Tissue Transglutaminase Is a Multifunctional BH3-only Protein
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m410938200
ISSN1083-351X
AutoresCarlo Rodolfo, Elisabetta Mormone, Paola Matarrese, Fabiola Ciccosanti, Maria Grazia Farrace, Elvira Garofano, Lucia Piredda, Gian María Fimia, Walter Malorni, Mauro Piacentini,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoTissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-XL. Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity. Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-XL. Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity. Tissue or type 2 transglutaminase (TG2, 1The abbreviations used are: TG2, tissue transglutaminase; CsA, cyclosporin a;Δψ, mitochondrial membrane potential; JC-1, 5–5′,6–6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide; MMP, mitochondrial membrane potential; PI, propidium iodide; STS, staurosporine; PLC, phospholipase C; HIV, human immunodeficiency virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; z-VAD, carbobenzoxy-valyl-alanyl-aspartyl; FCS, fetal calf serum; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; TMRM, tetramethylrhodamine ester; cyt c, cytochrome c; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; ANT, antennapedia. 1The abbreviations used are: TG2, tissue transglutaminase; CsA, cyclosporin a;Δψ, mitochondrial membrane potential; JC-1, 5–5′,6–6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide; MMP, mitochondrial membrane potential; PI, propidium iodide; STS, staurosporine; PLC, phospholipase C; HIV, human immunodeficiency virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; z-VAD, carbobenzoxy-valyl-alanyl-aspartyl; FCS, fetal calf serum; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; TMRM, tetramethylrhodamine ester; cyt c, cytochrome c; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; ANT, antennapedia. EC 2.3.2.13) is a multifunctional enzyme belonging to the transglutaminase family (1Lorand L. Graham R.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 140-156Crossref PubMed Scopus (1193) Google Scholar). Its primary enzymatic activity resides in a Ca2+-dependent reaction in which γ-carboxyamide groups of peptide-bound glutamine residues serve as acyl donors and primary amino groups of several compounds function as acceptor substrates (2Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). This reaction results in the post-translational modification of proteins by establishing ϵ(γ-glutamyl)lysine and N,N-bis(γ-glutamyl)polyamine isodipeptide linkages (3Folk J.E. Annu. Rev. Biochem. 1980; 49: 517-531Crossref PubMed Scopus (869) Google Scholar). Covalent TG2-dependent cross-linking leads to the polymerization of substrate proteins that can be dismantled only by the proteolytic degradation of the protein chains (4Fesus L. Thomazy V. Autuori F. Ceru M.P. Tarcsa E. Piacentini M. FEBS Lett. 1989; 245: 150-154Crossref PubMed Scopus (226) Google Scholar). While Ca2+ is a positive regulator of TG2 enzymatic activity, GTP is a negative one. It has been demonstrated that high intracellular GTP concentrations shift the enzyme activity from cross-linking to G-protein. At physiological intracellular GTP concentration, TG2 might act as the Gαh subunit of the GTP-binding protein (Gh) and form a ternary complex in association with the 50-kDa β subunit (Gβh) and the α1-adrenergic receptor (5Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar). Thus, the TG2/Gαh proves to be a multifunctional protein, which by binding GTP in a GαhGTP complex, can modulate receptor-stimulated phospholipase-C (PLC) activation. The binding of GTP to TG2/Gαh prevents the activation of the pro-apoptotic cross-linking activity (6Iismaa S.E. Chung L. Wu M.J. Teller D.C. Yee V.C. Graham R.M. Biochemistry. 1997; 36: 11655-11664Crossref PubMed Scopus (72) Google Scholar, 7Iismaa S.E. Wu M.J. Nanda N. Church W.B. Graham R.M. J. Biol. Chem. 2000; 275: 18259-18265Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 8Liu S. Cerione R.A. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Crossref PubMed Scopus (274) Google Scholar, 9Singh U.S. Cerione R.A. J. Biol. Chem. 1996; 271: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). It has been proposed that the fine modulation of TG2 enzymatic activity exerted by GTP, Ca2+, and nitric oxide allows cells to survive in the presence of high TG2 protein levels (10Bernassola F. Rossi A. Melino G. Ann. N. Y. Acad. Sci. 1999; 887: 83-91Crossref PubMed Scopus (35) Google Scholar). In addition, recent studies suggest that TG2 in its G protein configuration could lead to prevention of cell death (11Antonyak M.A. Singh U.S. Lee D.A. Boehm J.E. Combs C. Zgola M.M. Page R.L. Cerione R.A. J. Biol. Chem. 2001; 276: 33582-33587Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12Boehm J.E. Singh U. Combs C. Antonyak M.A. Cerione R.A. J. Biol. Chem. 2002; 277: 20127-20130Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 13Tucholski J. Johnson G.V. J. Neurochem. 2002; 81: 780-791Crossref PubMed Scopus (77) Google Scholar). In addition to these two main enzymatic activities, it has been proposed very recently that TG2 might also function as a protein-disulfide isomerase (14Hasegawa G. Suwa M. Ichikawa Y. Ohtsuka T. Kumagai S. Kikuchi M. Sato Y. Saito Y. Biochem. J. 2003; 373: 793-803Crossref PubMed Scopus (182) Google Scholar) and as a kinase (15Mishra S. Murphy L.J. J. Biol. Chem. 2004; 279: 23863-23868Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), depending on its subcellular localization. Under physiological conditions, TG2 is a ubiquitous enzyme, although high expression levels could be detected only in particular cell subsets (e.g. endothelial, mesangial, and smooth muscle cells). However, during both physiological (i.e. mammary gland regression, interdigital web shaping) and pathological (i.e. HIV infection, hepatitis) onset of apoptosis, a large increase in the enzyme synthesis and cross-linking activity could be observed (16Amendola A. Gougeon M.L. Poccia F. Bondurand A. Fesus L. Piacentini M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11057-11062Crossref PubMed Scopus (123) Google Scholar, 17Szegezdi E. Szondy Z. Nagy L. Nemes Z. Friis R.R. Davies P.J. Fesus L. Cell Death Differ. 2000; 7: 1225-1233Crossref PubMed Scopus (33) Google Scholar, 18Nardacci R. Ciccosanti F. Falasca L. Lo Iacono O. Amendola A. Antonucci G. Piacentini M. Cell Death Differ. 2003; 10: S79-S80Crossref PubMed Scopus (13) Google Scholar). During apoptosis, the drop of intracellular GTP concentration and the increase of intracellular Ca2+ levels are responsible for the activation of TG2 cross-linking activity, which results in the assembly of highly cross-linked intracellular protein polymers (4Fesus L. Thomazy V. Autuori F. Ceru M.P. Tarcsa E. Piacentini M. FEBS Lett. 1989; 245: 150-154Crossref PubMed Scopus (226) Google Scholar). This detergent-insoluble protein scaffold contributes to the stabilization of dying cells, before their clearance by phagocytosis. In addition to this "late" contribution to the apoptotic process, TG2 might act also as an upstream regulator of the mitochondrial pathway. In a previous report (19Piacentini M. Farrace M.G. Piredda L. Matarrese P. Ciccosanti F. Falasca L. Rodolfo C. Giammarioli A.M. Verderio E. Griffin M. Malorni W. J. Neurochem. 2002; 81: 1061-1072Crossref PubMed Scopus (117) Google Scholar), we showed that overexpression of TG2 in neural cells results in a 4-5-fold more rapid accomplishment of the death program, as compared with their parental counter-part that expresses low levels of TG2. We observed that mitochondria of TG2-overexpressing cells were greatly modified with respect to both their ultrastructure and physiology. Mitochondria appeared often clustered in discrete cytoplasmic regions, with few cristae and extremely electron-dense matrix. These ultrastructural modifications are coupled with a constitutive hyperpolarization of the organelles, a decrease of intracellular GSH level and an increased reactive oxygen species (ROS) production. The TG2-dependent hyperpolarization of mitochondria preceded and was separated from the PTP opening and the release of cytochrome c, which took place only upon induction of apoptosis. Prompted by these results, we investigated whether TG2 interaction/modification of proteins known to influence mitochondrial physiology and/or to control the mitochondrial apoptosis pathway might play a role in the observed commitment to death. To address these questions, we investigated the effect exerted by TG2 accumulation in mitochondria, focusing our attention to the possible interaction of TG2 with Bcl-2 family members. The choice of this class of protein was basically because of their well defined role in the regulation of the mitochondrial pathway of apoptosis (20Martinou J.C. Green D.R. Nat. Rev. Mol. Cell Biol. 2001; 2: 63-67Crossref PubMed Scopus (844) Google Scholar, 21Zamzami N. Kroemer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 67-71Crossref PubMed Scopus (882) Google Scholar). It has been widely accepted that the pro-apoptotic factors Bax and Bak are important regulators of the mitochondrial permeability transition and that other Bcl-2 family members could modulate their action. In particular, the anti-apoptotic Bcl-2 and Bcl-XL are able to inhibit Bax/Bak mitochondrial action, while the proapoptotic BH3-only subfamily are activators of Bax and Bak, also at the mitochondrial level (22Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 23Scorrano L. Korsmeyer S.J. Biochem. Biophys. Res. Commun. 2003; 304: 437-444Crossref PubMed Scopus (626) Google Scholar). Our results demonstrate for the first time that TG2 possesses a well defined and functional BH3 domain. Through this domain the enzyme might behave as a BH3-only protein, able to interact, even in the absence of any apoptotic stimuli, with pro-apoptotic members Bax and Bak, but not with anti-apoptotic Bcl-2 and Bcl-XL. Upon cell death induction, the interaction with Bax increased, and many mitochondrial proteins were post-translationally modified by TG2. It is worth noting that Bax acts as one of the major mitochondrial substrates, and its modification led to the formation of large molecular weight polymers. Cell Culture and Transfections—Human SK-n-BE(2) neuroblastoma cells and stably transfected TG2 derivatives (TGA) were grown as described (24Melino G. Annicchiarico-Petruzzelli M. Piredda L. Candi E. Gentile V. Davies P.J. Piacentini M. Mol. Cell. Biol. 1994; 14: 6584-6596Crossref PubMed Scopus (254) Google Scholar). For all experiments, cells were seeded in tissue culture flasks, multiwell plates, and chamber slides (NUNC), and allowed to attach overnight before treatment. The seeding density varied according to the type of experiment. Expression vectors for TG2 wild type (TG2-wt), deleted of the BH3 domain (TG2-ΔBH3), with cysteine 277 mutated to serine and with leucine 204 mutated to aspartic acid (TG2-LE), were generated by PCR and cloning in the pcDNA4/HisMax vector (Invitrogen). Deletion of the TG2-BH3 domain was accomplished by substitution of the nucleotides corresponding to the amino acids 204LKNAGRDCS212 with an EcoRI site via PCR. Mutations of the leucine 204 (CTG-GAG) and cysteine 277 (TGC-AGC) were also accomplished by PCR, with primers containing the mutations. Transient transfections of SK-n-BE(2) cells were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cytofluorimetric analysis of cell death induction by staurosporine (1 μm) was conducted 48 h after transfection. Synthetic Peptides and Cell Treatment—Peptides bearing the BH3 sequence of interest fused to antennapedia (ANT) sequence, as to allow cell entry, were designed as follows: ANT: NH2-RQIKIWFQNRRMKWKK-COOH; ANT-BH3 TG2: NH2-RQIKIWFQNRRMKWKKDVNPKFLKNAGRDCSR-COOH; ANT-BH3 Bax: NH2-RQIKIWFQNRRMKWKKKKLSECLKRIGDELDS-COOH; ANT-BH3 TG2 L-E; NH2-RQIKIWFQNRRMKWKKDVNPKFEKNAGRDCSR-COOH. All peptides were synthesized, with or without biotin at the N terminus, by Sigma-Genosys, purified by HPLC and controlled by mass spectroscopy. Upon arrival peptides were dissolved in Me2SO at 50 or 100 mm and stored in aliquots at -80 °C. SK-n-BE(2) cells were treated with 50 μm ANT-BH3 peptides for 30 min in culture medium without FCS, then supplemented with FCS, and incubation was continued for the indicated time period. Induction of Apoptosis by ANT-BH3 Peptides and Cell Viability Assessment—For cell viability assessment (CellTiter Proliferation Assay, Promega) 1-2 × 104 cells/well were seeded in 0.2 ml of complete culture medium in 96-well tissue culture plates. After 24 h, cells were washed with culture medium without FCS and treated with peptides (50 μm). At the indicated time, measurement of cell viability was determined following the manufacturer's protocol. For apoptosis evaluation and measurement of mitochondrial membrane potential (MMP) 2 × 106 cells were seeded in 25-cm2 tissue culture flasks and after 24 h treated as above. Quantitative flow cytometry evaluation of apoptosis was performed by double staining, using the FITC-conjugated annexin V/propidium iodide (PI) apoptosis detection kit (Eppendorf) and evaluation of DNA fragmentation in ethanol-fixed cells using PI (Sigma). For cell death inhibition, cells were treated 15 min with 1 mm cyclosporin A (CsA, Sigma) before adding BH3 peptides. Analysis of Mitochondrial Membrane Potential in Living Cells— Analysis of MMP in control and peptide-treated cells was conducted with the JC-1 probe. Briefly, cells were stained with 10 μm JC-1 (Molecular Probes) as previously described (25Cossarizza A. Cooper E.L. Quaglino D. Salvioli S. Kalachnikova G. Franceschi C. Biochem. Biophys. Res. Commun. 1995; 214: 503-510Crossref PubMed Scopus (40) Google Scholar) and analyzed by flow cytometry. Tetramethylrhodamine ester (1 μm, TMRM, Molecular Probes) was also used to confirm the data obtained using JC-1. Analysis of Cytochrome c Release in Living Cells and on Isolated Mitochondria—For cytochrome c release assays, 1 × 106 cells were seeded in 6-well plates and after 24 h were treated with 50 μm BH3 peptides as described above. At the indicated times, cells were harvested by trypsin treatment and collected by centrifugation. After washing three times in PBS, cells were suspended in 250 μl of Hypotonic Buffer (2 mm MgCl2, 10 mm KCl, 10 mm Tris-HCl, pH 7.6) supplemented with complete protease inhibitor mixture (Roche Applied Science), and incubated on ice for 10 min. Cells were homogenized with a Teflon homogenizer with B-type pestle as previously reported (26Zamzami N. Maisse C. Metivier D. Kroemer G. Methods Cell Biol. 2001; 65: 147-158Crossref PubMed Google Scholar) and, after addition of an equal volume of Mitochondria Buffer (400 mm sucrose, 10 mm TES, 400 μm EGTA, pH 7.2), centrifuged twice for 10 min at 900 × g at 4 °C. The supernatant was recovered and further centrifuged for 15 min at 17,000 × g at 4 °C. The pellet fraction was considered to be the mitochondria, and the supernatant was the cytosol. Release of cytochrome c was assessed by Western blot with monoclonal anti-cyt c antibody (65981A, BD PharMingen). Anti-COX subunit IV antibody (20E8-C12, Molecular Probes) was used as a control of mitochondrial isolation. For peptide treatments, mitochondria were isolated with a further 10 min centrifugation at 10,000 × g at 4 °C of the 900 × g supernatant, obtained as above. The mitochondrial pellet was suspended in swelling buffer (SB: 0.1 m sucrose, 0.5 m sodium succinate, 50 mm EGTA, pH 7.4, 1 m H3P04, 0.5 m MOPS, 2 mm rotenone), kept on ice, and used within 2 h from the preparation. Mitochondria (1 mg of protein/ml) were treated with 10 and 50 μm of different BH3 peptides in the presence or absence of 5 μg/ml cyclosporin a for 20 min at room temperature, followed by centrifugation (10,000 × g, 10 min, 4 °C). Proteins contained in the supernatants were then concentrated in 90% acetone (4 °C, 18 h; centrifugation at 17,000 × g, 20 min, 4 °C), and the precipitate was subjected to Western blot with anti-cytochrome c antibody. Swelling Experiments—For swelling experiments mitochondria, isolated as described above, were suspended in 1.5 ml of SB (0.5 mg protein/ml). As a general rule, 4 min after starting to record, the reagents to be tested were added. Total recording time was 20 min. As a positive control we used 300 μm CaCl2, which opens the protein transition pore causing high amplitude swelling, accompanied by a decrease of ΔΨ and an increase of outer membrane permeability. CsA (5 μg/ml) added before Ca2+ prevented ΔΨ loss. ΔΨ measurement was performed by cytofluorimetric analysis of TMRM (1 μm; Molecular Probes) incorporation. Low levels of TMRM incorporation (revealed by a decrease of red fluorescence) indicated a low ΔΨ. We tested the effect produced by different peptides (e.g. TG2-BH3, BAX-BH3, and TG2-BH3 L-E) on the ΔΨ either in the presence or absence of CsA. All samples were analyzed with a FACScan cytometer (Becton Dickinson) equipped with a 488 argon laser. To exclude debris, samples were gated based on light scattering properties in the side scattering (SSC) and forward scattering (FSC) modes. The red fluorescence emission of untreated mitochondria was considered as basal emission and recorded for 4 min; after this time reagents to be tested were added, and the effect was monitored for the following 16 min. Dot plots of red fluorescence emission as a function of the time, obtained in each condition, were statistically analyzed using CellQuest software to determine the percentage of mitochondria with depolarized membrane. Cyt c presence in the supernatants of swelling reactions, performed in the absence of TMRM, was assessed with a commercial ELISA kit (R&D System). The cyt c release was quantified and expressed as pg/ml in all tested samples, including the positive control. Co-precipitation between BH3 Domain Peptides and Bax—Mitochondria (0.5 mg) isolated from SK-n-BE(2) cells were treated with 5 and 10 μm biotinylated BH3 domain peptides for 20 min at room temperature, followed by centrifugation at 10,000 × g for 10 min at 4 °C to eliminate the supernatant. Pellets were lysed with CHAPS-IP buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 150 mm KCl, 1% CHAPS) for 30 min on ice and, after addition of 50 μl of prewashed streptavidin paramagnetic particles (MagneSphere, Promega), incubated for 2 h at 4 °C under gentle shaking. The beads were recovered and washed six times. Proteins attached to the beads were solubilized in sample buffer and after separation on 4-12% Nu-PAGE (Invitrogen) subjected to Western blot with anti-Bax antibody (N-20, Santa Cruz Biotechnology). For whole cell co-precipitation, cells were treated with 50 μm biotinylated peptides and, after lysis in CHAPS-IP buffer, peptides and interacting proteins were isolated as described above. Identification of Bax as a TG2 Protein Substrate—TG2-overexpressing cells (TGA) were grown in the presence of 5 mm 5-(biotinamido)pentylamine (EZ-link, Pierce) and treated for 2 h with 1 μm staurosporine (Sigma) in the presence or absence of 100 μm z-VAD (Promega). Cells were harvested and lysed in CHAPS-IP buffer, and biotinylated TG2 substrates were separated on 4-12% Nu-PAGE (Invitrogen). After transfer to nitrocellulose, TG2 substrates were revealed with streptavidin-horseradish-conjugated peroxidase (Amersham Biosciences, 1:3000). Mitochondria from the same cells were isolated and, after lysis in CHAPS-IP buffer, biotinylated TG2 protein substrates were purified by streptavidin paramagnetic particles (MagneSphere, Promega) following the manufacturer's protocol. Purified proteins were separated on 4-12% Nu-PAGE gels and revealed by Western blot with anti-Bax N-20 antibody. Immunoprecipitations—For immunoprecipitation experiments TG2-overexpressing cells (TGA), seeded in 175-cm2 tissue culture flasks, were treated with 1 μm staurosporine for 2 h, harvested with trypsin, and washed twice in PBS. Whole cell extracts were obtained by lysis with CHAPS-IP buffer for 30 min on ice and clearing at 14,000 × g at 4 °C for 10 min. For each reaction 500 μg of total protein were prein-cubated with 50 μl of prewashed Dynabeads-protein G (Dynal) for 1 h at 4 °C, with gentle shacking. Immunoprecipitations were performed by adding 5 μg of anti-TG2 (CUB 7402, NeoMarkers), or anti-Bax (N-20, Santa Cruz Biotechnology and 6A7, BD PharMingen) or anti-Bak (G-23, Santa Cruz Biotechnology) antibodies, and 50 μl of prewashed Dynabeads-protein G. After 4 h or overnight incubation, beads were recovered and washed six times with CHAPS-IP buffer. Immunoprecipitated proteins were detached from beads by boiling in sample buffer and separated on 4-12% Nu-PAGE. After transfer to nitrocellulose, membranes were incubated with anti-TG2 (0.2 μg/ml), anti-Bax (N-20, 0.2 μg/ml; 6A7, 1 μg/ml), and anti-Bak (0.2 μg/ml) antibodies for 1 h and then appropriate secondary horseradish peroxidase-conjugated secondary antibodies were added for 1 h. The signals were detected with Supersignal (Pierce). Immunofluorescence—For immunofluorescence experiments SK-n-BE(2) and TGA cells were seeded in 4-well chamber slides and treated or not with ANT-BH3 peptides, as described above. After washing with PBS, cells were fixed on paraformaldehyde (4% w/v) and picric acid (0.19% v/v) for 20 min at room temperature and washed three times in PBS. After permeabilization with PBS, 0.1% Triton X-100 for 10 min at room temperature and blocking in PBS, 0.1% Triton X-100, 10% FCS, primary antibody was added, and incubation was carried out for 1 h at room temperature. For indirect immunofluorescence anti-Bax (N-20, 2 μg/ml; 6A7, 1 μg/ml) and anti-Hsp60 (SPA-806, 1 μg/ml, StressGen) antibodies were used. Alexa Fluor-conjugated secondary antibodies (Molecular Probes) were used as suggested by the manufacturer. Direct labeling of anti-TG2 and anti-Bax (2D2, Santa Cruz Biotechnology) monoclonal antibodies with Alexa Fluor was performed with Zenon Mouse IgG Labeling Kit (Molecular Probes) following the manufacturer's instructions. Briefly, 6 μg of each monoclonal antibody were labeled with 30 μl of Zenon Mouse IgG labeling reagent for 5 min at room temperature, and then the reaction was blocked by addition of 30 μl of Zenon blocking reagent and further incubation for 5 min. The formed complex was added to permeabilized fixed cells, and incubation was carried out for 1 h in the dark. After washing three times with PBS, cells were subjected to a second fixation for 15 min at room temperature. Cells were washed three times with PBS and nuclei counterstained with Hoechst 33342 (Molecular Probes). The dried slides were mounted with Prolong Antifade Kit (Molecular Probes) and examined with a Nikon Eclipse TE200 epifluorescence microscope equipped with Coolsnap CCD camera. Images were assembled with Adobe Photoshop. TG2 Has a Functional BH3 Domain—TG2 protein selectively accumulates to high levels in cells undergoing cell death by apoptosis both in vivo and in vitro (27Piacentini M. Fesus L. Farrace M.G. Ghibelli L. Piredda L. Melino G. Eur. J. Cell Biol. 1991; 54: 246-254PubMed Google Scholar, 28Piacentini M. Melino G. Prog. Clin. Biol. Res. 1994; 385: 123-129PubMed Google Scholar). In keeping with this, we previously showed that TG2-overexpressing cells (TGA) were primed toward apoptosis, and their mitochondria were greatly modified both at the ultrastructural and functional levels (19Piacentini M. Farrace M.G. Piredda L. Matarrese P. Ciccosanti F. Falasca L. Rodolfo C. Giammarioli A.M. Verderio E. Griffin M. Malorni W. J. Neurochem. 2002; 81: 1061-1072Crossref PubMed Scopus (117) Google Scholar). On the basis of this evidence, we decided to further investigate the role exerted by TG2 at the mitochondrial level. We first analyzed the primary structure of TG2, in search of sequences that might be involved in its subcellular localization or responsible for specific protein-protein interactions. This analysis led us to the identification of an eight amino acid region (amino acids 204-212 in the enzyme's catalytic core) that shares more than 70% identity with the Bcl-2 BH3 domain (Fig. 1A). This sequence showed the presence of the highly conserved leucine, at position 204, and only two different amino acids out of eight. At first glance, the substitution of the aspartic acid 209, which is highly conserved in all other proteins of the Bcl-2 family, with arginine might be considered as relevant, given the opposite charge of the two amino acids. On the other hand, it might not be as drastic with respect to steric hindrance and folding properties. The other substitution, phenylalanine 211 with cysteine, occurs in an amino acid that seems not to be relevant for the domain-mediated interaction, at least as described for the other Bcl-2 family members (29Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (188) Google Scholar). In addition, by comparing the surrounding sequence to the 12 amino acid BH3 consensus found on Prosite (ca.expasy.org/prosite/), we found only one other difference consisting of a shift of the first amino acid of the domain. Computer-assisted analysis of the published crystal structure of TG2 (8Liu S. Cerione R.A. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Crossref PubMed Scopus (274) Google Scholar) revealed that the identified TG2 domain is structured into two α-helices, the conformation expected for the BH3 domain of Bcl-2 family proteins (30Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar), and exposed to the interaction with solvent (represented in red in Fig. 1B). Analyses carried out to identify other putative BH domains in TG2 primary sequence do not lead to the identification of regions showing relevant homology to them. In order to assess the relevancy and functionality of this identified domain, we generated two different TG2 mutants in which the BH3 domain had been either deleted (TG2 ΔBH3) or the leucine 204 mutated to glutamic acid (TG2 L-E), because this amino acid has been described to be critical for the homo-/heterodimerization of Bax (29Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (188) Google Scholar). Wild-type and mutated TG2s were expressed in SK-n-BE(2), by transient transfection, and sensitization to cell death induction was addressed. When we treated transfected cells with 1 μm staurosporine we observed that, as expected, expression of wild-type TG2 sensitized cells toward death. On the other hand, when the two mutants were expressed, this sensitization to death completely disappeared (Fig. 1C). We also observed that, as described (13Tucholski J. Johnson G.V. J. Neurochem. 2002; 81: 780-791Crossref PubMed Scopus (77) Google Scholar), TG2 bearing a mutation in the cysteine of the active site (C277S) was unable to sensitize cells toward cell death (data not shown). Taken together, these data suggest that the identified domain is a functional one and that TG2 sensitization to death relies on this BH3 domain, which proves to be necessary but not sufficient for priming cells toward apoptosis. In fact, the C277S mutant that retains the BH3 domain but lacks the transamidating activity indicates the TG2 enzymatic activity as necessary for the sensitization to death. Effect of TG2-BH3 Peptide in Vivo—To further assess whether the identified domain behaves as previously characterized BH3 domains, we decided to use synthetic peptides bearing the domain sequence fused to the C terminus of the antennapedia sequence (ANT-BH3 peptides), as described by others (31Vieira H.L. Boya P. Cohen I. El Hamel C. Haouzi D. Druillenec S. Belzacq A.S. Brenner C. Roques B. Kroemer G. Oncogene. 2002; 21: 1963-1977Crossref PubMed Scopus (80) Google Schol
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