Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol
Yi-Ping Ronga,b, Paul Barra, Vivien C. Yeec, Clark W. Distelhorsta,b,⁎
aDepartment of Medicine, Comprehensive Cancer Center and University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH 44106, USA
bDepartment of Pharmacology, Comprehensive Cancer Center and University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH 44106, USA
cDepartment of Biochemistry, Comprehensive Cancer Center and University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH 44106, USA
a b s t r a c ta r t i c l ei n f o
Received 12 September 2008
Received in revised form 28 October 2008
Accepted 29 October 2008
Available online 12 November 2008
Inositol 1,4,5-trisphosphate receptor
Bcl-2 is the founding member of a large family of apoptosis regulating proteins. Bcl-2 is a prime target for
novel therapeutics because it is elevated in many forms of cancer and contributes to cancer progression and
therapy resistance based on its ability to inhibit apoptosis. Bcl-2 interacts with proapoptotic members of the
Bcl-2 family to inhibit apoptosis and small molecules that disrupt this interaction have already entered the
cancer therapy arena. A separate function of Bcl-2 is to inhibit Ca2+signals that promote apoptosis. This
function is mediated through interaction of the Bcl-2 BH4 domain with the inositol 1,4,5-trisphosphate
receptor (IP3R) Ca2+channel. A novel peptide inhibitor of this interaction enhances proapoptotic Ca2+
signals. In preliminary experiments this peptide enhanced ABT-737 induced apoptosis in chronic
lymphocytic leukemia cells. These findings draw attention to the BH4 domain as a potential therapeutic
target. This review summarizes what is currently known about the BH4 domain of Bcl-2, its interaction with
the IP3R and other proteins, and the part it plays in Bcl-2's anti-apoptotic function. In addition, we speculate
on how the BH4 domain of Bcl-2 can be targeted therapeutically not only for diseases associated with
apoptosis resistance, but also for diseases associated with accelerated cell death.
© 2008 Elsevier B.V. All rights reserved.
Bcl-2 is one of the most important apoptosis regulators. It was first
identified through genetic analysis of B cell lymphomas two decades
ago and is a major target of novel therapeutic approaches for cancer
and other diseases [1–3]. In the most common type of human
lymphoma, the region of chromosome 18 encoding Bcl-2 translocates
to chromosome 14, downstream of the antibody heavy chain
enhancer. Thus, in these t(14; 18) positive cells, Bcl-2 expression is
driven by the IgH enhancer [4,5]. Bcl-2 is elevated in many cancers,
including breast cancer, colon cancer, prostate cancer, small cell lung
cancer, chronic lymphocytic leukemia and low-grade lymphomas. The
t(14; 18) translocation is the mechanism only in lymphomas and a
variety mechanisms contribute to Bcl-2 dysregulation and over-
expression in other types of cancer [2,6]. The Bcl-2 protein exerts its
oncogenic effects at least in part by inhibiting apoptosis. Impaired
apoptosis is a crucial step in tumorigenesis, neoplastic progression,
metastasis and chemotherapy resistance.
The Bcl-2 family is generally divided into two categories: pro-
survival Bcl-2 proteins including Bcl-2, Bcl-xL, A1 and Mcl1; and
proapoptotic Bcl-2 proteins including Bax/Bak, Bim, Bad, Bik and
Puma [4,5]. The Bcl-2 family members in these two categories can
interact with each other. For example, Bcl-2/Bcl-xL can interact with
Bim, thereby inhibiting Bim activation induced apoptosis. The balance
between pro-survival and proapoptotic Bcl-2 family proteins is a
major factor in determining whether or not cells undergo apoptosis in
response to cell stress. Disorders of the apoptotic machinerycan result
in either undesirable cell accumulation as in cancer, or a loss of cells as
seen in neurodegenerative, autoimmune and cardiovascular diseases
. Modulation of apoptosis has become a novel therapeutic concept.
As one of the major apoptosis regulators, Bcl-2 has attracted
considerable interest on the part of those involved in developing
innovative therapies for cancer [3,8]. These efforts have mainly
targeted the inhibitory interaction of Bcl-2 with proapoptotic
members of the Bcl-2 protein family, with the goal of disrupting this
interaction and thereby abrogating the antiapoptotic action of Bcl-2.
In additiontoinhibitingapoptosisbyinteracting withproapoptotic
Bcl-2 relatives, Bcl-2 also interacts with other apoptosis regulators
that are not members of the Bcl-2 family. These two different types of
interactions take place on different structural domains of the Bcl-2
protein. Interactions with non-Bcl-2 family members involve the BH4
domain, which is located near the N-terminus and is absent from
proapoptotic Bcl-2 family members. Here we review recent evidence
Biochimica et Biophysica Acta 1793 (2009) 971–978
⁎ Corresponding author. Department of Medicine, Comprehensive Cancer Center and
University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH
E-mail addresses: Yiping.Rong@case.edu (Y.-P. Rong), Clark.Distelhorst@case.edu
0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
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that the BH4 domain of Bcl-2 mediates an interaction between Bcl-2
and a number of factors involved in regulating cell growth and
survival, including the inositol 1, 4, 5-trisphosphate receptor (IP3R)
intracellular Ca2+channel. Ca2+signals play an important role in
mediating apoptosis [9–11]. By interacting with the IP3R Bcl-2
represses Ca2+signals that mediate apoptosis [12,13]. We propose
that interactions involving the BH4 domain of Bcl-2 may also prove to
be a worthwhile target for therapeutic development.
2. Structure of Bcl-2 proteins
Based on sequence alignment Bcl-2 family members share
considerable similarity in regions known as Bcl-2 homology domains
(BH domains) [14,15], shown in Fig. 1A. The antiapoptotic family
members including Bcl-2, Bcl-xL, and Bcl-w have four BH domains
(BH1-BH4), whereas the proapoptotic family members Bax and Bak
have only BH1, BH2 and BH3 domains. A third group that includes for
example Bim, Bad and Bik, only have a BH3 domain and thus are
referred to as BH3-only proteins.
The first X-ray and NMR structure of a Bcl-2 family protein to be
determined was that of Bcl-xL . The three dimensional structure of
Bcl-2 was subsequently determined by NMR spectroscopy of a Bcl-2/
Bcl-xL chimeric protein which contains a truncated version of the
unstructured loop between the BH4 and BH3 domains . The N-
located on the protein surface. This domain forms extensive hydro-
of the BH1, 2 and 3 domains . The C-terminus of Bcl-2 contains a
hydrophobic transmembrane region that inserts into intracellular
membranes, including mitochondria and endoplasmic reticulum (ER),
with the bulk of the Bcl-2 protein oriented on the cytoplasmic face of
these organelles. The BH1 and BH2 domains of Bcl-2 are critical for
heterodimerization with Bax and important for Bcl-2's pro-survival
function . The BH1, BH2 and BH3 domains form a hydrophobic cleft
on both the antiapoptotic Bcl-2/Bcl-xL proteins and proapoptotic
proteins, Baxand Bak. This bindingpocketontheantiapoptotic proteins
Bcl-2 and Bcl-xL can be occupied by the α-helixof interacting BH3-only
proteins, such as Bim and Bad. Through this mechanism Bcl-2/Bcl-xL
sequester the BH3-only proteins, thereby preventing them from
activating full-length proapoptotic proteins including Bax and Bak,
Bcl-xL are neutralized by the BH3-only proteins.
3. The antiapoptotic function of Bcl-2
One of the characteristics of Bcl-2 family proteins is their ability to
form heterodimers or homodimers, which contributes to the neutraliz-
ingcompetitionbetweenantiapoptotic and proapoptotic members.The
detailed mechanisms accountingfor Bcl-2's antiapoptotic action are not
fully understood and consequently there are many hypotheses. As
already noted above, considerable attention has focused on the direct
interactions between various Bcl-2 family members. It is generally held
that through this process, the antiapoptotic proteins Bcl-2 and Bcl-xL
prevent Bax and Bak from forming pores in the outer mitochondrial
membrane that release factors such as cytochrome c [18,19]. These
factors, in turn, activate caspases, proteases that function to dismantle
the cell during apoptosis. Also, as mentioned above, Bcl-2 and Bcl-xL
appear to sequester proapoptotic BH3-only proteins such as Bim and
Bad, preventing them from conveying a death signal. Thus, the major
current working models of apoptosis control are based on Bcl-2 family
members' mutual regulation [20–22]. Many Bcl-2 inhibitors intended
Although the roleof Bcl-2 family members in regulating mitochon-
drial membrane permeability have been generally emphasized in the
apoptosis literature, the roles of Bcl-2 family members on the
endoplasmic reticulum (ER) have received increasing attention in
Fig. 1. (A) BH domains of Bcl-2 family members. Bcl-2 family members share sequence similarity in Bcl-2 homology (BH) domains shown by different colored regions. Prosurvival
Bcl-2 family members, Bcl-2, Bcl-xL and Bcl-w have all four BH domains, whereas proapoptotic Bax subfamily members do nothave a BH4 domain. BH3-only subfamily members lack
all but a BH3 domain. Known α-helical regions are indicated. TM, transmembrane domain. (B) The binding regions of different proteins on the N-terminus of Bcl-2 (BH4 domain).
Y.-P. Rong et al. / Biochimica et Biophysica Acta 1793 (2009) 971–978
recent years, mainly focused on the role of Bcl-2 family members in
regulating Ca2+release from ER. This Ca2+release elevates cytoplas-
mic Ca2+, producing Ca2+signals that governmanycellular processes,
including proliferation, development, cell cycle and apoptosis. The Ca2
+ion is a versatile second messenger that regulates life and death
signals [23,24]. It induces apoptosis either by direct effects on
mitochondria or indirectly by activating or inducing other proapopto-
tic proteins, including Bim, Bad, proteases and endonucleases
[10,11,25]. Bcl-2 represses apoptosis by inhibiting Ca2+release from
ER. Two proposed mechanisms for how Bcl-2 regulates ER Ca2+,
brought out in the past decade, are the inhibition of IP3R channel
opening and the reduction of ER luminal Ca2+concentration
(reviewed in [9,26]).
Recent studies indicate that Bcl-2 and Bcl-xL interact with the
IP3R Ca2+channel on ER membrane and regulate its activity,
thereby inhibiting proapoptotic sustained Ca2+elevation without
interfering with prosurvival Ca2+oscillations [12,13,27–31]. Much
of this work has been performed in T cells, in which T cell receptor
activation (e.g., by antibody to the CD3 component of the T cell
receptor complex) can trigger apoptosis, mediated in part through
IP3-mediated Ca2+elevation. Intriguingly, a Bcl-2 mutant (G145A)
which does not form heterodimers with proapoptotic members of
the Bcl-2 family, such as Bax, still protects T cells from anti-CD3-
induced apoptosis , suggesting that Bcl-2 inhibits this form of
apoptosis by a mechanism other than that involving interactions
between Bcl-2 family members. One candidate mechanism is the
regulation of IP3-mediated Ca2+elevation, based on our evidence
that Bcl-2 inhibits anti-CD3-induced apoptosis by inhibiting IP3R-
mediated Ca2+release from the ER in T cells [12,27]. Thus, Bcl-2
can inhibit apoptosis independent of, or in addition to, its
association with Bax or other proapoptotic Bcl-2 family members
in those situations where IP3-mediated Ca2+elevation contributes
to apoptosis induction.
Although most recent reports emphasize the interaction of Bcl-2
with other family members, or more recently with the IP3R, Bcl-2 has
been reported to also interact with a number of other proteins with
the potential of regulating apoptosis, including protein phosphatase
1α, calcineurin, NF-κB, c-myc, FKBP38, Raf-1, NALP1, and Nur77/TR3.
Thus, a network of protein–protein interactions maycontribute to Bcl-
2's antiapoptotic function in cells.
4. The BH4 domain of Bcl-2/Bcl-xL is crucial for its
The BH4 domain is highly conserved among antiapoptotic proteins
and across species. Thus, there is considerable sequence homology
between the BH4 domains of Bcl-2 and Bcl-xL and swapping the BH4
function . The BH4 region is the only conserved domain among
Bcl-2 family members that is present only in those members with
antiapoptotic activity, including Bcl-2, Bcl-xL and Bcl-w, but absent
from proapoptotic family members (Fig. 1A). This fact alone suggests
that the BH4 domain plays a critical role in determining Bcl-2's
antiapoptotic potential. This concept is confirmed by evidence that
BH4 domain deletion or mutation eliminates the prosurvival activity
of Bcl-2 [34–36] without interfering with the ability of Bcl-2 to bind
BH3-only proteins with an affinity similar to that of full length Bcl-2
. Therefore, the BH4 domain-mediated antiapoptotic function of
Bcl-2 appears to be independent of its ability to dimerize with other
proapoptotic Bcl-2 family members. BH4 deleted Bcl-2 (ΔBH4 Bcl-2)
lacks protective function in multiple forms of apoptosis, including that
induced by IL-3 deprivation, staurosporine, γ-irradiation and dex-
amethasone [33,34]. Also, ΔBH4 Bcl-2 may possibly function as a
dominant negative inhibitor of Bcl-2 . Others have suggested that
ΔBH4 Bcl-2 functions like Bax to promote rather than inhibit cell
5. BH4 domain interacting proteins
The BH4 domain represents a critical region within the Bcl-2
molecule for the prevention of apoptosis through its ability to interact
with some of the following apoptotic regulation proteins.
Calcineurin is reported to interact with the BH4 domain of Bcl-2 in
baby hamster kidney cells, Jurkat cells and SUDHL-4 B-cell lymphoma
cells [38,39]. Amino acids 1–20 at the N terminus of Bcl-2 (Fig. 1B),
located in the BH4 domain, are sufficient and necessary to interact
with calcineurin. Bcl-2 sequesters active calcineurin from NF-AT,
thereby inhibiting calcineurin-dependent NF-AT signaling. Interest-
ingly, the proapoptotic protein Bax interferes with the interaction
between Bcl-2 and calcineurin. It is still not clear if the Bcl-2-
calcineurin interaction disrupts activation induced cell death in Tcells
or contributes to lymphoproliferation.
Calcineurin is reported to be anchored not only to Bcl-2 but also to
the IP3R. The interaction of calcineurin with the IP3R has been
reported and is thought to regulate the phosphorylation status of the
flux . The regulation of IP3R-mediated Ca2+signaling by
calcineurin has been most extensively characterized in the immune
system [41–43]. Also, in cortical and hippocampal slices of the brain
Billingsley and coworkers reported that calcineurin interacts with
both Bcl-2 and the IP3R, proposing that calcineurin shuttles between
these two proteins [44,45]. Thus, it is suggested that this triple protein
complex contributes to cell survival in primary neuronal cells.
However, others have questioned the relevance of calcineurin to
IP3R function . Thus, at present the role of calcineurin in the
function of both Bcl-2 and the IP3R remains uncertain and requires
The IP3R is not the only intracellular channel with which
antiapoptotic Bcl-2 family members interact. The BH4 domain of
Bcl-xL has been reported to interact with the voltage dependent anion
channel (VDAC), which is located on mitochondria andresponsible for
regulating mitochondrial membrane potential [46–48]. The interac-
tion of the BH4 domain with VDAC is required for Bcl-xL to inhibit
etoposide-induced cytochrome c release and preserve VDAC channel
activity and mitochondrial membrane potential . Although the N-
terminus of Bcl-xL (amino acids 2–19aa) coimmunoprecipitates with
VDAC, ΔBH4 Bcl-xL still interacts with VDAC, suggesting that the BH4
domain is not necessary for Bcl-xL–VDAC interaction . BH4
peptide's antiapoptotic function has been attributed to the inhibition
of VDAC channel activity, thereby preventing apoptotic mitochondrial
changes . But knocking down VDAC hasno effecton mitochondrial
permeability transition and cell death, indicating that VDAC is
dispensable for mitochondria-dependent cell death . This has
led some to question the importance of the VDAC channel in
mitochondria-mediated apoptosis, and hence to question whether
the interaction between Bcl-xL and VDAC contributes significantly to
Bcl-xL's antiapoptotic function.
Raf-1, a serine/threonine protein kinase in the MAPK/ERK signal
transduction pathway, plays important roles in cell cycle, apoptosis,
and differentiation [51,52]. It has been reported to interact with the
BH4 domain of Bcl-2 (residues 11–33) (Fig.1B) and in this manner to
regulate apoptosis [53,54], although this interaction may not be stable
in cellular extracts . One theory is that Bcl-2 targets Raf-1 to
mitochondria to block cell death by phosphorylating the BH3-only
Y.-P. Rong et al. / Biochimica et Biophysica Acta 1793 (2009) 971–978
proapoptotic proteinBad. Ras alsointeracts withRaf-1 andtargets it to
the plasma membrane . Thus, it is possible that competition
between Ras and Bcl-2 for binding to limiting amounts of Raf-1
determines Raf-1's localization, substrates and effects on cell survival
or death .
The BH4 domain of Bcl-2 is also reported to interact with activated
Ras and thereby block Ras-mediated apoptotic signaling . Ras is a
small GTPase oncogene involved in many cell processes, including
proliferation, differentiation and apoptosis, mainly through its effects
on signaling pathways regulated by MAP kinase, growth factors, and
Fas [58–60]. Deletion of the BH4 domain from Bcl-2 abrogates the
coimmunoprecipitation of Bcl-2 with Ras and eliminates the anti-
apoptotic effect of Bcl-2 in Fas-induced apoptosis.
Both Bcl-2 and Bcl-xL interact with the non-mammalian proapop-
totic protein CED-4, originally identified in C. elegans, and the BH4
domain appears required for this interaction . CED-4 enhances
CED-3-induced cell death and full length Bcl-xL, but not ΔBH4 Bcl-xL,
antagonizes the apoptotic activity of CED-4. Although Apaf-1 is a CED-
4 mammalian homologue, a predicted interaction between Bcl-2 and
Apaf-1 has not been supported experimentally [61,62].
Paxillin is a focal adhesion-associated adaptor protein, serving as a
docking protein to interact with focal adhesion and cytoskeleton or
signal transduction proteins. It is required in embryonic development
and plays critical roles in cell spreading and motility . Cell adhesion
determines tissue architecture during morphogenesis and inhibits
apoptosis [64–66]. Recent work by Sorenson showed that the BH4
domain of Bcl-2 interacts with paxillin in lysates from embryonic
kidney cells, HEK293 cells and NIH3T3 cells . Amino acids 17–31 in
the BH4 domain of Bcl-2 are necessary for the Bcl-2 interaction with
paxillin (Fig.1B). Tyrosines 21 and 28 in the BH4 domain are especially
critical for this interaction. A BH4 domain peptide is also sufficient to
interact with paxillin and disrupt nephrogenesis. Although how Bcl-2
regulates apoptosis by interactingwithpaxillin is still not understood,it
has been proposed that Bcl-2 protects cells from apoptosis caused by
loss of adhesion [67,68]. The focal adhesion kinase and paxillin complex
is thought to regulate cell adhesion and migration in an integrin-
mediated signaling pathway . Apoptosis controls inappropriate cell
positioning during three dimensional morphogenesis . Bcl-2 may
bypass integrin-mediated survival signals via interactions with the
paxillin/focal adhesion kinase complex, circumventing the need for
adhesion and thereby modulating cell adhesion and migration .
Nuclear factor κB (NF-κB), a transcription factor, plays an
important antiapoptotic function in mammalian cells [70,71]. NF-
κB activation is required for Bcl-2's antiapoptotic function in
ventricular myocytes . Also, the presence of Bcl-2-NF-κB
complexes has been confirmed in nuclear fractions of NIH3T3 cells
and it is thought that this interaction contributes to Bcl-2's roles in
cell cycle control and apoptosis . Full length Bcl-2 has been
shown to enhance NF-κB's DNA binding activity, but this activity is
lost when the BH4 domain is deleted from Bcl-2. Also, both the level
and activity of the NF-κB inhibitor IκBα were suppressed by Bcl-2
but not by ΔBH4 Bcl-2.
Recently we found that Bcl-2 interacts with all three subtypes of
IP3R, documented by multiple experimental approaches, including
coimmunoprecipitation, Blue Native Gel Electrophoresis, GST pull-
down and Fluorescence Resonance Energy Transfer [13,27]. The
interaction of Bcl-2 and Bcl-xL with the IP3R has been confirmed by
a number of laboratories [27–31]. Although Bcl-2 is widely known to
localize to mitochondria, it is also well documented on the ER where it
interacts with the IP3R, an IP3 sensitive intracellular Ca2+channel.
The IP3R transmits Ca2+from the ER lumen to the cytoplasm,
elevating cytoplasmic Ca2+concentration and thereby generating Ca2
+signals that mediate a wide range of cellular processes, including
apoptosis. Through its interaction with IP3R's, Bcl-2 inhibits IP3-
dependent opening of IP3R channels reconstituted in planar lipid
bilayers and also inhibits IP3-dependent Ca2+elevation induced by T
cell receptor (TCR) activation or by a cell permeant IP3 ester.
We recently mapped the Bcl-2 interacting site to an eighty amino
acid sequence within the regulatory and coupling domain of the IP3R,
and based on a twenty amino acid sequence within this region
developed an inhibitory peptide, referred to as peptide 2, that disrupts
the Bcl-2–IP3R interaction  (Fig. 2). By using peptide 2 to abrogate
the Bcl-2–IP3R interaction, we established that this interaction is
indeed necessary for Bcl-2's inhibitory effect on IP3-mediated Ca2+
elevation and apoptosis in lymphocytes following TCR activation.
Thus, the regulatory effect of Bcl-2 on IP3-induced Ca2+elevation
contributes to Bcl-2's antiapoptotic action by a mechanism different
from the well-known inhibitory effect of Bcl-2 on proapoptotic
members of the Bcl-2 protein family.
In recent unpublished studies, we found that the BH4 domain of
Bcl-2 is necessary and sufficient to interact with the IP3R. Moreover, a
peptide corresponding to the BH4 domain, when coupled with HIV-
TAT to facilitate its entry into cells, inhibits IP3-dependent Ca2+
elevation and apoptosis in lymphocytes following TCR activation
(unpublished data). These findings further indicate that Bcl-2's
inhibitory effect on IP3R-mediated Ca2+elevation and apoptosis is
independent of other BH domains or binding with other Bcl-2 family
In summary, the BH4 domain of Bcl-2 interacts with a number of
different factors and thereby plays an important role in apoptosis
inhibition. Although our laboratory has focused mainly on the interac-
tions of Bcl-2 and Bcl-xL with the IP3R, the other interactions discussed
Bcl-2 to regulate a wide range of signaling pathways, perhaps including
pathways not directly related to apoptosis.
Fig. 2. Bcl-2 interacts with the regulatoryand coupling domain of the IP3R. The locations of various structural and functional domains in the IP3R [96–98] are shown here, in addition
to the site within the regulatory and coupling domain of the IP3R where Bcl-2 interacts. The sequence of peptide 2, which inhibits the interaction of Bcl-2 with the IP3R, was derived
from the sequence of the Bcl-2 binding site on the IP3R .
Y.-P. Rong et al. / Biochimica et Biophysica Acta 1793 (2009) 971–978
6. Bcl-2 inhibitors
Many diseases can be attributed directly or indirectly to altered
apoptosis regulation. Targeting apoptosis has become an attractive
therapeutic strategy in the treatment of these disorders, especially
cancer which is often associated with resistance to apoptosis, often
due to elevated levels of Bcl-2 or other antiapoptotic Bcl-2 family
members. A number of proteins and small molecules designed to
trigger cell death have entered the clinic for use against cancer. Due to
Bcl-2's antiapoptotic and oncogenic function in cancer cells, Bcl-2
draws a lot of attention as one of the major targets in apoptosis
Many small molecule Bcl-2 inhibitors are in the pipeline.
Genasence (Genta), an antisense oligonucleotide targeted against
Bcl-2, can reduce Bcl-2's expression level [74,75]. It has entered Phase
III clinical trials for chronic lymphocytic leukemia (CLL) and meta-
static melanoma [76,77]. ABT-737 and ABT-263 by Abbott Pharma-
ceuticals , Obatoclax by Gemin X , and AT-101 by Ascenta 
are all BH3 only protein mimetics that bind to the hydrophobic groove
of Bcl-2/Bcl-xL and displace proapoptotic proteins from Bcl-2's
inhibitory grip. A small molecule Bcl-2 inhibitor by Infinity Pharma-
ceuticals also interrupts the interactions between Bcl-2 and its
proapoptotic binding partners. Some other natural and chemical
inhibitors of Bcl-2, including HA14-1 , BH3I-I , chelerythrine
, and gossypol  appear mainly to regulate the Bcl-2 interaction
with other Bcl-2 family members. As described before, the site
targeted by the novel therapeutics is the hydrophobic groove formed
by the BH1, BH2 and BH3 domains. Surprisingly, none of them target
the BH4 domain of Bcl-2 to antagonize Bcl-2's antiapoptotic function,
even thoughtheBH4 domaincontributes totheantiapoptoticfunction
of Bcl-2 as summarized as above.
The concept that the Bcl-2 BH4 domain may be a worthy target
arises in part from our studies of the Bcl-2–IP3R interaction, which is
mediated through the BH4 domain. As discussed earlier, a 20 amino
acid peptide (peptide 2), mimicking the Bcl-2 binding site sequence
on the IP3R, abrogates the Bcl-2–IP3R interaction, thereby reversing
Peptide 2 is derived from the sequence of IP3R that binds to the BH4
domain of Bcl-2. Peptide 2 does not interfere with the interaction of
Bcl-2 with Bim . Thus, peptide 2 targets the BH4 domain of Bcl-2
instead of the hydrophobic groove formed by BH1, 2 and 3 domains.
Peptide 2's proapoptotic effect makes it of potential value in cancer
As an initial test of this hypothesis, we investigated the effect of
peptide 2 in chronic lymphocytic leukemia (CLL), a common human
malignancy associated with elevated levels of Bcl-2. CLL cells are
“addicted toBcl-2”becausetheyhave elevated levelsof both BH3-only
proteins andBcl-2 . Consequently, CLL cells undergo apoptosis if the
ability of Bcl-2 to bind and inhibit BH3-only proteins is abrogated by
treatment with the BH3-mimetic ABT737 . Although peptide 2 by
itself was not toxic to primary CLL cells, peptide 2 significantly
enhanced ABT-737 induced apoptosis (Fig. 3). It would appear that a
combinatory inhibitory effect is achieved by simultaneously targeting
two different sites on Bcl-2. Although this preliminary study involved
CLL cells from only six patients, the results are promising enough to
cast light on the potential importance of the BH4 domain as a
therapeutic target. Also, it is not possible at this point to be sure
whether the mechanism of action of peptide 2 in this context is the
disruption of Bcl-2's interaction with the IP3R or one of the other BH4
domain interacting partners described above.
7. BH4 peptide has antiapoptotic function
Interestingly, although the BH4 domain has not previously been
targeted to reverse Bcl-2's antiapoptotic action, the BH4 domain
peptide is used to mimic Bcl-2 as an antiapoptotic reagent in many
apoptosis models. Shimizu et al used a TAT-BH4 peptide to facilitate
BH4 peptide entry into cells and found that the TAT-BH4 peptide
prevents Ca2+-induced loss of mitochondrial membrane potential and
cytochrome c release . Moreover, the TAT-BH4 peptide inhibited
X-ray- and VP-16 (Etoposide)-induced apoptosis, but not tunicamy-
cin-induced apoptosis in PC-12 or Hela cells. Injection of TAT-BH4
peptide into the peritoneum of C57BL6J mice conferred protection
againstX-ray irradiation-induced cell death in the small intestine .
Also, TAT-BH4 peptide suppressed anti-Fas induced fulminant
hepatitis  and improved ischemia–reperfusion-induced cardiac
dysfunction, therefore attenuating ischemia–reperfusion injury in the
rat heart . Other groups found that BH4 peptide inhibits oxidative
stress induced coronary endothelial cell apoptosis , human islet
cell apoptosis, staurosporine and serum deprivation-induced apopto-
sis [88,89], radiation-induced apoptosis in human T lymphocytes and
B cells , beta-amyloid peptide-induced apoptosis, and sepsis-
induced apoptosis . In addition, BH4 peptide's convenient delivery
into cells and antiapoptotic effect endow it protective roles in vivo in
spinal cord injury , islet transplantation , neurotoxicity and
hippocampal damage .
Recently, IP3R mediated Ca2+release has been implicated as
contributing to the pathogenesis of Alzheimer's disease . Inter-
estingly, TAT-BH4 peptide reduces the toxic effect of beta-amyloid
peptide on capillary endothelium . The mechanism of the
protective action of TAT-BH4 in Alzheimer's disease is not fully
elucidated. We have found TAT-BH4 peptide, like full length Bcl-2,
inhibits IP3R-mediated Ca2+release from the ER and apoptosis in T
cells (unpublished data). This raises the possibility that a similar
mechanism might be relevant in Alzheimer's disease.
In summary, there is considerable interest in development of
antiapoptotic therapies for diseases associated with accelerated cell
death, based on theBH4 domainofBcl-2 or Bcl-xL, eventhoughthe role
8. BH4 domain structure
In view of BH4 domain's importance as a potential target to
regulate Ca2+signaling and apoptosis, the concept of blocking BH4
domain interactions with other proteins involved in regulating or
Fig. 3. Peptide 2 enhances ABT-737-induced apoptosis in CLL cells. Lymphocytes were
freshly separated from heparinized peripheral blood obtained from adult patients with
chronic lymphocytic leukemia meeting standard diagnostic guidelines. We conformed
to all guidelines and regulations in accordance with Internal Review Board protocols
ICC2902/11-02-28 (Case Western Reserve University Cancer Center/University Hospi-
tals of Cleveland Ireland Cancer Center). Cells were separated bycentrifugation through
Ficoll–Hypaque and cultured in RPMI medium (10% fetal bovine serum) at a density of
2.0×106cells/ml. CLL cells were treated in vitro with 2 μM ABT-737 in the presence or
absence of 5 μM TAT-peptide 2 (pep2) or TAT-control peptide (ctrlpep). Cell death was
detected by trypan blue staining after 24 h treatment. Six individual experiments were
analyzed by the two-tailed Student's t test, using a significance level of pb0.05.
Y.-P. Rong et al. / Biochimica et Biophysica Acta 1793 (2009) 971–978
mediating apoptosis (e.g., IP3R, calcineurin, paxillin) by small
molecules or peptides, as illustrated by peptide 2, provides another
opportunity to design Bcl-2 inhibitors. Not only does the BH4
domain's role in apoptosis regulation provide an opportunity to
develop therapeutics designed to enhance apoptosis by abrogating
Bcl-2's antiapoptotic function, but the ability of the BH4 peptide by
itself to inhibit apoptosis encourages efforts to identify or engineer
molecules that mimic the BH4 domain and thus have antiapoptotic
function similar to that of full length Bcl-2/Bcl-xL. These would likely
prove to be useful in diseases associated with accelerated cell death.
Based on what is known about the structure and location of the BH4
domain in Bcl-2 these goals should be feasible.
The BH4 domain is an α-helical region located at the N-terminus
of Bcl-2. Based on conservation and surface accessibility, the Bcl-2
BH4 domain residues predicted to be most like involved in binding
other proteins are D10 and R12 (on one face) and H20, Y21, Q25, R26,
and Y28 (on the opposite face) (Fig. 4). These residues are conserved
in BH4 domains of seven of the closest mammalian Bcl-2 family
member sequences and have highly accessible exposed side chains
available for specific binding to other molecules. A positive and a
negative residue are located on one face, and positively charged
residues on the opposite face which may contribute to ionic or
hydrogen bond interactions. The residues R12, I14, V15, Y18, I19 and
L23 have been reported indispensable for the anti-apoptotic activity
of Bcl-2 [33,36].
Notably, the hydrophobic groove formed by the BH1-3 domains,
responsible for interaction with BH3-only proteins and targeted
therapeutically by BH3-mimetics, is separate from the BH4 domain
in the three dimensional structure of Bcl-2. Thus, it will be interesting
to explore targeting the predicted binding sites of the BH4 domain to
develop therapeutics that act by a different mechanism to inhibit Bcl-
2's antiapoptotic activity.
The BH4 domain is present only in antiapoptotic members of the
Bcl-2 protein family and thus is a major distinguishing feature that
separates the antiapoptotic and proapoptotic family members at a
molecular level. Although this is the case, exploring the BH4 domain
for its therapeutic potential has lagged far behind the concentrated
efforts focused on the interaction of antiapoptotic family members
with proapoptotic family members mediated through the BH1, BH2
and BH3 domains. The BH4 domain is not only a promising target for
small molecule therapeutics intended to reverse the antiapoptotic
functions of Bcl-2 and Bcl-xL, but also a useful model for therapeutics
intended to inhibit apoptosis based on considerable evidence that a
BH4 peptide itself inhibits proapoptotic Ca2+signals and apoptosis in
a variety of settings.
The BH4 domain-mediated interactions with IP3R and other
apoptotic regulators is another dimension of Bcl-2's function that is
not sufficiently understood. The development of peptide inhibitors or
small molecule peptide mimetics directed at the BH4 domain of Bcl-2
will be one way to further elucidate the function of the BH4 domain.
This is illustrated by our recent development of peptide 2 based on
detailed analysis of the interaction of Bcl-2 with the IP3R. The use of
peptide 2 has already established the importance of the Bcl-2–IP3R
interaction in regulating Ca2+signals and apoptosis, and even
suggested potential therapeutic utility of targeting the BH4 domain
for treatment of diseases associated with elevated Bcl-2. Perhaps in
the future BH4 domain-based Bcl-2 targeting strategies combined
with BH3 mimetic strategies will inhibit Bcl-2's function more
efficiently than either alone in cancer therapy. Alternatively, perhaps
therapeutics based on the intrinsic antiapoptotic activity of the BH4
domain will be useful for treatment of diseases associated with
accelerated cell death.
We thank Stephen Tahir, Abbott Laboratories, for providing ABT-
737. This work was supported by funding from NIH grants CA085804
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