribute to high-affinity binding. However, with the exception of
`
`304 Molecular Cell 37, February 12, 2010 ª2010 Elsevier Inc.
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`Molecular Cell
`
`Review
`
`survival, although it is not known if this is due to MCL-1’s antia-
`poptotic effects (Opferman et al., 2005).
`Bak/ and bax/ mice display mild phenotypes compared to
`the combined bak/ bax/ mice. While the bak/ mouse does
`not display any striking developmental or homeostatic defects,
`the bax/ mouse displays T and B cell hyperplasia and repro-
`ductive abnormalities in both sexes. However, it was not until
`the two null alleles were combined that it was suggested that
`the two proteins play redundant roles in apoptosis. The vast
`majority of bak/ bax/ mice are embryonic lethal due to an
`inability to eliminate excess cells during development. The few
`mice that survive to birth (<10%) display a myriad of
`apoptosis-related phenotypes,
`including lymphadenopathy,
`splenomegaly, and interdigital webbing.
`Genetic deletion of the BH3-only proteins revealed tissue-
`and/or stimulus-specific roles in development and tissue
`homeostasis. bid/ mice are resistant
`to anti-CD95/Fas-
`induced liver injury but are otherwise developmentally normal.
`bim/ mice have lymphoid and myeloid cell hyperplasia (and
`observable splenomegaly,
`lymphadenopathy, and systemic
`lupus erythematosus), and lymphoid cells from these mice are
`resistant to cytokine withdrawal. The notion that BH3-only
`proteins function in specific tissues and pathways is also sup-
`ported by the deletion of hrk, which is required for nerve growth
`factor withdrawal-induced apoptosis in sensory neurons.
`However, deletion of other BH3-only proteins, such as PUMA,
`revealed a broader role in regulating MOMP and apoptosis.
`Mice deficient in puma are markedly resistant to hematopoietic
`and gastrointestinal apoptosis following irradiation, and
`puma/ MEFs and lymphocytes are resistant to cytokine with-
`drawal and glucocorticoids. Further analyses indicate a role for
`PUMA in ER stress, ischemia/reperfusion, and bacterial/viral/
`fungal infections.
`The removal of one bim allele rescues the renal phenotype and
`survival of bcl-2/ mice, whereas loss of both bim alleles further
`corrects the hypopigmentation phenotype. These observations
`suggest
`that basal
`levels of BIM are sufficient
`to induce
`apoptosis in the absence of BCL-2. To dissect the mechanisms
`of BIM-mediated apoptosis in vivo, the BIM BH3