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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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.

 
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. 1AGo, 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. 1AGo). 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. 1BGo) 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. 2Go, 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. 2Go), 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.

 
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. 3AGo, 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. 3AGo).



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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. 3AGo. 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.

 
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. 3BGo, 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. 3BGo, 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. 4Go, 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. 3Go legend. The cell-killing activity of BOD-ES and its 5A mutant with the same BH3 domain alteration was also investigated for comparison.

 
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. 5Go). 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.

 
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. 6Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 14–3-3 after phosphorylation of serine residues in its 14–3-3 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 {gamma}-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. 2Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 5–30 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 1–328 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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