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• W1980236481 abstract "Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L. Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L. Granzyme B (GrB), 1The abbreviations used are: GrB, granzyme B; IAP, inhibitor of apoptosis; XIAP, x-linked inhibitor of apoptosis; MEF, mouse embryo fibroblast; Mcl-1L, myeloid cell leukemia-1; Ab, antibody; mAb, monoclonal antibody; Z-VAD-Fmk, benzoyloxycarbonyl-VAD-fluoromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; nt, nucelotide(s); UTR, untranslated region; ORF, open reading frame; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ad, adenovirus; pfu, plaque-forming unit(s); MIB, mitochondrial buffer; Ac-IETD-CHO, acetyl-Ile-Glu-Thr-Asp-aldehyde; VDAC, voltage dependent anion-selective channel. 1The abbreviations used are: GrB, granzyme B; IAP, inhibitor of apoptosis; XIAP, x-linked inhibitor of apoptosis; MEF, mouse embryo fibroblast; Mcl-1L, myeloid cell leukemia-1; Ab, antibody; mAb, monoclonal antibody; Z-VAD-Fmk, benzoyloxycarbonyl-VAD-fluoromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; nt, nucelotide(s); UTR, untranslated region; ORF, open reading frame; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ad, adenovirus; pfu, plaque-forming unit(s); MIB, mitochondrial buffer; Ac-IETD-CHO, acetyl-Ile-Glu-Thr-Asp-aldehyde; VDAC, voltage dependent anion-selective channel. the prototype member of the granzyme family of serine proteases, shares substrate specificity with caspases for cleaving after aspartate residues (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar). GrB has been reported to cleave caspases, including caspase-3, -6, -7, -8, and -10 in vitro. It has been assumed that GrB has multiple entry points for initiating the caspase-dependent apoptotic cascade. However, GrB also activates cell death and apoptotic morphology in the presence of short peptide fluoromethyl ketones, which are potent caspase inhibitors, but do not inactivate GrB (2Lieberman J. Nat. Rev. Immunol. 2003; 3: 361-370Crossref PubMed Scopus (547) Google Scholar). These observations led to the notion that GrB may be capable of initiating an alternate death pathway in the presence of viral or cellular inhibitors of caspases. This concept is supported by the identification of caspase substrates that are also processed by GrB, including the sensor for initiation of DNA damage repair, poly-(ADP)ribose polymerase (3Froelich C.J. Hanna W.L. Poirier G.G. Duriez P.J. D'Amours D. Salvesen G.S. Alnemri E.S. Earnshaw W.C. Shah G.M. Biochem. Biophys. Res. Commun. 1996; 227: 658-665Crossref PubMed Scopus (91) Google Scholar); the nuclear mitotic apparatus protein (4Andrade F. Roy S. Nicholson D. Thornberry N. Rosen A. Casciola-Rosen L. Immunity. 1998; 8: 451-460Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar); inhibitor of caspase-activated DNase to liberate the caspase-activated DNase from its complex with the inhibitor (5Thomas D.A. Du C. Xu M. Wang X. Ley T.J. Immunity. 2000; 12: 621-632Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar); T cell receptor-ζ chain, which is essential for T cell signaling (6Wieckowski E. Wang G.Q. Gastman B.R. Goldstein L.A. Rabinowich H. Cancer Res. 2002; 62: 4884-4889PubMed Google Scholar); the catalytic subunit of DNA-dependent protein kinase, which is involved in repairing double-stranded DNA breaks (4Andrade F. Roy S. Nicholson D. Thornberry N. Rosen A. Casciola-Rosen L. Immunity. 1998; 8: 451-460Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar); and the nuclear-envelope intermediate-filament protein, lamin B (7Zhang D. Beresford P.J. Greenberg A.H. Lieberman J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5746-5751Crossref PubMed Scopus (130) Google Scholar).A potential role for mitochondria in the response to GrB has been indicated by the protection conferred by overexpression of Bcl-2, a member of a family of apoptotic regulators that resides mainly on the cytoplasmic side of intracellular membranes (8Roberts D.L. Goping I.S. Bleackley R.C. Biochem. Biophys. Res. Commun. 2003; 304: 513-518Crossref PubMed Scopus (16) Google Scholar). Recent studies have identified Bid as a direct substrate for GrB, and thereby as a direct link to a mitochondrial apoptotic cascade mediated by GrB (9Sutton V.R. Davis J.E. Cancilla M. Johnstone R.W. Ruefli A.A. Sedelies K. Browne K.A. Trapani J.A. J. Exp. Med. 2000; 192: 1403-1414Crossref PubMed Scopus (301) Google Scholar, 10Alimonti J.B. Shi L. Baijal P.K. Greenberg A.H. J. Biol. Chem. 2001; 276: 6974-6982Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Heibein J.A. Goping I.S. Barry M. Pinkoski M.J. Shore G.C. Green D.R. Bleackley R.C. J. Exp. Med. 2000; 192: 1391-1402Crossref PubMed Scopus (269) Google Scholar). Bid, a cytosolic BH3-only Bcl-2 family member, is cleaved by caspase-8, lysosomal proteases, or GrB. Although cleaved at different sites, each of the resultant truncated Bids translocates to the mitochondrial outer membrane, where it triggers the release of the pro-apoptotic proteins cytochrome c, SMAC/DIABLO, HtrA2/Omi, endonuclease G, and AIF (12Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (93) Google Scholar). Although in a cell-free system GrB-cleaved Bid is a potent inducer for the release of mitochondrial apoptotic proteins, a recent study questions whether cleavage of Bid by GrB occurs directly and independently of caspase activation under physiologic conditions (13Metkar S.S. Wang B. Ebbs M.L. Kim J.H. Lee Y.J. Raja S.M. Froelich C.J. J. Cell Biol. 2003; 160: 875-885Crossref PubMed Scopus (128) Google Scholar, 14Froelich C.J. Metkar S.S. Raja S.M. Cell Death Differ. 2004; 11: 369-371Crossref PubMed Scopus (20) Google Scholar). Despite this controversy regarding the direct role of GrB-cleaved Bid, the need for the mitochondrial amplification of the caspase pathway in GrB-mediated apoptosis is well established, particularly in tumor or viral infected target cells with increased expression of cellular or viral inhibitors of apoptosis (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar, 2Lieberman J. Nat. Rev. Immunol. 2003; 3: 361-370Crossref PubMed Scopus (547) Google Scholar, 8Roberts D.L. Goping I.S. Bleackley R.C. Biochem. Biophys. Res. Commun. 2003; 304: 513-518Crossref PubMed Scopus (16) Google Scholar). Cellular inhibitors of apoptosis (IAP), such as XIAP or cIAP1/2 that are overexpressed in numerous types of tumors (15Tamm I. Kornblau S.M. Segall H. Krajewski S. Welsh K. Kitada S. Scudiero D.A. Tudor G. Qui Y.H. Monks A. Andreeff M. Reed J.C. Clin. Cancer Res. 2000; 6: 1796-1803PubMed Google Scholar) directly block caspase-3, -7, and -9 (16Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1566) Google Scholar). Consequently, mitochondrial secreted antagonists of IAP, SMAC/DIABLO and HtrA2/Omi, are required to relieve the inhibited caspases (17Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 18Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 19Goping I.S. Barry M. Liston P. Sawchuk T. Constantinescu G. Michalak K.M. Shostak I. Roberts D.L. Hunter A.M. Korneluk R. Bleackley R.C. Immunity. 2003; 18: 355-365Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar).The mitochondrial function of tBid is dependent on the expression of either Bax and/or Bak (20Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3317) Google Scholar) and can be inhibited by overexpression of the anti-apoptotic proteins Bcl-2 and Bcl-XL (21Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar). Although a role for GrB-cleaved Bid in the physiologic induction of a mitochondrial apoptotic cascade is controversial, it has been documented that a deficiency in both Bax and Bak attenuates the mitochondrial response to GrB (13Metkar S.S. Wang B. Ebbs M.L. Kim J.H. Lee Y.J. Raja S.M. Froelich C.J. J. Cell Biol. 2003; 160: 875-885Crossref PubMed Scopus (128) Google Scholar, 22Wang G.-Q. Wieckowski E. Goldstein L.A. Gastman B.R. Rabinovitz A. Gambotto A. Li S. Fang B. Yin X.-M. Rabinowich H. J. Exp. Med. 2001; 194: 1325-1337Crossref PubMed Scopus (66) Google Scholar). Furthermore, embryonic fibroblasts (MEFs) from Bid–/– mice or Bax/Bak double knockout mice still have disrupted mitochondrial transmembrane potential in response to GrB (23Thomas D.A. Scorrano L. Putcha G.V. Korsmeyer S.J. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14985-14990Crossref PubMed Scopus (102) Google Scholar). These findings imply that one or more cytosolic mediators other than Bid may act as a link between GrB and the mitochondria. Alternatively, GrB may act directly on mitochondrial outer membrane proteins providing an explanation for the ability of GrB to disrupt the mitochondrial transmembrane potential in a caspase- and Bid-independent manner (10Alimonti J.B. Shi L. Baijal P.K. Greenberg A.H. J. Biol. Chem. 2001; 276: 6974-6982Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 13Metkar S.S. Wang B. Ebbs M.L. Kim J.H. Lee Y.J. Raja S.M. Froelich C.J. J. Cell Biol. 2003; 160: 875-885Crossref PubMed Scopus (128) Google Scholar).Mitochondrial response to apoptotic stimuli is determined by the balance between pro- and anti-apoptotic Bcl-2 family members. Anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-XL, and Mcl-1L (myeloid cell leukemia-1) protect against mitochondrial apoptotic events, whereas pro-apoptotic Bcl-2 family members, including Bax, Bak, and BH3-only proteins, promote the release of apoptogenic proteins from the mitochondria. Mcl-1 is an anti-apoptotic Bcl-2 family protein that was discovered as an early induction gene during myeloblastic leukemic cell differentiation (24Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (873) Google Scholar). The biological significance of Mcl-1 has been elucidated by recent studies demonstrating that a Mcl-1 deficiency results in peri-implantation embryonic lethality (25Rinkenberger J.L. Horning S. Klocke B. Roth K. Korsmeyer S.J. Genes Dev. 2000; 14: 23-27Crossref PubMed Google Scholar). Increased expression of endogenous full-length Mcl-1 is associated with the maintenance of cell viability and decreased expression with cell death (24Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (873) Google Scholar, 26Lomo J. Smeland E.B. Krajewski S. Reed J.C. Blomhoff H.K. Cancer Res. 1996; 56: 40-43PubMed Google Scholar, 27Vrana J.A. Bieszczad C.K. Cleaveland E.S. Ma Y. Park J.P. Mohandas T.K. Craig R.W. Cancer Res. 2002; 62: 892-900PubMed Google Scholar). A short splice variant of Mcl-1, Mcl-1S, has recently been identified as a BH3 domain only pro-apoptotic protein (28Bae 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, 29Bingle 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 (157) Google Scholar). Despite numerous studies on the induction of Mcl-1, scarce information is available regarding the mechanisms involved in its down-regulation. Based on the effects of proteasome inhibitors, two recent studies proposed that Mcl-1L is degraded by proteasomes in response to UV or actinomycin D, in HeLa or multiple myeloma cell lines, respectively (30Zhang B. Gojo I. Fenton R.G. Blood. 2002; 99: 1885-1893Crossref PubMed Scopus (344) Google Scholar, 31Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (516) Google Scholar).Our studies, described below, are the first to report that Mcl-1L is a direct substrate for caspase-3 and GrB. We have also identified Bim, a BH3-only protein, as a high affinity binding partner for Mcl-1. The Mcl-1L/Bim cooperation may constitute an alternative mitochondrial apoptotic pathway that could be activated by the direct effect of GrB on the mitochondria, independent of Bid.EXPERIMENTAL PROCEDURESReagents—Anti-human Mcl-1 Abs were from Oncogene (Boston, MA; Ab-2, mouse clone RC13 generated against recombinant Mcl-1L), and from Santa Cruz Biotechnology (Santa Cruz, CA; Ab-1, mAb generated against recombinant Mcl-1L; and Ab-3, polyclonal Ab generated against 20-amino acid residue synthetic peptide of human Mcl-1L). Anti-β-actin mAb (clone AC-15) was purchased from Sigma (St. Louis, MO); anti-cytochrome c oxidase IV (Cox IV) Ab was from Molecular Probes (Eugene, OR); rabbit anti-Bid Ab was a generous gift from Dr. Xiaodong Wang (Southwestern Medical Center, Dallas, TX). Anti-caspase-3, caspase-8, cytochrome c, and XIAP were from BD Pharmingen; anti-Bim Ab were from ProSci (Poway, CA) and from Apoptech (San Diego, CA, clone 14A8); anti-SMAC mAb from Apoptech (clone 10G7); anti-AIF, Bcl-2, and Bcl-XL were from Santa Cruz Biotechnology; Z-VAD-Fmk and Ac-IETD-CHO were from ICN (Aurora, OH). [35S]Methionine, Protein A-Sepharose beads, and Protein G-Sepharose beads were from Amersham Biosciences (Piscataway, NJ).Cell Lines, Cell Lysates, and Cell Extracts—Jurkat T leukemic cell line and the HeLa tumor cell line were obtained from American Type Culture Collection (ATCC, Rockville, MD). Jurkat cells were grown in RPMI 1640 medium containing 10% fetal calf serum, 20 mm HEPES, 2 mm l-glutamine, and 100 units/ml each of penicillin and streptomycin. The Bak-deficient Jurkat clonal cell line was obtained from wild-type Jurkat cells (22Wang G.-Q. Wieckowski E. Goldstein L.A. Gastman B.R. Rabinovitz A. Gambotto A. Li S. Fang B. Yin X.-M. Rabinowich H. J. Exp. Med. 2001; 194: 1325-1337Crossref PubMed Scopus (66) Google Scholar). HeLa cells were grown in Dulbecco's modified Eagle's medium containing 15% fetal calf serum, 20 mm l-glutamine, and 100 units/ml each of penicillin and streptomycin. Cell lysates were prepared with 1% Nonidet P-40, 20 mm Tris base, pH 7.4, 137 mm NaCl, 10% glycerol, 1 mm PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin. Cellular lysates were obtained as the supernatants resulting from centrifugation at 14,000 × g at 4 °C for 30 min. To prepare cell extracts for GrB or caspase-3 reactions, cultured Jurkat cells were washed twice with phosphate-buffered saline and then resuspended in ice-cold buffer (20 mm HEPES, pH 7.0, 10 mm KCl, 1.5 mm MgCl2, 1 mm sodium EDTA, 1mm sodium EGTA, 1 mm dithiothreitol, 250 mm sucrose, and protease inhibitors). After incubation on ice for 20 min, cells (2.5 × 106/0.5 ml) were disrupted by Dounce homogenization. Nuclei were removed by centrifugation at 650 × g for 10 min at 4 °C.Molecular Cloning of Mcl-1L—Total RNA was isolated from HeLa cells using RNA STAT-60 Reagent (Tel-Test “B”, Inc.). Reverse transcription was carried out with 5 μg of total RNA using the oligonucleotide primer Mcl-R, 5′-TACAGCTTGGAGTCCAACTGC-3′, which is complementary to nucleotides (nt) 1779–1799 in the 3′-untranslated region (UTR) of the Mcl-1L mRNA sequence and SuperScript III RNase H– reverse transcriptase (Invitrogen). PCR was performed using the GC-Rich PCR System kit (Roche Applied Science). Amplicons containing the entire open reading frame (ORF) of Mcl-1L were generated using the forward primer Mcl-F, 5′-CTGGCAATGTTTGGCCTCAAA-3′, which corresponds to nt 658–678 and thus extends by 6 nt into the 5′-UTR and the reverse primer Mcl-R (see above). The Mcl-1L amplicon was size-selected and purified using a 1% agarose gel and the QIAquick gel extraction kit (Qiagen). Amplicons were subcloned into the vector pCR3.1 by utilizing the Eukaryotic TA Expression kit (Invitrogen). Mcl-1L clones were confirmed by automated DNA sequence analysis (University of Pittsburgh DNA Sequencing Core Facility) of randomly picked colonies.Generation of Mcl-1S—We produced a Mcl-1S splice variant cDNA clone based on the published sequence (GenBank™ accession number AF203373) utilizing deletion mutagenesis of Mcl-1L cDNA by overlap extension using PCR. To generate the deletion site, two primers were designed as follows: the forward primer Mcl-10, 5′-GGCCTTCCAAGGATGGGTTTGTGGAGTTCTTCC-3′, which corresponds to nt 1340–1352 and 1601–1621, and the reverse primer Mcl-11, 5′-CCACAAACCCATCCTTGGAAGGCCGTCTCGTGG-3′, which is complementary to nt 1612–1601 and 1352–1331. These primers overlap the sequence region (nt 1353–1600) that is deleted from Mcl-1L to generate the Mcl-1S variant. PCR was carried out with Mcl-1L cDNA and primer pairs Mcl-F (see above) and Mcl-11 and Mcl-R (see above) and Mcl-10, respectively, using the Expand Long Template PCR system (Roche Applied Science). The deletion mutant amplicons (0.5 μl of each) were combined in a subsequent PCR reaction using primers Mcl-F and Mcl-R to produce the putative Mcl-1S amplicon. This amplicon was then gel-purified and subcloned as above for Mcl-1L. Sequence analysis (as above) of randomly picked clones confirmed the Mcl-1S sequence.Molecular Cloning of Human BimEL—Total RNA was isolated from Jurkat T cells as described for Mcl-1L. First strand cDNA synthesis was carried out using SuperScript II RNase H– reverse transcriptase and oligo(dT)12–18 primer (Invitrogen). PCR was performed with the Expand Long Template PCR system (Roche Applied Science) and a forward and reverse primer pair specific for the amino and carboxyl-terminal ends, respectively, of BimEL, BimL, and BimS ORFs. The sequence of the forward Bim primer is 5′-GCCACCATGGCAAAGCAACCTTCTGAT-3′, whereas the reverse primer is 5′-TCAATGCATTCTCCACACCAG-3′. Reaction products were separated on a 1% agarose gel, and a band corresponding in size (∼600 bp) to the BimEL ORF was excised and DNA extracted as described for Mcl-1L. The putative BimEL amplicon was subcloned into the vector pCR3.1 as above. Random clones were sequenced also as above to confirm the presence of the BimEL ORF.Preparation of His-tagged BimL—Mouse amino-terminal histidine-tagged BimL was expressed from a recombinant plasmid produced by ligating an NdeI-XhoI-digested BimL amplicon produced with the Expand Long Template PCR system kit (Roche Applied Science) utilizing mouse BimL cDNA and the primers BimL-F, 5′-GGAATTCCATATGGCCAAGCAACCTTCT-3′, and BimL-R, 5′-CCGCTCGAGTCAATGCCTTCTCCATACCAG-3′, into the NdeI-XhoI-digested bacterial expression vector pET-14b (Novagen). Escherichia coli strain BL21(DE3) cells were transformed and cultured at 37 °C in Terrific Broth. The induction of expression was started at an A600 of 0.8–1.0 by the addition of 0.4 mm, isopropyl β-d-thiogalactoside with continued incubation of the culture at 37 °C for 2–3 h. The bacterial pellets were resuspended and sonicated in a buffer containing 5 mm imidazole, 500 mm NaCl, and 20 mm Tris-HCl, pH 7.9. After centrifugation, the cleared supernatants were passed through a His-Bind nickel agarose affinity chromatographic column pre-charged with 50 mm NiSO4 (Novagen). The columns were washed with wash buffer containing 60 mm imidazole, 500 mm NaCl, and 20 mm Tris-HCl, pH 7.9. His-tagged Bim was eluted with elution buffer containing 400 mm imidazole, 500 mm NaCl, and 20 mm Tris-Cl, pH 7.9, and were further purified using a Sephadex G-50 column equilibrated with phosphate-buffered saline. A single major band was detected by SDS-PAGE stained with Coomassie Blue.GrB-induced Apoptosis—CTL-free apoptosis was induced by incubation of target cells with GrB (33–330 nm) and replication-deficient adenovirus type V (Ad; 10–100 pfu/ml) for 6 h. The cells were then washed to remove excess exogenous GrB. To avoid the enzymatic activity of GrB during the lysis procedure, the GrB inhibitor, Ac-IETD-CHO (500 μm) was added to the lysis buffer.Cellular Fractionation and Mitochondria Purification—To obtain an enriched mitochondrial fraction, Jurkat or HeLa cells were suspended in mitochondrial buffer (MIB) composed of 0.3 m sucrose, 10 mm MOPS, 1 mm EDTA, and 4 mm KH2PO4, pH 7.4, and lysed by Dounce homogenization as previously described (32Petit P.X. O'Connor J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Crossref PubMed Scopus (213) Google Scholar, 33Matsko C.M. Hunter O.C. Rabinowich H. Lotze M.T. Amoscato A.A. Biochem. Biophys. Res. Commun. 2001; 287: 1112-1120Crossref PubMed Scopus (65) Google Scholar). Briefly, nuclei and debris were removed by a 10-min centrifugation at 650 × g, and a pellet containing mitochondria was obtained by two successive spins at 10,000 × g for 12 min. To obtain the S-100 fraction, the postnuclear supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C. To obtain the enriched mitochondrial fraction, the mitochondria containing pellet was resuspended in MIB and layered on a Percoll gradient consisting of four layers of 10, 18, 30, and 70% Percoll in MIB. After centrifugation for 30 min at 15,000 × g, the mitochondrial fraction was collected at the 30/70 interface. Mitochondria were diluted in MIB containing 1 mg/ml bovine serum albumin (at least a 10-fold dilution required to remove Percoll). The mitochondrial pellet was obtained by a 40-min spin at 20,000 × g and used immediately. Purity was assessed by electron microscopy and by enzyme marker analysis (33Matsko C.M. Hunter O.C. Rabinowich H. Lotze M.T. Amoscato A.A. Biochem. Biophys. Res. Commun. 2001; 287: 1112-1120Crossref PubMed Scopus (65) Google Scholar). For enzyme analysis, the following enzymes were assayed: aryl sulfatase (lysosomes/granules); N-acetyl-β-d-glucosaminidase, α-l-fucosidase, and β-glucuronidase (lysosome); lactate dehydrogenase (cytosol); cytochrome oxidase or monoamine oxidase (mitochondria); thiamine pyrophosphatase (Golgi); NADH oxidase (endoplasmic reticulum); and dipeptidyl peptidase IV (plasma membrane). The purity was assessed at 95%, with ∼5% or less contamination from the microsomal fraction. When indicated, mitochondria were pelleted then incubated in 0.1 m Na2CO3, pH 11.5, for 20 min on ice to remove loosely attached proteins (34Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). The alkali treatment was not associated with the release of mitochondrial intermembrane proteins, such as SMAC or AIF. Following alkali treatment, supernatants and mitoplasts were separated by centrifugation and boiled in SDS sample buffer. The fractions were analyzed by immunoblotting.Release of Mitochondrial Apoptogenic Proteins—Purified mitochondria (50 μg of protein) were incubated with His-BimL at various doses as indicated, in 25 μl of MIB at 37 °C for 30 min. Mitochondria were pelleted by centrifugation at 10,000 × g for 10 min. The resulting supernatants or mitochondria were mixed with SDS sample buffer and analyzed by SDS-PAGE and immunoblotting for the presence of mitochondrial apoptogenic proteins.In Vitro Transcription-Translation—Mcl-1L, Mcl-1S, and BimEL cDNAs were expressed in the TnT T7 transcription-translation reticulocyte lysate system (Promega). Each coupled transcription-translation reaction contained 1 μg of plasmid DNA in a final volume of 50 μl in a methionine-free reticulocyte lysate reaction mixture supplemented with 35S-labeled methionine according to the manufacturer's instructions. After incubation at 30 °C for 90 min, the reaction products were immediately used or stored at –70 °C.In Vitro Cleavage Reaction with Caspase-3 or GrB—In vitro cleavage reactions were performed in total volume of 20 μl. The reaction buffer consisted of 20 mm HEPES (pH 7.4), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 20% glycerol, 1 mm PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Each reaction also contained 3 μl of reticulocyte lysate containing 35S-labeled Mcl-1L, Mcl-1S, or BimEL and reticulocyte lysate minus plasmid in the presence or the absence of recombinant caspase-3 (5–100 nm) or GrB (33–330 nm) for 20 min at 37 °C. The reactions were terminated by addition of SDS loading buffer and boiled for 5 min.Immunoprecipitation—For Mcl-1 and Bim immunoprecipitation experiments cells (5–10 × 106) and mitochondria (200 μg of protein) were lysed in 1% CHAPS buffer (20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm PMSF, and 1% CHAPS). The lysates were precleared with Protein A- or G-Sepharose beads and incubated with anti-Mcl-1 or anti-Bim Abs at 4 °C for 4 h. The immune complexes were then precipitated with Protein A- or G-Sepharose beads at 4 °C overnight. The pellets were washed four times with the appropriate lysis buffer and boiled for 5 min in SDS sample buffer.Western Blot Analysis—Proteins in cell lysates, cell extracts, mitochondria, or S-100 were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, as previously described (35Johnson D.E. Gastman B.R. Wieckowski E. Wang G.Q. Amoscato A. Delach S.M. Rabinowich H. Cancer Res. 2000; 60: 1818-1823PubMed Google Scholar). Following probing with a specific primary Ab and horseradish peroxidase-conjugated secondary Ab, the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL).RESULTSBid-dependent and Bid-independent Mechanisms of Mitochondrial Release of Apoptogenic Proteins in Response to Direct Application of GrB—GrB has been reported to have a direct effect on mitochondria that results in the release of cytochrome c (10Alimonti J.B. Shi L. Baijal P.K. Greenberg A.H. J. Biol. Chem. 2001; 276: 6974-6982Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Such GrB dose-dependent release of apoptogenic proteins, including, cytochrome c, SMAC, and AIF (Fig. 1A) may be mediated by full-length Bid that in addition to its cytoplasmic localization, is also associated with purified mitochondria. However, full-length Bid is loosely attached to purified mitochondria, because it is entirely removed by treatment with alkali, and therefore it is probably not anchored to the mitochondrial outer membrane (Fig. 1B). Exposure of purified mitochondria to GrB results in the processing of mitochondria-associated Bid as indicated by its reduced level of detection (Fig. 1C). To investigate the significance of mitochondria-attached full-length Bid in the response to direct application of GrB, we utilized liver mitochondria purified from Bid–/– mice. Direct application of various doses of GrB (33–330 nm) to murine wild-type liver mitochondria resulted in a significant release of cytochrome c (Fig. 1D). We confirmed that some full-" @default.
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• W1980236481 cites W1970932773 @default.
• W1980236481 cites W1973363947 @default.
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• W1980236481 cites W1981231641 @default.
• W1980236481 cites W1984547206 @default.
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• W1980236481 cites W2002973070 @default.
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• W1980236481 cites W2019412595 @default.
• W1980236481 cites W2026391442 @default.
• W1980236481 cites W2029612038 @default.
• W1980236481 cites W2031475867 @default.
• W1980236481 cites W2047394489 @default.
• W1980236481 cites W2054998458 @default.
• W1980236481 cites W2057942504 @default.
• W1980236481 cites W2068384792 @default.
• W1980236481 cites W2070156993 @default.
• W1980236481 cites W2076020247 @default.
• W1980236481 cites W2078934458 @default.
• W1980236481 cites W2080389159 @default.
• W1980236481 cites W2084318835 @default.
• W1980236481 cites W2092759765 @default.
• W1980236481 cites W2098160574 @default.
• W1980236481 cites W2102443458 @default.
• W1980236481 cites W2102954334 @default.
• W1980236481 cites W2103699489 @default.
• W1980236481 cites W2110077740 @default.
• W1980236481 cites W2125963106 @default.
• W1980236481 cites W2128229887 @default.
• W1980236481 cites W2132411546 @default.
• W1980236481 cites W2132740145 @default.
• W1980236481 cites W2144951893 @default.
• W1980236481 cites W2146983373 @default.
• W1980236481 cites W2152247516 @default.
• W1980236481 cites W2157554916 @default.
• W1980236481 cites W2166219468 @default.
• W1980236481 cites W2166601968 @default.
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