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• W1996954871 abstract "Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. BidS contains the N-terminal regulatory domains of Bid without the BH3 domain; BidEL corresponds to full-length Bid with additional N-terminal sequence; and BidES contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies BidEL induces apoptosis, whereas BidS abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. BidES induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli. Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. BidS contains the N-terminal regulatory domains of Bid without the BH3 domain; BidEL corresponds to full-length Bid with additional N-terminal sequence; and BidES contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies BidEL induces apoptosis, whereas BidS abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. BidES induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli. Apoptosis, or programmed cell death, is an evolutionarily conserved program of changes in cell biochemistry and structure (1.Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6173) Google Scholar) leading to the loss of cellular functions and to engulfment and removal by phagocytes (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar). This process is essential during normal development, in homeostasis, and also in disease pathogenesis (3.Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6162) Google Scholar). Apoptosis occurs following activation of specific caspases that amplify apoptotic signals in a cascade of proteolysis leading to cleavage of specific substrates. Caspase activation may be initiated in a number of ways, including ligation of cell surface death receptors (4.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4526) Google Scholar) or activation of the apoptosome (a large multiprotein complex containing caspase-9) by cytochrome c released from mitochondria (5.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Cytochrome c release from mitochondria is in turn regulated by the interaction of pro- and anti-apoptotic members of the Bcl-2 family of proteins at the mitochondrial surface (6.Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Mammalian Bcl-2 family members share homology with the prototypic protein Bcl-2 and with the nematode CED-9 protein, across four conserved regions, termed the Bcl-2 homology domains (BH1-4). Bcl-2 proteins can be broadly divided into three groups according to their ability to either induce or inhibit apoptosis and their structural relationship to Bcl-2 (7.Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). In addition to the BH3 domain, all anti-apoptotic Bcl-2 proteins described to date contain BH1, BH2, and sometimes BH4 domains. Pro-apoptotic family members either share the multidomain structure (e.g. Bax and Bak) or contain only the BH3 domain (e.g. Bim and Bid). Bcl-2 proteins containing only the BH3 domain have been suggested to play an important role in initiating mitochondrial-mediated apoptosis. Bid is a 22-kDa “BH3-only” protein with similarity to other Bcl-2 family proteins only within the death-inducing BH3 region (8.Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (801) Google Scholar). Bid has a unique role in signaling of apoptosis, because it links the death receptor signaling pathway to the mitochondrial signaling pathway mediated by Bcl-2 proteins. Death receptor ligation activates caspase-8, which cleaves Bid, freeing the C-terminal moiety (tcBid) to cooperate in the release of cytochrome c from mitochondria. Examination of the crystal structure of Bid reveals that the hydrophobic face of the BH3 domain, which is exposed in other family members, is enclosed by the N-terminal of Bid and only exposed following proteolytic cleavage of Bid (9.McDonnell J.M. Fushman D. Milliman C.L. Korsmeyer S.J. Cowburn D. Cell. 1999; 96: 625-634Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 10.Chou J.J. Li H. Salvesen G.S. Yuan J. Wagner G. Cell. 1999; 96: 615-624Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). There appears to be a second functional domain in Bid, in the N terminus, termed the BH3B domain (11.Tan K.O. Tan K.M. Yu V.C. J. Biol. Chem. 1999; 274: 23687-23690Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which has activity to suppress the apoptogenic activity of the BH3 domain. The region between these two opposing domains is extremely sensitive to proteolytic cleavage, containing cleavage sites for caspases, granzyme B, and various lysosomal proteases. The action of these proteases leads to the creation of two cleavage products (termed tnBid and tcBid). tcBid translocates to mitochondria, where it interacts with other Bcl-2 family proteins to bring about the release of cytochrome c (12.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 13.Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar). We and others have shown that alternative gene splicing may regulate the function of other Bcl-2 family members (14.Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2908) Google Scholar, 15.Jiang Z.H. Wu J.Y. Proc. Soc. Exp. Biol. Med. 1999; 220: 64-72Crossref PubMed Google Scholar, 16.Bingle C.D. Craig R.W. Swales B.M. Singleton V. Zhou P. Whyte M.K. J. Biol. Chem. 2000; 275: 22136-22146Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 17.Bae J. Leo C.P. Hsu S.Y. Hsueh A.J. J. Biol. Chem. 2000; 275: 25255-25261Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The structure of Bid, with opposing Bcl-2 homology domains, would potentially be readily regulatable by alternative gene splicing. We therefore investigated the functional regulation of this gene at the molecular level. Antibodies, Reagents, and Cell Lines—All chemicals were of analytical reagent grade and were purchased from Sigma unless stated otherwise. Culture media (Hanks' balanced salt solution, RPMI 1640, and Dulbecco's modified Eagle's medium), LipofectAMINE, and Optimem were from Invitrogen. The antibody against Bid has been described previously (12.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar) and was the kind gift of Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). zVAD.fmk was from Bachem (St. Helen's, UK). Preprepared protein samples were from the BioChain Institute (Hayward, CA). CH-11 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Cos, HeLa, Jurkat, HL 60, 293T, and HepG2 cells were from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 supplemented with 10% fetal calf serum (Invitrogen). mRNA from NB4 cells was kindly supplied to us by Dr. M. Lanotte (Paris, France) from cells cultured and stimulated as previously described (18.Lanotte M. Martin-Thouvenin V. Najman S. Balerini P. Valensi F. Berger R. Blood. 1991; 77: 1080-1086Crossref PubMed Google Scholar). mRNA from neutrophil precursor populations was kindly supplied to us by Dr. J. Cowland (Copenhagen, Denmark) from samples purified from bone marrow and peripheral blood as previously described (19.Bjerregaard M.D. Jurlander J. Klausen P. Borregaard N. Cowland J.B. Blood. 2003; 101: 4322-4332Crossref PubMed Scopus (129) Google Scholar). PCR—RNA was extracted using the RNeasy kit (Qiagen). PCR was performed according to standard protocols. The primers used were BidF4 (exon 2, CTG GGA GAC GCT GCC TCG), BidF7 (exon 3, TGC TGG GAA ACT GTT GAG TG), BidF10 (exon 6, CAA GTG CTG AGG AAG AAA CG), BidR2 (exon 9, AGC CAG TCA CAC TTC TGG AAC), GAPDH 1The abbreviations used are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RACE, rapid amplification of cDNA ends; GFP, green fluorescent protein. forward (AAC TTT GGT ATC GTG GAA GGA C), and GAPDH reverse (TGG TCG TTG AGG GCA ATG). 5′ RACE was performed using the SMART™ RACE kit (BD Biosciences Clontech, Palo Alto, CA), using a modification of the protocol to include reduction of nonspecific base pair interaction (GC-Melt™; BD Biosciences Clontech). Gel extraction (Qiaquick; Qiagen), cloning (pCRII-TOPO; Invitrogen), and isolation of plasmid DNA (Concert Rapid; Invitrogen) were performed using commercially available kits according to the manufacturers' instructions. Preprepared mRNA (Premium RNA) was from BD Biosciences Clontech, and cDNA was prepared using standard reverse transcription techniques. Generation of Bid Expression Constructs—To generate the pCR3.1 (Invitrogen) expression constructs, PCR fragments were generated using the reverse primer, GGA TCC TCA GTC CAT CCC ATT TCT GGC TAA (exon 9), and the relevant forward primers, AAG CTT AGC CAC CAT GGA CTG TGA GGT CAA CAA C (Bid and BidS, exon 4), AAG CTT AGC CAC CAT GTG CAG CGG TGC TGG GGT CA (BidEL, exon 3), and AAG CTT AGC CAC CAT GGA CCG TAG CAT CCC TCC GG (BidES, exon 7) using the appropriate construct in pCRII-TOPO as a template. The PCR product obtained was purified, and was cloned directly into pCR3.1. Correct orientation and reading frame were confirmed by sequencing. The inserts were subcloned by restriction enzyme digestion from these vectors into suitable pEGFP vectors (Living Colors; Clontech). pcDNA3.1.tBid.myc-His was generated by subcloning from pCR3.1.tBid into pcDNA3.1(+)myc-His (Invitrogen). pCR3.1.FLAGBcl-2 and pCR3.1.FLAGBax were generated by subcloning from the appropriate pEF FLAG puro vector, kindly supplied to us by Dr. D. Huang (Walter and Eliza Hall Institute, Melbourne, Australia). Transfection—A549 cells maintained in RPMI and 10% fetal calf serum were seeded at a concentration of 2 × 105/ml into 6-well plates and were transfected using LipofectAMINE in Optimem. The day after transfection, the presence of green fluorescent protein (transfection control) was examined by fluorescence microscopy. HepG2 cells were maintained in Dulbecco's modified Eagle's medium and 10% fetal calf serum and seeded at a concentration of 2 × 105/ml into 6-well plates. Transfection was with the calcium phosphate method (20.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 2nd Ed. Harvard Medical School, Cambridge, MA1992Google Scholar). Western Blot Analysis—The cell lysates were prepared as previously described (21.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: A8.40-A8.55Google Scholar), and in vitro translation was performed using the TnT quick-coupled transcription/translation system (Promega UK, Southampton, UK) according to the manufacturer's instructions. The protein samples were loaded on polyacrylamide gels such that protein from 1 × 106 cells (or in the case of preprepared samples, 50 μg of protein) was loaded per lane. Electrophoresis, transfer, and probing were performed using standard methods. Confocal Microscopy and Cell Staining—The cells were examined under magnifications in the range of 100× to 1000- using a Molecular Dynamics CLSM 2010 coupled to a Nikon Diaphot microscope. Excitation was with the 488- or 568-nm line of the krypton-argon laser. Mitochondrial staining was with Mitotracker Red 580 (Molecular Probes, Eugene, OR) and was performed according to the manufacturer's instructions. Golgi staining was performed with BODIPY TR ceramide analogue (Molecular Probes) according to the manufacturer's instructions. Nuclear staining was performed using propidium iodide on formaldehyde fixed cells at a concentration of 1 μg/ml. Endoplasmic reticulum staining was with rhodamine 6G chloride (Molecular Probes) and was performed according to the published protocol (22.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Statistical Analysis—Where appropriate the results are expressed as the means ± S.E. of the number (n) of independent experiments, with each experiment performed in duplicate. Statistical analysis was performed by one-way analysis of variance with Bonferroni (see Figs. 5, A and C, and 7) and Dunnett (see Fig. 5B) post-test correction for multiple comparisons using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Significance was assumed at levels of p < 0.05.Fig. 7Interaction of Bid-isoforms with other Bcl-2 family proteins. A, A549 cells transfected with the vectors expressing the inserts shown were assessed for rates of apoptosis, as described under “Experimental Procedures”. BidL and BidEL significantly increased rates of apoptosis compared with empty vector control (p < 0.001 in each case), that is inhibited by co-transfection with Bcl-2 (p < 0.001 compared with either isoform alone), consistent with functional interaction between these proteins in vivo. In contrast, the pro-apoptotic effects of BidES were not significantly abrogated by Bcl-2. B, A549 cells transfected with the vectors expressing the inserts shown were assessed for rates of apoptosis, as described under “Experimental Procedures.” Bax shows significantly increased rates of apoptosis compared with empty vector control (p < 0.01). This can be abrogated by co-transfection with Bcl-2 (p < 0.05 compared with Bax alone) but not by co-transfection with BidS or BidES (p > 0.5 compared with Bax alone).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of Novel Bid-related Proteins in Human Cells—A search of the known sequence for human Bid against the genomic sequence of chromosome 22 identified five coding exons analogous to those coding for murine Bid (23.Yin X.M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar). In addition, a sixth noncoding exon is present, which lies ∼25 kilobase pairs 5′ of the Bid gene, which we have designated as exon 2 (Fig. 1). This exon has been previously recognized to be part of the Bid gene (24.Sax J.K. Fei P. Murphy M.E. Bernhard E. Korsmeyer S.J. El-Deiry W.S. Nat. Cell Biol. 2002; 4: 842-849Crossref PubMed Scopus (343) Google Scholar). A second previously undescribed 5′ exon of the Bid gene exists within an expressed sequence tag (accession number AA338833) that maps to chromosome 22 just 5′ of exon 2 and that we have designated exon 1. To identify mRNA species utilizing exon 1 or 2 in association with other bid exons, 5′ RACE was performed. Neutrophil mRNA was chosen as the source of transcripts, because of our interest in death receptor signaling in these cells (25.Renshaw S.A. Timmons S.J. Eaton V. Usher L.R. Akil M. Bingle C.D. Whyte M.K. J. Leukocyte. Biol. 2000; 67: 662-668Crossref PubMed Scopus (58) Google Scholar, 26.Renshaw S.A. Parmar J.S. Singleton V. Rowe S.J. Dockrell D.H. Dower S.K. Bingle C.D. Chilvers E.R. Whyte M.K. J. Immunol. 2003; 170: 1027-1033Crossref PubMed Scopus (151) Google Scholar) and because transcripts of alternatively spliced products were abundant in myeloid cell lines in preliminary experiments (data not shown). Using 5′ RACE, a number of alternatively spliced transcripts were isolated, including transcripts utilizing two further previously undescribed exons. The third of these novel exons we refer to as exon 3, the genomic locus of which is located 3′ of exon 2. mRNA transcripts contained exon 3 as the most 5′ exon, and this was identified either in continuity with exons 4, 5, 7, 8, and 9, or in alternatively spliced isoforms “missing” exon 4 or exons 4 and 5. The fourth novel exon, which we refer to as exon 6 was isolated between sequences corresponding to exons 2, 4, and 5 and exons 7, 8, and 9. Translation of these novel transcripts creates three potential novel proteins, which we have named BidS (accession number AY005151) for “short,” BidEL (accession number AF250233) for “extra long,” and BidES for “extra short.” BidES is generated from a number of splice variants that share the common feature of loss of the first AUG together with the potential for translation from an internal AUG within exon 7 of the Bid gene. A summary of all the splice variants of bid isolated by PCR, RACE, and searching the GenBank™ data base is shown in Fig. 1A. For the sake of clarity, we will use “BidL” to refer specifically to the previously described gene product of the Bid gene (8.Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (801) Google Scholar) and “Bid” where the reference is to a more general property of the gene and its products. Putative protein products of these splice variants are shown in Fig. 1B. BidEL is predicted to have an additional 45 amino acids at the N terminus that do not contain any recognized conserved domains. BidS contains only the unopposed regulatory BH3-B domain, omitting the pro-apoptotic BH3 domain. There are an additional 63 amino acids at the C terminus of BidS that are entirely novel and that contain no recognized conserved domains. BidES contains the final 99 amino acids of BidL, beginning with the terminal amino acids of the BH3 domain and including the sequence likely to be involved in mitochondrial targeting of Bid (27.Lutter M. Fang M. Luo X. Nishijima M. Xie X. Wang X. Nat. Cell Biol. 2000; 2: 754-761Crossref PubMed Scopus (404) Google Scholar). Differential Expression of Bid Isoforms—Using specific primers, as described under “Experimental Procedures,” PCR was performed using a commercially available RNA panel (Premium RNA; Clontech) to screen a variety of tissues. Using primers in exons 2 and 9 of bid (BidF4, BidR2), a three-banded pattern was seen in all of the tissues studied (Fig. 2A). Sequencing of cDNA extracted from these bands confirmed that these correspond to cDNA for BidL and two transcripts for BidES (designated BidES (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar) and BidES (3.Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6162) Google Scholar) in Fig. 1A). Using primers in exons 3 and 9 (BidF7 and BidR2), transcripts encoding BidEL were detected predominantly in tissues with large numbers of hemopoetic cells (e.g. spleen and bone marrow). BidEL was also expressed at high levels in cerebral and cerebellar cortex, as might be expected given the importance of Bid in regulating neuronal cell death (28.Plesnila N. Zinkel S. Le D.A. Amin-Hanjani S. Wu Y. Qiu J. Chiarugi A. Thomas S.S. Kohane D.S. Korsmeyer S.J. Moskowitz M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15318-15323Crossref PubMed Scopus (225) Google Scholar). Transcripts corresponding to cDNA for BidES (4.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4526) Google Scholar) and BidES (5.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) were also detected. PCR using primers to exon 6 and 9 (BidF10 and BidR2) shows expression of cDNA for BidS in a similar distribution to cDNA for BidEL. In addition, a range of transformed cell lines was screened for the presence of mRNA for Bid isoforms. mRNA for BidL, BidES (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar), and BidES (3.Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6162) Google Scholar) were seen in Cos, HeLa, HL60, Jurkat, and 293T cells. mRNA for BidS was seen in all of the above cell lines, and mRNA for BidEL was seen only in Jurkat cells (Fig. 2B). There is therefore evidence of endogenous mRNAs expressing all of the isoforms identified by RACE. To demonstrate endogenous protein expression, we screened a panel of primary tissues by Western blotting. Immunoreactive bands of appropriate size were seen for BidEL in spleen, placenta, and pancreas; for BidS in lung, pancreas, and spleen; and for BidES in lung and pancreas (Fig. 3A). Transformed cell lines with high levels of mRNA expression of various Bid isoforms were also screened for Bid protein expression. Both HeLa cells and 293T cells contain an immunoreactive band that migrates at the same size as in vitro translated BidS (Fig. 3B). Jurkat cells contain a low intensity protein band of ∼27 kDa on Western blotting with an anti-Bid antibody that migrates at an identical size to BidEL overexpressed in the 293T cell line (Fig. 3B) and to in vitro translated BidEL (data not shown). We were thus able to identify protein bands on Western blotting that correspond to either in vitro translated or transfected BidEL and BidS, confirming endogenous expression of these proteins. Recently, a role for BidL in regulation of myeloid homeostasis, including peripheral blood neutrophil numbers, has been demonstrated using Bid-/- mice (29.Zinkel S.S. Ong C.C. Ferguson D.O. Iwasaki H. Akashi K. Bronson R.T. Kutok J.L. Alt F.W. Korsmeyer S.J. Genes Dev. 2003; 17: 229-239Crossref PubMed Scopus (148) Google Scholar). We therefore examined expression of Bid isoforms in two models of neutrophil maturation. NB4 cells are members of a myeloid leukemia cell line that, following treatment with retinoic acid, undergo biochemical and functional differentiation along the neutrophil lineage, such that by 24 h these cells share many characteristics with mature neutrophils (18.Lanotte M. Martin-Thouvenin V. Najman S. Balerini P. Valensi F. Berger R. Blood. 1991; 77: 1080-1086Crossref PubMed Google Scholar, 30.Ruchaud S. Duprez E. Gendron M.C. Houge G. Genieser H.G. Jastorff B. Doskeland S.O. Lanotte M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8428-8432Crossref PubMed Scopus (109) Google Scholar). With progressive maturation, NB4 cells expressed higher levels of BidEL (Fig. 4). BidS levels did not consistently change in these experiments. In addition, we studied BidL, BidEL, and BidS expression in myeloid precursors extracted from bone marrow by density gradient centrifugation (19.Bjerregaard M.D. Jurlander J. Klausen P. Borregaard N. Cowland J.B. Blood. 2003; 101: 4322-4332Crossref PubMed Scopus (129) Google Scholar, 31.Cowland J.B. Borregaard N. J. Immunol. Methods. 1999; 232: 191-200Crossref PubMed Scopus (65) Google Scholar). Cells extracted in this way represent progressively more mature neutrophil precursors and are here compared with circulating neutrophils from the same donor. Mature neutrophils express more of both BidEL and BidS (Fig. 4), in part paralleling the data from NB4 cells. Thus, not only are transcripts for BidEL and BidS expressed in a variety of primary tissues, they are differentially regulated between tissue types and, during maturation of a single cell type, the neutrophil granulocyte. Effects of Bid Isoforms on Cellular Apoptosis—Based on the predicted protein structure determined for the Bid isoforms (Fig. 1B), each of the three novel isoforms would be predicted to behave in different ways to modulate the activity of Bid following cleavage and activation. We hypothesized that BidS, containing only the inhibitory BH3B domain, could act as a naturally occurring inhibitor of tcBid-induced apoptosis. BidEL is identical to BidL at the C terminus, contains the BH3 domain, and differs only in the N-terminal fragment. BidEL might therefore induce apoptosis in a similar way to BidL, but the additional N-terminal sequence might influence the subcellular localization of BidEL or perhaps alter the cleavage and activation of BidEL. Because BidES, which again lacks a BH3 domain, corresponds to the portion of Bid that localizes to the mitochondrial surface, we hypothesized that BidES might interrupt the action of cleaved BidL at the mitochondria. To test these hypotheses we first assessed the potential of BidS, BidEL, and BidES to modulate apoptosis in an overexpression assay. Apoptotic morphology was assessed in A549 cells transfected with the following constructs: pCR3.1.empty vector, pCR3.1.BidEL, pCR3.1.BidS, pCR3.1.BidL, or pCR3.1.BidES as described under “Experimental Procedures” (16.Bingle C.D. Craig R.W. Swales B.M. Singleton V. Zhou P. Whyte M.K. J. Biol. Chem. 2000; 275: 22136-22146Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In keeping with our predictions, transfection with constructs expressing BidL or BidEL induced high levels of apoptotic morphology, whereas BidS did not (Fig. 5A). BidS did not protect against cell death caused by the transfection process. In contrast to the hypothesis based on predicted structure, BidES, which contains no functional BH3 homology domains, appeared to induce apoptosis, albeit to a lesser degree than BidL and BidEL. We then tested the ability of the novel isoforms to interfere with BidL signaling. To simulate the specific effects of Bid activation, the cells were transfected with a construct expressing tcBidL (pCR3.1.tcBidL). This mimics the effects of isolated BidL cleavage, without direct activation of caspase pathways, as might occur with death receptor ligation. The ability of BidS and BidES to inhibit tcBidL-induced cell death was tested in comparison with a construct expressing the N terminus of BidL (pCR3.1.tnBidL), which has previously been shown to inhibit the effects of tcBidL (32.Kudla G. Montessuit S. Eskes R. Berrier C. Martinou J.C. Ghazi A. Antonsson B. J. Biol. Chem. 2000; 275: 22713-22718Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). BidS and BidES both significantly inhibited tcBidL-induced apoptosis to a similar degree to tnBidL (Fig. 5B). These studies also included an investigation of a further construct, corresponding to the N-terminal fragment of BidEL, to assess the effects of the additional N-terminal sequence on the ability of the cleavage product tnBidEL to inhibit tcBidL-induced apoptosis. tnBidEL, unlike tnBidL, did not significantly inhibit tcBidL-induced apoptosis (Fig. 5B). We further explored the ability of BidS to inhibit apoptosis by investigating whether BidS could inhibit Fas-mediated apoptosis in a “type II” cell, which requires Bid activity to complete the apoptotic program following Fas ligation. HepG2 cells are a hepatocellular line, with a type II phenotype (23.Yin X.M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar). Transient transfection with pCR3.1.BidS protected HepG2 cells from CH-11 (an anti-Fas agonistic antibody)-induced apoptosis (Fig. 5C), consistent with a role for BidS in direct inhibition of tcBidL-induced apoptosis. Subcellular Distribution of Bid Isoforms—Because the function of Bid is thought to relate to the ability of the active C-terminal cleavage fragment to translocate from cytoplasm to mitochondria, we investigated the subcellular localization of the various Bid isoforms using GFP fusion proteins. Transfection of a construct expressing GFP-BidL into A549 cells showed the expected diffuse distribution (Fig. 6a). When the cells were transfected with constructs for GFP-BidS, the fluorescence was similarly distributed throughout the cell (Fig. 6b), suggesting that the additio" @default.
• W1996954871 created "2016-06-24" @default.
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• W1996954871 creator A5009580916 @default.
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• W1996954871 date "2004-01-01" @default.
• W1996954871 modified "2023-09-28" @default.
• W1996954871 title "Three Novel Bid Proteins Generated by Alternative Splicing of the Human Bid Gene" @default.
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