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J. Biol. Chem., Vol. 279, Issue 52, 54783-54792, December 24, 2004
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From the
Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy, the Department of Drug Research and Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanità, 00161 Rome, Italy, and the ¶Laboratory of Cell Biology and E.M., INMI-IRCCS Lazzaro Spallanzani, 00149 Rome, Italy
Received for publication, September 23, 2004 , and in revised form, October 12, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-carboxyamide groups of peptide-bound glutamine residues serve as acyl donors and primary amino groups of several compounds function as acceptor substrates (2). This reaction results in the post-translational modification of proteins by establishing 
(-glutamyl)lysine and N,N-bis(-glutamyl)polyamine isodipeptide linkages (3). 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 (4). 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 G
h subunit of the GTP-binding protein (Gh) and form a ternary complex in association with the 50-kDa 
subunit (Gh) and the 1-adrenergic receptor (5). Thus, the TG2/Gh proves to be a multifunctional protein, which by binding GTP in a GhGTP complex, can modulate receptor-stimulated phospholipase-C (PLC) activation. The binding of GTP to TG2/Gh prevents the activation of the pro-apoptotic cross-linking activity (6-9). 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 (10). In addition, recent studies suggest that TG2 in its G protein configuration could lead to prevention of cell death (11-13).
In addition to these two main enzymatic activities, it has been proposed very recently that TG2 might also function as a protein-disulfide isomerase (14) and as a kinase (15), 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 (16-18). 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 (4). 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 (19), 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 (20, 21). 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 (22, 23).
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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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BH3), 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 x 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 x 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 (25) 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 x 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 (26) 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 x g at 4 °C. The supernatant was recovered and further centrifuged for 15 min at 17,000 x 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 x g at 4 °C of the 900 x 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 x g, 10 min, 4 °C). Proteins contained in the supernatants were then concentrated in 90% acetone (4 °C, 18 h; centrifugation at 17,000 x 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 
and an increase of outer membrane permeability. CsA (5 µg/ml) added before Ca2+ prevented loss. 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 . We tested the effect produced by different peptides (e.g. TG2-BH3, BAX-BH3, and TG2-BH3 L-E) on the 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 x 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 x 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (29). 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.
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-helices, the conformation expected for the BH3 domain of Bcl-2 family proteins (30), 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 BH3) 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 (29). 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 (13), 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 (31). The antennapedia sequence delivers peptides through the plasma membrane, thus allowing the study of the intracellular effect of the domain of interest (32, 33). We generated also a set of peptides with biotinylation at the N terminus to allow their purification by streptavidin paramagnetic particles (see "Experimental Procedures"). As the experimental model, we used the neuroblastoma SK-n-BE(2) cell line, which has low levels of endogenous TG2 (see Fig. 5C). Cells were treated with 50 µM peptides bearing the ANT sequence alone, as a negative control, the ANT-BH3 Bax sequence, as positive control, and ANT-BH3 TG2 sequence. Time course analysis of cell viability, as assessed by MTS assay (Fig. 2A), showed that both the BH3 peptides induced cell death that reached about 80% of the treated cells after 6 h of incubation. We assessed also phosphatidylserine exposure and propidium iodide (PI) incorporation after peptide treatment. Fig. 2B shows a representative experiment in which cells were treated with different BH3 peptides for 6 h in the presence or absence of mitochondrial membrane permeability transition pore inhibitor CsA (34). It is interesting to note that CsA is able to inhibit both phosphatidylserine externalization and PI uptake in TG2-BH3 peptide-treated cells, while it had little or no effect on Bax-BH3 peptide-treated cells (Fig. 2B). In addition, TG2-BH3 peptides bearing the mutation of the leucine 204 (TG2 LE) did not induce phosphatidylserine externalization nor PI incorporation, suggesting that this amino acid was important for the functionality of the BH3 domain. These data were consistent with what we previously observed in TG2-overexpressing cells (TGA) (19). After staurosporine treatment, they rapidly die by apoptosis via the mitochondrial pathway, and this phenotype could be reverted by CsA. In that report, we hypothesized that the observed sensitivity of TGA cells toward apoptosis might be related to the hyperpolarized state of their mitochondria, which rapidly undergo MMP loss and cytochrome c release (19). To address whether this mitochondrial behavior might be related to the presence of the identified TG2-BH3 domain, we analyzed the mitochondrial status in SK-n-BE(2) prior to and after treatment with ANT-BH3 peptides. We did not detect any hyperpolarization of mitochondria (Fig. 2C). This result was probably caused by the fact that both TG2- and Bax-BH3 peptides were able to induce a very rapid decrease, after 1 h, of the mitochondrial membrane potential (). In keeping with what we previously observed in TGA cells, pretreatment with CsA completely inhibited the mitochondrial depolarization in cells treated with the TG2-BH3 peptide, whereas it had a negligible effect on Bax-BH3-treated cells. The different sensitivity to CsA inhibition suggests that TG2-BH3 might induce mitochondrial membrane permeabilization by a mechanism different from that of Bax-BH3. The translocation of the pro-apoptotic Bcl-2 family member Bax to mitochondria has been described as a relevant step toward mitochondrial membrane permeabilization (20, 21), We investigated whether the TG2-BH3 peptide was able to induce the activation/translocation of Bax protein. In cells treated for 1 h with 50 µM TG2-BH3 peptide, we observed the Bax conformational change, detected by labeling with specific 6A7 monoclonal antibody (Fig. 2D), as well as translocation of Bax to mitochondria, deduced from the punctate staining visible after labeling overall of Bax with polyclonal N-20 antibody (Fig. 2E). To address whether this Bax translocation was related to mitochondrial membrane depolarization we analyzed the ability of TG2- and Bax-BH3 peptides to induce cyt c release. Fig. 2F shows that cyt c release is induced by both peptides, and it takes place at the same time of collapse. Interestingly, the TG2-BH3 peptide is as effective as Bax-BH3 peptide in inducing cyt c release in vivo. Data presented so far were consistent with the mitochondria-regulated cell death observed in TGA cells (19), which also showed an increase of the intracellular ROS. When we checked the ROS production levels, we did not observe any variation after treatment with either TG2- or Bax-BH3 peptides (data not shown), suggesting that this event might be a consequence of the sustained mitochondrial hyperpolarization observed in TGA cells.
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TG2 Interacts with Bax and Bak—On the basis of the above described data we hypothesized that TG2 might be able to modulate cytochrome c release by interacting with Bcl-2 family members and that this interaction might be modulated during cell death induction and progression. In order to address this hypothesis, we performed immunoprecipitations on TG2-overexpressing cells before and after cell death induction with 1 µM STS for 2 h. Whole cell extracts were prepared in CHAPS-IP buffer, to preserve native forms of Bax and Bak (35), and we looked for co-precipitations of TG2 with Bcl-2 family members using antibodies directed against TG2, Bax, and Bak. Fig. 4A shows that, even under steady state conditions, TG2 co-precipitates with Bax, and this interaction is enhanced after cell death induction. The interaction between Bax and TG2, as well as its increase upon cell death induction, was confirmed by immunoprecipitation with the anti-Bax N-20 antibody (Fig. 4B). Moreover, when we used as bait the anti-Bax 6A7 antibody, specific for Bax conformational change, we observed that TG2 co-precipitated with the Bax that has undergone the conformational change, and, also in this case, the interaction between the two proteins increased after apoptosis induction (Fig. 4C). These data indicate that TG2 interacts with Bax in living cells and that this interaction is enhanced upon induction of apoptosis. We also checked the ability of TG2 to interact with other members of the Bcl-2 family, and we observed co-precipitation with Bak (Fig. 4A); but in this case, there were no variations in the amount of protein associated before and after induction of cell death. Interestingly, no binding with the anti-apoptotic members Bcl-2 or Bcl-XL had been detected (data not shown).
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-Bax 2D2 antibody (Fig. 5D) results in diffuse staining, corresponding to the cytosolic monomeric form, as well as in punctate aggregated staining that might correspond to the multimeric forms observed by Western blot (Fig. 5C). Staining of Bax with the monoclonal -Bax 6A7 antibody, which recognizes the conformational change of Bax, results in the same punctate staining observed with the -Bax 2D2 antibody. These data are in accordance with what we observed in immunoprecipitation experiments (Fig. 4C), where, even under steady state conditions, the -Bax 6A7 antibody co-precipitates Bax and TG2. Taken together this evidence confirms that there is an interaction between TG2 and Bax, but also indicates that TG2 might post-translationally modify Bax via the formation of SDS insoluble cross-links, which result in the appearance of stable Bax polymers at the mitochondrial level. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helix, as for all other BH3 domains, and exposed on the protein surface. Expression of mutated TG2, in which the domain was completely deleted or the leucine 204 mutated to aspartic acid, results in a complete abolishment of the sensitization to death observed when wild type TG2 was expressed. Cell permeable TG2-BH3 peptide induces, both in vivo and on isolated mitochondria, swelling, depolarization, and cytochrome c release at a rate comparable to that of the Bax-BH3 peptide. The only observed difference between the two peptides relies on the complete inhibition exerted by the permeability transition pore inhibitor CsA on the TG2-BH3 peptide. In addition, TG2-BH3 peptide induces an early conformational change, coupled with translocation to mitochondria, of Bax. Co-precipitation assays demonstrated its direct interaction with Bax. By immunoprecipitation assay, we confirmed that the TG2 protein itself is able to interact with pro-apoptotic members of the Bcl-2 family Bax and Bak, even in the absence of any apoptotic stimuli. As far as Bax is concerned the interaction with TG2 proves to be increased upon cell death induction, whereas we did not observe any change in the ratio of Bak that associates with TG2. It is interesting to note that the conformational change that Bax undergoes after apoptosis induction has no effect on its ability to interact with TG2, but rather seems to be causative of the increased association of the two proteins. In addition, we found out that in TG2-overexpressing cells cytosolic Bax seemed to be mainly in the monomeric form, whereas in mitochondria it appeared also in a conformationally changed and aggregated form, similar to the one described by Youle and co-workers (51). From our data, it is not possible to state unequivocally whether the conformationally changed/aggregated Bax interacts with cytosolic rather than mitochondrial TG2 fraction or vice versa. Nevertheless, as the TG2-BH3 peptide is able to induce the Bax conformational change, we could speculate that the TG2-Bax interaction takes place in the cytosol and might promote the Bax conformational change/aggregation and translocation to mitochondria. It is worth noting that in TG2-overexpressing cells, about half of total TG2 protein is localized on the mitochondria, where after apoptosis induction it cross-links many protein substrates, among which is Bax itself. This TG2-dependent Bax cross-linking leads to the formation of Bax polymers with an apparent electrophoretic mobility that suggests they result from the oligomerization of multiple molecules of Bax. This kind of Bax aggregation is very similar to the one previously described (38, 39), and Bax multimerization has been proposed as the mechanism by which it might form pores on the outer mitochondrial membrane that lead to the release of cytochrome c. In this view, TG2 cross-linking action might stabilize the aggregated Bax, resulting in a stably open pore on the outer mitochondrial membrane. We also observed interaction of TG2 with Bak, but this kind of interaction does not seem to change after induction of apoptosis, and we observed the presence of Bak multimers to a lesser extent than Bax multimers. The ability of TG2 to interact with the pro-apoptotic members of the Bcl-2 family Bax and Bak supported the biological significance of the domain in the whole protein context. The lack of interaction with the anti-apoptotic members of the Bcl-2 family (we never observed interaction of TG2 with Bcl-2 or Bcl-XL) prompted us to hypothesize a model for TG2 regulation of Bax activation. Under physiological Ca2+ concentration, TG2 is inactive as a cross-linking enzyme. We might speculate that the interaction between TG2 and Bax in the cytosol is responsible for the further localization of both proteins in mitochondria, where they might be in close proximity. In this configuration TG2-Bax interaction might be anti-apoptotic, as it blocks Bax action on mitochondria and prevents cytochrome c release. After apoptosis induction, the early increase of calcium concentration at the mitochondrial level activates the cross-linking activity of TG2, which by polymerizing Bax stabilizes its poreforming conformation. This action results in a more rapid release of cytochrome c compared with cells that do not have high TG2 levels. This hypothesis seems to be confirmed by the complete abolition of the priming effect toward apoptosis, observed in transfections of TG2 in which the BH3 domain was either deleted or mutated (L204E). In this view, TG2 seems to act both as a regulator of the apoptotic response upstream to mitochondria and as an accelerator of the process at the mitochondrial level. This evidence also suggests that TG2 behaves as a Bid-like BH3-only protein, as proposed by Letai et al. (39). TG2 action at the mitochondrial level seems not to be limited to this stabilizing effect on Bax, because we previously observed that mitochondria of TG2-overexpressing cells were hyperpolarized, and there was an increase in ROS levels. In contrast with these observations, the TG2-BH3 peptide was not able to induce either hyperpolarization of mitochondria or increases in ROS levels. We have evidence that these effects are the consequence of a different and more complex action exerted by TG2 on some mitochondrial membrane proteins.2
In conclusion we found that TG2 might contribute to the control of the mitochondrial pathway of apoptosis by interacting with and post-translationally modifying pro-apoptotic members of the Bcl-2 family. The presence of a BH3 domain in TG2, which is of large mass size (80 kDa) and possesses different enzymatic activities (cross-linking, G-protein, PDI), suggests the existence of a new class of BH3-only proteins.
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FOOTNOTES |
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* This work was supported in part by Grants QLG1-CT-1999-00739 and QLK3-CT-2002-02017 from the European Community, PRIN-2002 and FIRB-2001 from Ministero dell'Istruzione, dell'Università e della Roma, Ricerca Corrente e Finalizzata from the Ministry of Health and Associazione Italiana per la Ricerca sul Cancro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
|| To whom correspondence should be addressed: University of Rome Tor Vergata, Dept. of Biology, Via della Ricerca Scientifica, 00133 Rome, Italy. Tel.: 390672594234; Fax: 39062023500; E-mail: mauro.piacentini{at}uniroma2.it.
1 The abbreviations used are: TG2, tissue transglutaminase; CsA, cyclosporin a;
, 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.
2 P. G. Mastroberardino, M. G. Farrace, I. Viti, F. Pavone, M. R. Wieckowski, G. M. Fimia, F. Fratini, C. Rodolfo, B. Devreese, G. Melino, J. Van Beeumen, R. Rizzuto, and M. Piacentini, submitted manuscript.
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