BOD (Bcl-2-Related Ovarian Death Gene) Is an Ovarian BH3 Domain-Containing Proapoptotic Bcl-2 Protein Capable of Dimerization with Diverse Antiapoptotic Bcl-2 Members
Sheau Yu Hsu,
Patty Lin and
Aaron J. W. Hsueh
Division of Reproductive Biology Department of Gynecology and
Obstetrics Stanford University School of Medicine Stanford,
California 94305-5317
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ABSTRACT
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Using the yeast two-hybrid protein-protein
interaction system to search for genes capable of forming dimers with
the antiapoptotic protein Mcl-1, we have isolated BOD (Bcl-2-related
ovarian death agonist) from an ovarian fusion cDNA library.
The three variants of BOD (long, medium, and short) have an open
reading frame of 196, 110, and 93 amino acids, respectively; all of
them contain a consensus Bcl-2 homology 3 (BH3) domain but lack other
BH domains found in channel-forming Bcl-2 family proteins. In the yeast
cell assay, BOD interacts with diverse antiapoptotic Bcl-2 proteins
[Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and Epstein-Barr virus
(EBV) BHRF-1] but not with different proapoptotic Bcl-2 proteins (BAD,
Bak, Bok, and Bax). After overexpression in mammalian Chinese hamster
ovary (CHO) cells, BOD induces apoptosis that can be prevented by the
baculoviral caspase inhibitor P35. The cell-killing activity of BOD is
also antagonized in cells cotransfected with the antiapoptotic Bcl-w
protein, which showed high affinity for BOD in the two-hybrid assay.
Furthermore, mutagenesis studies showed that BOD mutants with
alterations in the BH3 domain lose cell-killing ability, suggesting
that the BH3 domain is important for the mediation of cell killing by
BOD. BOD mRNA is ubiquitously expressed in ovary and multiple other
tissues. The BOD gene is also conserved in diverse mammalian species.
Identification of BOD expands the group of proapoptotic Bcl-2 proteins
that only contains the BH3 domain and allows future elucidation of the
intracellular mechanism for apoptosis regulation in ovary and other
tissues.
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INTRODUCTION
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In multicellular organisms, apoptosis ensures the elimination of
superfluous cells including those that are generated in excess, have
already completed their specific functions, or are harmful to the whole
organism (1). During reproductive life, 99% of the ovarian follicles
endowed during early life undergo apoptosis, and the process is
regulated by diverse hormones (2); thus, the ovary serves as a valuable
model for studying the regulation of cell death by diverse
extracellular and intracellular signaling mechanisms.
A growing body of evidence suggests that the intracellular death
program activated during apoptosis is similar in different cell types
and conserved during evolution (1, 3, 4). The protooncogene Bcl-2 was
isolated at the breakpoint of the t(14, 18) chromosomal translocation
associated with follicular B cell lymphoma (5, 6). Overexpression of
the Bcl-2 protein suppresses apoptosis induced by a variety of agents
both in vitro and in vivo (7). Subsequent studies
identified a number of Bcl-2-related proteins possessing several
conserved BH (Bcl-2 homology) domains important for homo- or
heterodimerization between family members (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In addition, a
C-terminal membrane-anchoring region important for subcellular
localization is found in some members. Based on their differential
ability to regulate apoptosis, the Bcl-2-related proteins can be
separated into anti- and proapoptotic members, and the balance between
these counteracting proteins presumably determines the cell fate (1, 8, 9). Based on studies in mice with deletion of different Bcl-2-related
proteins (20, 21, 22) and studies on differential interactions among
multiple Bcl-2-related proteins (18, 23), it is becoming clear that the
balance of cell survival or apoptosis is maintained by different
combinations of Bcl-2 family proteins in a tissue-, dimerization-, and
circumstance-specific manner. In addition, Bcl-2 family proteins,
represented by Bcl-xL, could regulate the activities of downstream
apoptotic effectors, capases, by forming a functional complex with
Apaf-1 and caspase 9 (24). Furthermore, Bcl-2 proteins containing the
BH1, BH2, and BH3 domains have been shown to form ion channels and
regulate osmotic changes in mitochondria and other subcellular
compartments, leading to the release of cytochrome c, an important
cofactor for caspase activation (25, 26, 27, 28, 29). In contrast to the
membrane-bound Bcl-2 proteins, several soluble Bcl-2 proteins such as
BAD (Bcl-xL/Bcl-2-associated death promoter) and BID (BH3 interacting
domain death agonist), containing only the BH3 domain, are
likely to function as adaptor proteins linking the membrane-bound
family proteins and cytoplasmic signaling molecules (13, 15, 30, 31).
In the ovary, overexpression of Bcl-2 in transgenic mice led to the
suppression of follicle cell apoptosis and subsequent formation of
teratoma of germ cell origin (32), whereas deletion of the proapoptotic
Bax gene resulted in the accumulation of apoptotic follicular cells
(21). These data suggest that the Bcl-2 family proteins have important
roles in the regulation of follicular atresia. In preliminary studies,
we found that an antiapoptotic Bcl-2 family protein Mcl-1, but not
Bcl-2 itself, was highly expressed in ovarian follicles, suggesting
that Mcl-1 could regulate ovarian follicle atresia. Using Mcl-1 as bait
to screen an ovarian fusion cDNA library in the yeast two-hybrid
system, we isolated Bok (Bcl-2-related ovarian killer), a new
proapoptotic Bcl-2 family member expressed mainly in the ovary, uterus,
and testis (18). In the present study, we report the isolation of
another proapoptotic protein, Bcl-2-related ovarian death agonist
(BOD), using the Mcl-1 bait in the yeast two-hybrid screen. BOD encodes
a protein containing a consensus BH3 domain known to be important for
the heterodimerization of Bcl-2 proteins and the cell-killing activity
of proapoptotic Bcl-2 members. Unlike Bok, BOD shows a wide
heterodimerization property by binding to diverse anti- but not
proapoptotic Bcl-2 proteins. In addition, BOD is expressed in a variety
of tissues and could play regulatory roles on cell death in diverse
cell lineages. Future characterization of the role of BOD in apoptosis
could provide new understandings on intracellular mechanisms of cell
death regulation in the ovary and other tissues.
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RESULTS
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Screening for Mcl-1-Interacting Proteins and the Isolation of
Full-Length BOD
Using the antiapoptotic protein Mcl-1 as bait, we screened an
ovarian fusion cDNA library (33) and isolated two candidate clones
encoding overlapping fragments of a 60-amino-acid open reading frame
(ORF) with a region showing high homology to the BH3 domain and
flanking sequences of BAD (13). Based on these clones, specific primers
downstream of the putative stop codon were designed to construct a
sublibrary enriched with cDNAs for the candidate gene. Subsequent
colony screening of this sublibrary allowed the isolation of
full-length ORF of three BOD-splicing variants. These clones encoded
polypeptides of 192, 110, and 93 amino acids in length and were named
as BOD-L (long), BOD-M (medium), and BOD-S (short), respectively (Fig. 1A
). GenBank accession numbers for BOD-L,
BOD-M, and BOD-S are AF065433, AF065432, and AF065431,
respectively.

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Figure 1. Characteristics of Three Rat BOD Splicing Variants
and Comparison of BH3 Domains in Different Bcl-2 Proteins
A, Amino acid sequences of three different rat BOD variants (BOD-L,
BOD-M, and BOD-S). The longest ORF of BOD predicts a protein of 196
amino acids in length, whereas BOD-M and BOD-S encode proteins of 110
and 93 amino acids, respectively. The methionine residues in the start
site are circled, whereas potential phosphorylation
sites are indicated by asterisks. B, Comparison of BH3
domains and flanking sequences of BOD, Bcl-xL, and BAD. Shaded
residues are identical in at least two of the three BH3 domain
sequences compared. r, Rat, h, human.
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The longest ORF of BOD (BOD-L) encoded a novel protein with a predicted
molecular mass of 22.3 kDa and an isoelectric point (pI) of 6.4.
Hydrophobicity analysis indicated that the C terminus of BOD variants
contained a stretch of hydrophobic residues flanked by charged
residues, suggesting the possible existence of a transmembrane domain
(Fig. 1A
, lightly hatched residues). However, the
length of this hydrophobic region is short (15 amino acids), suggesting
the putative transmembrane domain in BOD is atypical. The N-terminal
region of BOD-L is rich in proline and serine residues, and multiple
potential phosphorylation sites were found along the entire length of
BOD-L (Fig. 1A
). Nucleotide sequence analysis further suggested that
BOD-M is a splicing variant of BOD-L, whereas BOD-S could derive from
the usage of an alternative transcription starting site because its
translation start site is preceded by nucleotide sequences different
from that of BOD-L and BOD-M (data deposited in the GenBank).
Comparison of DNA sequences with known genes in the GenBank using the
BLAST server indicated that BOD is a novel member of the Bcl-2 family
of proteins showing only the conserved BH3 domains but lacking the BH1,
BH2, and BH4 domains found in channel-forming Bcl-2 proteins. The core
sequence of the BH3 domain found in BOD (LRRIGDE) is the same as that
of rat and mouse Bax, but the flanking sequences are different.
Comparison of the BH3 domain and flanking sequences in BOD, Bcl-xL, and
BAD (Fig. 1B
) indicated that, in addition to the core sequence
(LRRIGDE), the flanking regions are also partially conserved.
During the preparation of our manuscript, the mouse Bim gene was
isolated in an expression screen for proteins capable of binding Bcl-2
from a lymphoma cell line (19). Based on sequence similarity, the
present rat BOD gene is likely the ortholog of mouse Bim. However, the
shortest splicing variant of rat BOD (BOD-S) is shorter than any of the
reported Bim variants.
BOD Heterodimerized with Different Antiapoptotic Bcl-2 Proteins
Using the yeast two-hybrid system, interactions between BOD and
different anti- and proapoptotic Bcl-2 proteins were studied. As shown
in Fig. 2
, BOD-L, BOD-M, and BOD-S
interacted strongly with diverse antiapoptotic proteins including
Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and the Epstein-Barr viral-derived
BHRF-1. In contrast, no interaction was observed between different BOD
variants and several proapoptotic Bcl-2 proteins (BAD, Bak, Bok, and
Bax). Because our original screening indicated that the C-terminal 60
amino acids of BOD are sufficient for interaction with Mcl-1 in the
yeast two-hybrid system, a truncated construct containing only the
C-terminal 70 amino acids of BOD was also tested for interaction with
different Bcl-2 family proteins. As expected, this extra short
construct (named BOD-ES) showed strong interactions with all
antiapoptotic proteins tested (Fig. 2
), suggesting that the C-terminal
BH3 domain-containing region is the functional motif for BOD to
interact with other Bcl-2 proteins. To demonstrate that the lack of
interactions between BOD and proapoptotic Bcl-2 proteins was not due to
the killing of yeast cells by these death agonists, we also tested the
growth of yeast cells cotransformed with different proapoptotic
proteins and Bcl-xL or Mcl-1. Although all the proapoptotic members
tested showed negligible interaction with BOD, they interacted strongly
with Bcl-xL or Mcl-1 (data not shown).

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Figure 2. BOD Binds to Diverse Antiapoptotic but not
Proapoptotic Bcl-2 Proteins in the Yeast Two-Hybrid System
Upper panel, Yeast cells were grown in the selective
media containing 5 mM 3-aminotriazole and without Trp, Leu,
and His. Prominent growth could be seen in yeast colonies coexpressing
BOD-L, BOD-M, BOD-S, or BOD-ES fused to the GAL4-binding domain
together with Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, or EBV BHRF1 fused to
the GAL4 activation domain. Minimal growth of yeast colonies was found
in cells that express the same BOD expressing vectors together with
BAD, Bak, Bok, or Bax fused to the GAL4 activation domain. Lower
panel, Growth of yeast colonies transformed with the same
vector pairs maintained in a nonselective media.
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Induction of Apoptosis after Overexpression of BOD in CHO Cells:
Blockage by Baculovirus Apoptosis Inhibitor P35 and the Antiapoptotic
Bcl-w Protein
To investigate the role of BOD on apoptosis, a
ß-galactosidase cotransfection assay was used to examine BOD
activity (18). CHO cells were transiently transfected with various
expression vectors together with a 1/20 fraction of the pCMV-ß-gal
plasmid. After 24 h, cells were stained with X-gal to identify
transfected blue cells for the estimation of surviving cells. As shown
in Fig. 3A
, transfection with expression
plasmids encoding different BOD-splicing variants (BOD-L, BOD-M, and
BOD-S) and BOD-ES using the ß-galactosidase cotransfection assay
resulted in a loss of greater than 98% of viable cells as compared
with the control group transfected with an empty vector. In contrast,
cells transfected with the plasmid with different BOD variant cDNAs in
reverse orientation remained viable (Fig. 3A
).

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Figure 3. Induction of Apoptosis after Overexpression of BOD
in CHO Cells: Suppression of BOD Action by the Caspase Inhibitor P35
and the Antiapoptotic Bcl-w Protein
A, Quantitative analysis of cell killing by BOD. CHO cells were
transiently transfected with pcDNA3 expression vectors containing cDNA
for BOD-L, -M, -S, or -ES (2.1 µg DNA/35-mm dish) or the same cDNAs
in reverse orientation. The pCMV-ß-gal expression vector (0.1
µg/dish) was included to monitor transfected cells. The number of
ß-gal-expressing cells (mean ± SEM, n = 3) was
determined at 24 h after transfection. Data from cells transfected
with BOD variants are presented as percentage of viable cells as
compared with the control group. Similar results were obtained in four
separate experiments. B, Blockage of apoptosis induced by BOD-L or -ES
after cotransfection with the baculoviral caspase inhibitor P35 or the
antiapoptotic protein Bcl-w. Cells were cotransfected with BOD-L or -ES
with or without an expression plasmid encoding P35 or Bcl-w using
procedures as described for Fig. 3A . The number of ß-gal-expressing
cells was determined at 24 h after transfection. Cells were
transfected with a total of 2.12 µg plasmid DNA including 2.02 µg
of pcDNA3 expression constructs and 0.1 µg of the pCMV-ß-gal
reporter. In groups receiving two different pcDNA3 expression plasmids,
0.02 µg of the BOD expression vector and 2 µg of the P35 or Bcl-w
expression vector were used.
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To demonstrate that the observed apoptosis was mediated by
caspase family of proteases, cells were cotransfected with plasmids
encoding BOD with or without the baculovirus-derived serpin inhibitor
P35 (34, 35). As shown in Fig. 3B
, induction of apoptosis by BOD-L and
BOD-ES was reduced after cotransfection with P35 (P <
0.01), suggesting the involvement of a caspase-mediated proteolysis
cascade. In contrast, transfection with the P35 expression vector alone
did not affect cell survival. To test the ability of antiapoptotic
Bcl-2 proteins to modulate BOD-induced apoptosis, CHO cells were
cotransfected with vectors encoding BOD and Bcl-w (36), a Bcl-2 protein
showing high affinity for different BOD variants in the yeast
two-hybrid assay. As shown in Fig. 3B
, the ability of BOD-L or BOD-ES
to induce apoptosis was reduced after cotransfection with the
expression vector encoding Bcl-w (P < 0.01).
To further study the role of the putative BH3 region of BOD in its
cell- killing ability, we mutated the BH3 region in the shortest
splicing variant (BOD-S) that is still capable of inducing apoptosis.
As shown in Fig. 4
, mutations of the core
BH3 sequence in BOD-S from LRRIGDE to AAAAADE (BOD-S 5A) completely
abolished its proapoptotic activity in transfected CHO cells.
Furthermore, we generated the same mutations in the truncated BOD-ES
with only 70 amino acids in the C-terminal sequence of BOD (BOD-ES 5A)
and found that this mutant also lost its proapoptotic activity.

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Figure 4. Mutations in the BH3 Domain of BOD Abolish Its
Cell-Killing Capability
Quantitative analysis of cell-killing activity of wild-type and mutant
BOD. Schematic representation of BH3 domain sequences in wild-type and
mutant BOD are shown in the top panel. CHO cells were
transiently transfected with pcDNA3 expression vectors containing cDNA
for wild-type BOD-S or a BH3 domain mutant (BOD-S 5A; 2.1 µg DNA/35
mm dish). Proapoptotic activity of these constructs was determined as
described in the Fig. 3 legend. The cell-killing activity of BOD-ES and
its 5A mutant with the same BH3 domain alteration was also investigated
for comparison.
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Expression of BOD mRNA in Human and Rat Tissues
Northern blot analysis revealed that the BOD mRNAs are widely
expressed in different human and rat tissues (Fig. 5
). One main transcript with a size of
approximately 5.5 kb was found in diverse tissues of both species. In
addition to the major transcript, a less prominent transcript of 3.0 kb
was detected in the rat spleen and human leukocyte, whereas a band of
1.3 kb was also detected in the testis of human and rat. These
different mRNA species could result from the use of alternative
polyadenylation sites and/or the alternative splicing of the BOD
gene.

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Figure 5. Expression of BOD mRNA Transcripts in Diverse Human
and Rat Tissues
For Northern blot analysis, poly (A)+-selected RNA from
different tissues of human (left) and rat
(right) (Tissue blots; CLONTECH) were hybridized with a
32P-labeled BOD cDNA probe. After washing, the blots were
exposed to x-ray films at -70 C for 5 days. Specific BOD transcripts
are indicated by arrows. Subsequent hybridization with a
ß-actin cDNA probe was performed to estimate nucleic acid loading (8
h exposure; lower panels). Sp, Spleen; Th, thymus; Pr,
prostate; Te, testis; Ov, ovary; In, intestine; Co, colon; Le,
leukocyte; He, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu,
skeletal muscle; Ki, kidney.
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Conservation of BOD among Different Mammalian Species
Conservation of the BOD gene among diverse vertebrates was
investigated by using Southern blot hybridization of genomic DNA from
different species. The rat cDNA probe hybridized strongly with specific
genomic DNA bands from all mammalian species studied, but not with DNA
from chicken (Fig. 6
). These data suggest
that the BOD gene is well conserved in mammals during evolution.

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Figure 6. Conservation of BOD in Diverse Vertebrate Species
Southern blot analysis of genomic DNA from different vertebrate species
was performed. Genomic DNA was digested with the EcoRI
enzyme and probed with a rat BOD cDNA probe. After hybridization at 60
C using ExpressHyb hybridization solution (CLONTECH), the membrane was
washed under medium stringency conditions (0.5% SDS, 0.2 x SSC
at 55 C) before exposure.
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DISCUSSION
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We have identified BOD as an ovarian Bcl-2-related, BH3
domain-only protein capable of inducing apoptosis presumably after
heterodimerization with antiapoptotic Bcl-2 proteins. The BOD gene has
several splicing variants and is expressed in diverse tissues based on
its mRNA expression pattern. In addition to Mcl-1, which was used as
the bait to isolate BOD, different BOD isoforms interact with diverse
antiapoptotic Bcl-2 proteins in the yeast two-hybrid assay. Similar to
another BH3-only proapoptotic protein BAD, but distinct from the BID,
BOD does not interact with proapoptotic Bcl-2 proteins. Together with
Bok, BAD, and Mcl-1, BOD belongs to a subgroup of Bcl-2 proteins
expressed in the ovary and is likely to be important in the regulation
of ovarian follicle atresia.
Recent studies suggested that the region spanning BH1 and BH2 domains
of Bcl-2 proteins is important for pore formation in the artificial
membrane and could function as ion channels in the mitochondria as well
as other subcellular membrane organelles (3). Furthermore, the
amphipathic BH3 domain in proapoptotic Bcl-2 proteins might regulate
apoptosis by binding to a hydrophobic cleft formed by the conserved
BH1, BH2, and BH3 domains found in the antiapoptotic Bcl-2 proteins,
represented by Bcl-2 and Bcl-xL (37, 38). Mutations in the BH3 domain
of several proapoptotic proteins abolished their heterodimerization
with antiapoptotic partners and dampened their cell-killing activity
(37, 39, 40, 41, 42, 43). In addition, polypeptides containing minimal BH3 domain
sequences of Bax and Bak are capable of binding to antiapoptotic
proteins (39, 43) and inducing apoptosis in transfected cells or
cell-free systems (44). The presence of the conserved BH3 domain in all
the BOD variants and the observed loss of cell killing in BOD mutants
with alterations in the BH3 domain underscore the importance of this
region for apoptosis induction.
It is becoming clear that the proapoptotic Bcl-2 proteins can be
divided into two subgroups: one with BH1, BH2, and BH3 domains and one
with the BH3 domain only. Although both subgroups could dimerize with
antiapoptotic Bcl-2 proteins to regulate apoptosis, all proteins in the
first subgroup (Bax, Bak, and Bok) have transmembrane-anchoring regions
and could regulate mitochondrial cytochrome c release and the
subsequent activation of caspases (27, 28). In contrast, proteins in
the second subgroup probably initiate apoptosis mainly through
dimerization with antiapoptotic Bcl-2 proteins to antagonize their
function. BOD belongs to the second subgroup of proapoptotic Bcl-2
proteins and shows a wide heterodimerization pattern, capable of
interacting with diverse antiapoptotic Bcl-2 proteins of mammalian and
viral origins. The broad expression and interaction profile of BOD
suggests that it could serve as an apoptosis mediator in diverse cell
lineages. Among proteins in the second subgroup, BAD is known to
function as a cytoplasmic adaptor protein capable of interacting with
other upstream signaling molecules in the cytoplasm (30, 31). The
soluble proapoptotic BAD protein binds to widely distributed
cytoplasmic protein 143-3 after phosphorylation of serine residues in
its 143-3 binding sites (30, 31). Because insulin-like growth factor
I and insulin activate the Akt kinase capable of phosphorylating BAD,
BAD phosphorylation is an important mechanism by which upstream
survival factors suppress apoptosis (42, 45, 46, 47, 48). In contrast, the
soluble BID normally locates in the cytoplasm and signals apoptosis by
binding to the membrane-bound proapoptotic protein Bax (15). Although
the exact role of BOD in apoptosis regulation requires further study,
the lack of a channel-forming domain in BOD and its preferential
interaction with antiapoptotic Bcl-2 proteins in the yeast two-hybrid
assay suggest that BOD, like BAD, may also function as an adaptor
protein for upstream signals and promote apoptosis by interacting with
antiapoptotic Bcl-2 proteins. Future studies on BOD interaction with
upstream cytoplasmic proteins are of interest.
Recently, a proapoptotic protein Bim was identified based on expression
cloning of Bcl-2-binding proteins from a mouse lymphoma cell line (19).
This mouse protein has three splicing variants, all of which contain a
shared C-terminal BH3 domain. Sequence comparison indicated that rat
BOD-L and BOD-M represent the orthologs of Bim-splicing variants Bim-EL
and Bim-S, respectively. However, the BOD gene encodes a shorter
variant (BOD-S) in the rat ovary having only 93 amino acids of the C
terminus of BOD-L, whereas the shortest Bim variants (Bim-S) are 110
amino acids in length. Of interest, both BOD-S and a truncated BOD
construct (BOD-ES), containing only 70 amino acids in the C terminus,
are still capable of inducing apoptosis, consistent with the finding
that the short form of Bim is the most potent isoform in apoptosis
induction after interleukin-3 deprivation or
-irradiation of a tumor
cell line (19). Although these data suggest that C-terminal sequences,
including the consensus BH3 domain, are important for the proapoptotic
activity of BOD, the N-terminal sequences that are unique to BOD-L and
BOD-M could be important for posttranslational regulation of these BOD
variants. The future isolation of the BOD/Bim gene will
elucidate the splicing mechanisms leading to the derivation of
different mRNA variants.
In contrast to Bim, which does not bind virally derived antiapoptotic
protein E1B 19 k and Epstein-Barr virus (EBV) BHRF-1 in a
coprecipitation test (19), BOD heterodimerizes with all known mammalian
antiapoptotic Bcl-2 proteins and the viral-derived BHRF-1 (Fig. 2
). It
is possible that the yeast two-hybrid assay is more sensitive than the
protein coprecipitation test used to study Bim function. In addition,
it has been reported that Bim is colocalized with Bcl-2 and possibly
anchored to membrane fractions through its C-terminal hydrophobic
region (19). However, sequence analysis of BOD indicated that the
hydrophobic sequence in the putative transmembrane region is
exceedingly short, and the importance of this C terminus region
in BOD action requires further study. Because BOD is ubiquitously
expressed in diverse tissues and shows a wide heterodimerization
property, BOD could regulate apoptosis in a wide spectrum of tissues by
interacting with diverse antiapoptotic Bcl-2 proteins.
The present identification of a proapoptotic protein BOD based on its
heterodimerization with the antiapoptotic Mcl-1 protein provides
further understanding of genes involved in the decision step of ovarian
follicle apoptosis. The yeast two-hybrid approach used here and in
earlier studies (18, 31) serves as an experimental paradigm to
elucidate protein-protein interactions between diverse tissue-specific
Bcl-2 protein pairs in the decision of cell fate. In the ovary, the
antiapoptotic protein Mcl-1 is believed to heterodimerize with BOD
and/or other proapoptotic Bcl-2 proteins (Bax, Bok, and BAD), and the
ratio of these protein pairs could regulate downstream events
including binding to Apaf-1 or other mammalian Ced-4 homologs and the
release of mitochondrial cytochrome c, leading to the activation of
caspases as executioners of apoptosis. It is envisioned that further
studies on the identification and hormonal regulation of
tissue-specific Bcl-2 proteins and their heterodimerization protein
partners could unravel intracellular mechanisms underlying
apoptosis.
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MATERIALS AND METHODS
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Two-Hybrid Screen of Mcl-1-Binding Proteins and Isolation of
Full-Length BOD cDNA Variants
We isolated multiple clones of BOD cDNA based on their ability
to interact with rat Mcl-1 in an HF7c yeast reporter strain after the
screening of a GAL4-activation domain-tagged ovarian Matchmaker
cDNA library prepared from 27-day-old Sprague-Dawley rats primed for
36 h with PMSG (CLONTECH Laboratories, Inc., Palo Alto, CA) (33).
The full-length rat Mcl-1 cDNA was fused to the binding domain
of GAL4 in a yeast shuttle vector pGBT9 to serve as the bait, and a
cDNA library screen was performed using a two-step procedure. In the
first step, yeast cells were transformed with the bait cDNA and
selected on plates deficient in tryptophan. In the second step,
selected cells were further transformed with the library cDNAs, and
clones harboring interacting proteins for Mcl-1 were selected in plates
lacking tryptophan, leucine, and histidine. Positive transformants were
then selected for growth in media containing 30 mM
3-aminotriazole. Individual activation domain-fusion cDNAs in positive
yeast cells were retrieved after transformation of HB101 strain
Escherichia coli cells with the yeast DNA extract. Among the
positive clones sequenced, two clones contained cDNAs encoding a
60-amino acid ORF with a conserved BH3 domain found in other Bcl-2
family proteins.
Nucleotide sequences of the putative Bcl-2-related cDNA fragments were
used to design primers to prepare a cDNA sublibrary enriched with
clones containing the 5'-end sequence of the candidate cDNA. To allow
5'-end extension, RT was performed using rat ovarian mRNA preparations
and a specific primer downstream of the termination codon of the
putative ORF found in the novel cDNAs. After second-strand synthesis,
the enriched cDNA pool was tailed at 5'-ends with adaptor sequences to
allow further PCR amplification. The sublibrary was then used as a
template for PCR amplification of upstream sequences using internal
primer pairs. PCR products were fractionated using agarose gels, and
those with strong hybridization signals to the original cDNA fragments
were subcloned into the pUC18 vector. After screening of the sublibrary
based on colony hybridization using the original cDNA fragment as a
probe, clones with extended 5'-end sequences of the putative
Bcl-2-related protein were isolated for DNA sequencing. Using this
procedure, cDNAs encoding the complete ORF of the BOD and several
putative splicing variants were isolated.
Construction of Expression Vectors Encoding BOD Variants and
Mutants
Using Pfu or Vent DNA polymerase, different BOD mutants were
generated by oligonucleotide-directed, two-step PCR mutagenesis (18),
whereas the truncated BOD mutants were derived by PCR amplification
using specific primers. Wild-type and mutant cDNAs were subcloned into
the pGBT9 expression vector for yeast cell studies or into the pcDNA3
expression vector (Invitrogen, Inc., San Diego, CA) for mammalian cell
studies. The authenticity of wild-type and mutant constructs was
confirmed by dideoxy sequencing.
Binding between BOD and Different Bcl-2 Family Members
Interactions between BOD and different Bcl-2 family members were
assessed in yeast cells using the pGBT9 GAL4-binding domain and pGADGH
GAL4-AD vectors. Specific binding of different protein pairs in yeast
was evaluated based on the activation of the GAL1-HIS3 reporter gene.
Wild-type and mutant BOD cDNAs were subcloned in pGBT-9, whereas all
other Bcl-2 related proteins were expressed as Gal-AD fusion proteins
using the pGADGH vector. A minimum of six independent transformants
with each pair of hybrid cDNAs were analyzed for the expression of
GAL1-HIS3 reporter gene. For GAL1-HIS3 reporter expression, cells were
grown in a medium lacking leucine, tryptophan, and histidine but
containing 530 mM 3-aminotriazole to inhibit endogenous
histidine production.
Analysis of Apoptosis after Transient Transfection of CHO
Cells
Apoptosis was monitored after transfection of different
cDNAs as previously described (18). Briefly, CHO cells were plated
at a density of 2 x 105 cells per well in DMEM/F12
supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 2 mM glutamine. One day later, cells were
transfected using the lipofectamine procedure (Life Technologies,
Gaithersburg, MD) with the empty pcDNA3 expression vector or the same
vector containing different cDNAs, together with 1/20 fractions of an
indicator plasmid pCMV-ß-gal to allow the identification of
transfected cells. Inclusion of 20-fold excess expression vectors as
compared with the pCMV-ß-gal reporter plasmid ensured that most of
the ß-galactosidase-expressing cells also expressed the
protein(s) under investigation. Cells were incubated with plasmids in a
serum-free medium for 12 h, followed by the addition of FBS to a
final concentration of 5%. After an additional culture for 12 h,
cells were fixed by 0.25% glutaraldehyde and stained with X-gal [0.4
mg/ml in buffer containing 150 mM NaCl, 100 mM
Na2HPO4, 1 mM MgCl2,
3.3 mM
K4Fe(CN)6·3H2O, and
3.3 mM K3Fe(CN)6, pH 7.0] for
6 h at 37 C to detect ß-galactosidase expression. The number of
viable blue cells were counted by microscopic examination. Data are
expressed as the percentage (mean ± SEM) of viable
cells as compared with the control group based on the counting of six
independent samples (at least 500 cells per 35-mm dish) from three or
more separate experiments. Statistical differences among treatment
groups were analyzed using one-way ANOVA and Scheffe F-test.
Northern and Southern Blot Analysis
For mRNA analysis, the BOD cDNA probe (nucleotides 1328 of the
BOD-L ORF) was radiolabeled with 32P using random priming.
Blots containing poly(A)+ RNA from various adult human and rat tissues
(CLONTECH) were hybridized with the BOD probe at 60 C before washing to
a final stringency of 0.1x saline sodium citrate (SSC) and
0.5% SDS at 65 C. To estimate mRNA loading, the blots were
subsequently probed with a ß-actin cDNA probe. For studies of
cross-species conservation of the BOD gene, a Zoo blot (CLONTECH)
containing EcoRI-digested genomic DNA from different
vertebrates was probed with a 32P-labeled cDNA probe
corresponding to the 5'-end sequences of BOD-L (nucleotides -180 to
+40 of BOD-L). The blot was washed to a final stringency of 0.5% SDS
and 0.2x SSC at 55 C before exposure.
 |
ACKNOWLEDGMENTS
|
---|
The GenBank submission number for rat BOD-L, BOD-M, and BOD-S
cDNAs are AF065433, AF065432, and AF065431, respectively. We
thank Dr. Lois K. Miller (University of Georgia, Athens, GA) for the
gift of P35 cDNA. We also thank the following individuals for the
provision of cDNAs for different Bcl-2 proteins: M. Cleary (Stanford,
CA; Bcl-2); S. Cory (Victoria, Australia; Bcl-w); G. Chinnadurai (St.
Louis, MO; Bfl-1); T. Chettenden (Cambridge, MA; Bak); A. Rickinson
(Birmingham, England; BHRF1); and C. Thompson (Chicago, IL;
Bclx-L).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317.
This study was supported by NIH Grant HD-31566.
Received for publication April 3, 1998.
Revision received May 15, 1998.
Accepted for publication May 19, 1998.
 |
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