Deterin, a New Inhibitor of Apoptosis from Drosophila melanogaster
2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês
10.1074/jbc.m000369200
ISSN1083-351X
AutoresGrace Jones, Davy Jones, Lei Zhou, Hermann Steller, Yanxia Chu,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoDeterin, a new apoptosis inhibitor fromDrosophila melanogaster, possesses an unusual structure of only a single baculovirus inhibitor of apoptosis (IAP)-type repeat and no RING finger motif. The biochemical actions of deterin are demonstrated in SF9 and S2 cell transfection assays, in which the expressed protein acts in the cytoplasm to inhibit or deter cells from apoptosis otherwise induced by the caspase-dependent apoptosis activator reaper or by cytotoxicants. A loss of function phenotype for deterin of cell death was indicated by transfections with either a dominant negative deterin mutant or with inhibitory RNA (RNAi) for deterin. The dominant negative C-terminal fragment that antagonized antiapoptotic activity of deterin did not affect antiapoptotic activity of DIAP1 or p35. Both the baculovirus IAP-type repeat (BIR) domain and the α-helical C-terminal domain are necessary in both SF9 and S2 cells for deterin to manifest its activity to prevent cell death. The approximately 650-base deterin transcript is present in embryos, third instar larvae, and late stage nurse cells of adult females. The deterin transcript is distributed throughout early stage embryos, whereas in later stage embryos it becomes progressively restricted to the central nervous system and gonads. Whereas the nematode survivin-type IAP has thus far been implicated only as a mitotic regulator,Drosophila deterin constitutes the first invertebrate member of the survivin-type IAP group to exhibit apoptosis-inhibitory activity. Deterin, a new apoptosis inhibitor fromDrosophila melanogaster, possesses an unusual structure of only a single baculovirus inhibitor of apoptosis (IAP)-type repeat and no RING finger motif. The biochemical actions of deterin are demonstrated in SF9 and S2 cell transfection assays, in which the expressed protein acts in the cytoplasm to inhibit or deter cells from apoptosis otherwise induced by the caspase-dependent apoptosis activator reaper or by cytotoxicants. A loss of function phenotype for deterin of cell death was indicated by transfections with either a dominant negative deterin mutant or with inhibitory RNA (RNAi) for deterin. The dominant negative C-terminal fragment that antagonized antiapoptotic activity of deterin did not affect antiapoptotic activity of DIAP1 or p35. Both the baculovirus IAP-type repeat (BIR) domain and the α-helical C-terminal domain are necessary in both SF9 and S2 cells for deterin to manifest its activity to prevent cell death. The approximately 650-base deterin transcript is present in embryos, third instar larvae, and late stage nurse cells of adult females. The deterin transcript is distributed throughout early stage embryos, whereas in later stage embryos it becomes progressively restricted to the central nervous system and gonads. Whereas the nematode survivin-type IAP has thus far been implicated only as a mitotic regulator,Drosophila deterin constitutes the first invertebrate member of the survivin-type IAP group to exhibit apoptosis-inhibitory activity. inhibitors of apoptosis β-galactosidase green fluorescent protein phosphate-buffered saline, pH 7.4, 0.1% Tween polymerase chain reaction RNA-mediated interference reverse transcription baculovirus IAP-type repeat Programmed cell death is a process that specifies the elimination of superfluous or otherwise unwanted cells and tissues (1.Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar, 2.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1363) Google Scholar, 3.Vaux D.L. Korsmeyer S.J. 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In some tissues, these apoptotic cells are phagocytosed intact, without an acute inflammatory reaction. These phenomena are in contrast to cell death by necrosis, in which cells swell and lyse to release cellular contents, inducing an inflammatory reaction. The various diverse stimuli that induce apoptosis can be external to the cell (e.g. UV, antibiotics, hormones, and liposomal cytotoxicants) or endogenous (e.g. transcriptional regulators and DNA damage due to free radicals). These apoptotic stimuli feed into apoptotic pathways of the cell by routes not well understood, although many pathways appear to converge into common or at least overlapping biochemical machinery. These pathways include activators that can activate special effector proteolytic enzymes, caspases, that in turn cleave and activate other caspases, which then cleave end target molecules; the molecular death of these target molecules causes the apoptotic death of the cell (8.Yuan J. Shaham S. Ledoux S. Ellis H.M. 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There are also other caspase inhibitors, such as certain inhibitors of apoptosis (IAP)1 proteins, first discovered in baculoviruses (12.Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar) and then identified in both vertebrate and invertebrate systems (13.Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 14.Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1057) Google Scholar, 15.Duckett C.S. Nava V.E. Gedrich R.W. Clem R.J. van Dongen J.L. Gilfillan M.C. Shiels H. Hardwick J.M. Thompson C.B. EMBO J. 1996; 15: 2685-2689Crossref PubMed Google Scholar, 16.Liston P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat Farahani R. McLean M. Ikeda J. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (872) Google Scholar, 17.Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar). These proteins are typically characterized by BIR type repeats and a RING finger motif (18.Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2285) Google Scholar, 19.LaCasse E.C. Baird S. Korneluk R.G. MacKenzie A.E. Oncogene. 1998; 17: 3247-3249Crossref PubMed Scopus (947) Google Scholar). However, there is much that we do not yet understand about the role and action of the IAPs generally in apoptosis and normal cell physiology. Structurally, the function of the characteristic BIR type repeat in IAPs is also little understood. Roy et al. (20.Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar) report that for the vertebrate c-IAP-1 and c-IAP-2, the BIR region alone could bind to caspases and block caspase activation. However, the inhibitory effect was greater when the RING finger was also present. More paradoxically, Hauser et al. (21.Hauser H.P. Bardroff M. Pyrowolakis G. Jentsch S.A. J. Cell Biol. 1998; 141: 1415-1422Crossref PubMed Scopus (210) Google Scholar) report a ubiquitin conjugating complex component that has a single BIR and no RING finger and no activity to inhibit apoptosis. Similarly, both an insect virus (AcIAP, Ref. 22.Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar) and an unrelated mammalian virus (23.Neilan J.G. Lu Z. Kutish G.F. Zsak L. Burrage T.G. Borca M.V. Carrillo C. Rock D.L. Virology. 1997; 230: 252-264Crossref PubMed Scopus (52) Google Scholar) encode BIR-containing proteins that have no demonstrated function to inhibit apoptosis. Recently, a vertebrate protein, survivin, has been reported from humans and mice that represents a new subgroup of IAPs that contain only a single BIR and no RING finger (24.Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1747) Google Scholar, 25.Kobayashi K. Hatano M. Otaki M. Ogasawara T. Tokuhisa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1457-1462Crossref PubMed Scopus (231) Google Scholar). A great deal of excitement about survivin-like IAPs has been engendered by the findings that survivin is expressed in a wider variety of cancers than any other known apoptosis inhibitor (26.Jaattela M. Exp. Cell Res. 1999; 248: 30-43Crossref PubMed Scopus (583) Google Scholar). An apparently homologous single BIR-containing protein from C. elegans, BIR1, has also been described recently (27.Fraser A.G. Claerwen J. Evan G.I. Hengartner M.O. Current Biology. 1999; 9: 292-301Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The studies on vertebrate survivin demonstrated antiapoptotic activity of survivin in cell transfection assays and also suggested a possible role in cell division due to the localization of survivin to mitotic spindles and its expression in proliferating but not quiescent tissues. In contrast, genetic studies on BIR1 molecule in C. elegans indicated a role in cell division but did not detect antiapoptotic activity. These disparate findings raise the important question as to whether the antiapoptotic activity of this protein group is restricted to vertebrates, in contrast to a role solely in cell division in invertebrates (28.Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). To date, two Drosophila IAPs have been reported, DIAP1 and DIAP2, that contain the conventional multiple BIR and RING finger motifs (13.Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 16.Liston P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat Farahani R. McLean M. Ikeda J. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (872) Google Scholar, 29.Uren A. Coulson E.J. Vaux D.L. Trends Biochem. Sci. 1998; 23: 159-162Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Apoptosis induced by intracellular regulators reaper, grim, and hid (30.Grether M.E. Abrams J.M. Agapit E.J. White K. Steller H. Genes Dev. 1995; 9: 1694-1708Crossref PubMed Scopus (594) Google Scholar, 31.White K. Grether M.E. Abrams J.M. Young L. Farrell K. Steller H. Science. 1994; 264: 677-683Crossref PubMed Scopus (897) Google Scholar, 32.Chen P. Nordstrom W. Gish B Abrams J.M. Genes Dev. 1996; 10: 1773-1782Crossref PubMed Scopus (362) Google Scholar), and extracellular toxicants, can be inhibited by these IAPs (13.Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 33.Vucic D. Kaiser W.J. Miller L.K. J. Biol. Chem. 1998; 273: 33915-33921Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 34.Harvey A.J. Soliman H. Kaiser W. Miller L.K. Cell Death Differ. 1997; 4: 733-744Crossref PubMed Scopus (44) Google Scholar). Yet, to date, no IAP-type proteins have been published from insects that possess the single BIR/no RING finger structure. We report here the gene and cDNA for a new insect IAP, deterin, that has a single BIR and no RING finger motif. We also demonstrate that this new insect IAP exerts a distinct action to deter apoptosis, that a loss of function phenotype for deterin is cell death, and that its transcript exists in particular tissues of multiple developmental stages. As the first invertebrate member of the survivin-type IAP group to exhibit apoptosis-inhibitory activity, Drosophila deterin demonstrates that antiapoptotic activity of survivin-type IAPs is not confined to the vertebrate kingdom. The cDNA clone for deterin was isolated from a Drosophila melanogaster embryo cDNA library kindly provided by Roger Brent. The sequenced cDNA was radiolabeled and used to screen D. melanogaster P1 genomic library filters (Genomesystems). The two most strongly positive hybridizing clones corresponded to independent P1 clones both associated with the 90A region of the D. melanogaster third chromosome. The inserts in these two clones were excised, recovered, and subjected to restriction digestion and probing with the radiolabeled cDNA, and the restriction pattern confirmed the overlap of the two clones. An approximately 3-kbp BamHI fragment was detected to completely contain the cDNA probe, and the deterin gene within this fragment was sequenced in both directions. During this study, a cDNA was reported into the Flybase as isolated from a late larval-prepupal library (AI260030). The reported protein coding sequence for that corresponding cDNA matched exactly the sequence of the cDNA we obtained from the embryonic library. Mutant constructs were prepared by PCR amplification of respective coding fragments and cloning into pIE1-4 vector. Deterin deleted for its C terminus (Det-NBIR) was prepared by deleting amino acids 111–153. The construct with the C terminus alone (Det-Cterm) contained the coding region for amino acids 108–153. In the Det-RING construct, the DIAP1 RING motif residues 391–425 were placed after residue 153 of deterin. For exchange of the C terminus of deterin with that of survivin (Det-SurvCterm), residues 111–153 of deterin were replaced with residues 98–142 of human survivin. All constructs were cloned into the pIE1-4 insect cell expression vector. Salivary glands were dissected, fixed to slides, and denatured in a method similar to that described by Pardue (35.Pardue M.-L. Goldstein L.S.B. Fryberg E.A. Methods in Cell Biology, Drosophila melanogaster: Practical Uses in Cell and Molecular Biology. Academic Press, San Diego, CA1994: 334-350Google Scholar). The probe (biotinylated deterin cDNA) was added to hybridization solution (formamide/SSC/dextran sulfate) and incubated with the prepared chromosomes overnight at 37 °C. The slides were washed with 2× SSC and then phosphate-buffered saline, and the hybridized probe was detected with Vectastain reagents (Vector). The EcoRI/XhoHI deterin cDNA fragment was subcloned into Bluescript and then excised with BamHI/XhoHI (the latter end filled in with Klenow) and ligated into the BamHI/NruI sites of pIE1-4 vector, in which expression of the encoded protein is driven in insect cells by a baculovirus promoter ie1(Novagen). A fusion construct was prepared by placing the coding sequence for green fluorescent protein (GFP) in frame with deterin.D. melanogaster reaper coding sequence (30.Grether M.E. Abrams J.M. Agapit E.J. White K. Steller H. Genes Dev. 1995; 9: 1694-1708Crossref PubMed Scopus (594) Google Scholar) was inserted by PCR cloning into the BamHI/NruI sites of pIE1-4. The D. melanogaster apoptosis inhibitor DIAP1 reported by Hay et al. (13.Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar), which was initially named threadbut which has been referred to in the literature by numerous abbreviations (DIAP1, DIAP-1, dIAP1, etc.), is referred to here by the abbreviation used in the original paper by Hay et al. (13.Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar). The coding sequence of DIAP1, reaper, the procaspases DCP1 and DRONC, and baculoviral p35 were also each cloned into the BamHI andNotI sites of pIE1-4. The control plasmid expressing β-galactosidase under the constitutive control of hsp70 promoter (phsp-β-gal) was as described (38.Jones G. O'Mahony P. Chang S. Schachtschabel U. Gene. 1996; 173: 209-214Crossref PubMed Scopus (14) Google Scholar). Spodoptera frugiperda SF9 cells were maintained at 25 °C in SF900 SFM media (Life Technologies, Inc.). D. melanogaster S2 cells were maintained at 25 °C in Schneider's Drosophila medium, (Life Technologies, Inc.) with 10% fetal calf serum. The pIE1-4-deterin construct (above), and the pIE1-4 vector only, were each separately and stably transformed into SF9 cells by cotransfection with pIE1-neo plasmid, and selection with neomycin, as per manufacturers instructions (Novagen). For transfections involvingDrosophila reaper-mediated apoptosis, 106 cells were transferred to 6 well culture dishes (Corning) in 1 ml media, and transfected with 3 μl of Cellfectin (Life Technologies, Inc.), with 0.2 μg of reporter phsp-β-gal or the pIE1-4-GFP construct, as noted, and with either 2 μg of pIE1-4-reaper and/or 6 μg of pIE1-4-deterin, pIE1-4-GFP-deterin, pIE1-4-DIAP1, or pIE1-4-p35. In addition, pIE1-4 vector was added in an amount sufficient to equalize the total amount of DNA transfected in each treatment to 8.2 μg. To assess deterin activity against exogenous apoptotic activation, liposomal conditions for induction of caspase-dependent apoptosis were used (39.Ebert O. Finke S. Salahi A. Herrmann M. Trojaneck B. Lefterova P. Wagner E. Kircheis R. Huhn D. Schriever F. Schmidt-Wolf I.G. Gene Ther. 1997; 4: 296-302Crossref PubMed Scopus (57) Google Scholar, 40.Aramaki Y. Takano S. Tsuchiya S. FEBS Lett. 1999; 460: 472-476Crossref PubMed Scopus (81) Google Scholar, 41.Imamura Y. Kemura Y. Matsumoto Y. Ueoka R. Biol. Pharm. Bull. 1997; 20: 1119-1121Crossref PubMed Scopus (18) Google Scholar). We determined that these conditions of S2 and SF9 cells were an excess of liposomal reagent relative to numbers of treated cells of 3 μl of Cellfectin to 0.5 × 106cells/ml. For RNAi experiments, sense and antisense transcripts were synthesized from the corresponding cDNA in Bluescript SK+ vector, using T3 or T7 RNA polymerase. The resulting transcript preparations were mixed, briefly boiled, and then annealed. The given RNAi was subjected to the same transfection procedures as transfected DNAs. For all transfections, 5 h after addition of reagents, the appropriate medium was added to achieve a total volume of 2 ml in each well, and 24–48 h later (as noted), the cells were either stained for β-galactosidase activity (2 mm MgCl2, 5 mMK4Fe(CN)6-3H2O, 5 mm K3Fe(CN)6, 0.2% 5-bromo-4-chloro-3-indoyl β-d-galactoside) or fluorescence-activated cell sorter analyzed for the GFP reporter, as described above. For the β-galactosidase reporter, the number of positive reporter (blue X-gal staining) cells in the consecutive fields of view in a transect across the widest diameter of each well were counted (typically several hundred to several thousand). The standard method for assessing cell viability used previously for survivin and other IAP-type molecules was also used here (22.Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar, 24.Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1747) Google Scholar, 32.Chen P. Nordstrom W. Gish B Abrams J.M. Genes Dev. 1996; 10: 1773-1782Crossref PubMed Scopus (362) Google Scholar, 42.Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3025) Google Scholar, 44.Duckett C.S. Nava V.E. Gedrich R.W. Clem R.J. Van Dongen J.L. Gilfillan M.C. Shiels H. Hardwick J.M. Thompson C.B. EMBO J. 1996; 15: 2685-2694Crossref PubMed Scopus (525) Google Scholar, 45.Claveria C. Albar J.B. Serrano A. Buesa J.M. Barbero J.L. Martinez-A C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar) (i.e. measurements are made within a time course such that the number of surviving cells (those not excluding trypan blue or those expressing the reporter) have had insufficient time to significantly proliferate); this procedure thereby prevents any bias towards showing a higher than actual number of surviving viable cells. This procedure was additionally verified in the present studies by preliminary tests, which showed little cell division by transfected SF9 or S2 cells during the time course under the conditions used and which showed that there was no increase in DNA synthesis during the time course in cells overexpressing deterin relative to control cells. In all transfection experiments, each treatment was repeated at least three times. Data are reported as mean and standard error. RNA was extracted from 0–4-h-old embryos, 4–8-h-old embryos, and late feeding stage final instar larvae, as described (46.Andres A.J. Thummel C.S. Goldstein L.S.B. Fyrberg E.A. Methods in Cell Biology, Drosophila melanogaster: Practical Uses in Cell and Molecular Biology. Academic Press, San Diego, CA1994: 565-573Google Scholar). Embryos or larvae were homogenized in SDS lysis buffer and digested with proteinase K, and the nucleic acids were extracted with phenol and then with phenol/chloroform and ethanol-precipitated. After fractionation on formaldehyde-containing agarose gels, the nucleic acids were transferred to Nytran membrane (Schleicher and Schuell) and probed with [α-32P]dCTP-labeled deterin cDNA; radiolabeling was performed by the random primer method. Signals were visualized by autoradiography. RNA samples from embryos or larvae were also used as the RT-PCR template, using primers corresponding to the 5′ end and 3′ end of the protein-coding region, respectively. Templates for RT-PCR were RNAs extracted from 0–12 h old embryos, late feeding stage third instar larvae, and mated females. For the transcript corresponding to the cloned deterin cDNA, the expected PCR product using these primers is 459 base pairs in length. In order to confirm the presence of endogenous Drosophila deterin transcripts inDrosophila S2 cells, cultured cells were collected and boiled for 5 min, the debris was pelleted, and aliquots of the supernatant were used as template in various PCRs using combinations of primers designed from cDNA designed to yield diagnostic and different size transcripts in each reaction. Confirmation of the presence of Drosophila deterin transcripts inSpodoptera SF9 cells stably transformed with aDrosophila deterin-expressing construct was performed on the cultured cells in a similar manner. The presence of overexpressed deterin protein in these SF9 cells was also confirmed by immunoblotting cellular proteins after SDS-polyacrylamide gel electrophoresis using rabbit polyclonal antibodies prepared against bacterial recombinantDrosophila deterin. Adult female ovaries were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, permeabilized with 4 μg/ml proteinase K, refixed, prehybridized, and then hybridized in hybridization solution: 50% deionized formamide, 5× SSC, 100 mg/ml each of sonicated salmon sperm DNA and yeast type X tRNA (Sigma), 50 mg/ml heparin, and 1%Tween. Hybridization was performed overnight at 65 °C, with either sense or antisense riboprobe of the deterin cDNA, labeled by incorporation of digoxigenin-labeled UTP. After washings at 65 °C, alkaline-phosphatase conjugated antidigoxigenin antibody (BMB; 2 μl per 4 ml of phosphate-buffered saline/0.1% Tween) was added for 1 h of incubation at room temperature. The preparation was washed with pH 9.0 Tris-buffered saline/0.1% Tween and then stained with alkaline phosphatase in the same buffer. Mixed stage embryos were dechorionated with bleach; agitated betweenn-heptane and 4% formaldehyde, 0.1 mNa2PO4; passed sequentially through methanol, ethanol, xylene/ethanol, and methanol; and finally fixed in 5% formaldehyde in phosphate-buffered saline, pH 7.4, 0.1% Tween (PBT)). After the embryos were changed to PBT, they were digested with 40 μg/ml proteinase K (BMB) for 8–10 min and then postfixed in 5% formaldehyde. The formaldehyde was washed out with PBT, and the embryos were prehybridized for 1–2 h at 55 °C in the same hybridization solution as described above. Hybridization in the same buffer overnight at 55 °C was performed with the same sense or antisense probes as used for the ovary in situ hybridizations. The next day, the embryos were washed with PBT, incubated with alkaline-phosphatase-conjugated antidigoxigenin antibody, and stained by methods similar to that described above for the ovaries. The encoded sequence for deterin, a 153-amino acid protein, contains a BIR-type repeat that is typical of BIRs found in IAP-type apoptosis inhibitory proteins (Fig.1). However, it possesses a single BIR and no RING finger motif, whereas most IAP-type proteins encode two or three BIR repeats, and most have a RING finger. In this structure, the encoded protein is most similar to a human and mouse apoptosis inhibitor, survivin and TIAP, respectively (25.Kobayashi K. Hatano M. Otaki M. Ogasawara T. Tokuhisa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1457-1462Crossref PubMed Scopus (231) Google Scholar, 42.Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3025) Google Scholar), although there is considerable divergence in structure in the N-terminal 25 amino acids, including a 13-residue insertion, and little sequence homology over the C terminus (Fig. 1 B). Cotransfection of SF9 cells with a deterin-expressing plasmid rescued the cell viability from the apoptotic effects of reaper, the insect cell death activator (Fig.1 D). The gene encoding deterin was isolated in order to assess indications from gene structure of the functional organization of the protein. Thedeterin cDNA was used for polytene chromosome in situ hybridization, and a single specific signal was detected at band 90A1–A2, on the right arm of the third chromosome (not shown). The cDNA was also used to probe a P1 filter genomic library. Both of two overlapping, approximately 80-kilobase pair specific hybridizing clones were shown by restriction mapping to encode the same singledeterin sequence consistent with the in situresults. On this basis, it does not appear that sequences closely similar to the single detected deterin gene are located at loci outside of 90A1–A2. The transcription start site has not been mapped, but the protein coding sequence of the cDNA is the same as an expressed sequence tag sequence deposited into the BDGP Flybase data base (accession number AI260030). The protein coding sequence is interrupted by two introns of 77 and 61 base pairs and is flanked in the mature transcript by a 5′ untranslated region and a 3′ untranslated region of 91 bases (Fig. 1 A). The locations of the introns may also be informative in relation to the functional structure of the deterin protein. Despite the divergence in primary structure of the human survivin and the D. melanogaster deterin, the first intron in both genes is inserted in essentially identical positions (Fig. 1 B, closed arrow). Both first introns begin either immediately before (deterin) or immediately after (survivin) the codon for the basic amino acid at the beginning of the most conserved motif between the two protein sequences (↓50KMAEAGFYW58 for deterin, 37R↓MAEAGFIH45 for survivin). In addition, predictive secondary structural analysis for the encoded deterin protein suggests that the first exon encodes a protein region that is predominated by α-helical conformation (Fig. 1 C). In contrast, the second exon is predicted to encode a region nearly devoid of α-helical structure and instead has a primarily coiled coil conformation, along with most of the β-form present in the protein. Furthermore, the second intron is positioned at exactly the residue (Val111) predicted to mark both the end of the BIR repeat and the beginning of a final domain with a high helical density (Fig.1, B and C). This final domain was confirmed (as described below) as possessing a discrete biological activity relating to cell death. Nurse cells undergo apoptosis only after they have completed their function of delivering molecules and cytoplasmic components to the developing oocyte (48.Foley K. Cooley L. Development. 1998; 125: 1075-1082Crossref PubMed Google Scholar). The specific developmental timing of this apoptosis suggests a mechanism to prevent premature apoptosis in oocytes prior to that time. We analyzed the abundance of the deterin transcript in the ovaries of the adult female by in situ hybridization. The level of deterin mRNA increased sharply in later stage nurse cells, especially during stages 10 and 11 (Fig.2 D). The transcript accumulates in the darkly staining cytoplasm of the nurse cells, shortly before the dumping of cytoplasmic contents into the developing oocyte. The presence of abundant deterin transcript in the nurse cells just prior to dumping of their cytoplasmic contents into the oocyte suggested that the transcript may be found in the embryo. In situ hybridization detected the deterin transcript distributed throughout the embryo prior to stage 4 (not shown), consistent with a putative
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