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1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In a previous study (Toriumi et al. 2008), we examined the effect of expanded polyalanine tracts fused with yellow fluorescent protein (YFP) on cultured cell lines. Cells expressing the YFP-fused expanded polyalanine tracts exhibited cytotoxicity and aggregate formation like that seen in polyalanine diseases. We speculated that the polyalanine tract itself possesses properties that bring about cell death by a mechanism common to the polyalanine diseases. We also found that polyalanine tracts are associated with mitochondria and that expression of polyalanine tracts decreases the mitochondrial membrane (MM) potential, a step considered key in the initial apoptotic process (Crompton 1999). In response to an apoptotic signal, mitochondria decrease their membrane potential, which increases the permeability of their outer membranes and allows the release of various apoptogenic factors that normally reside in the intermembrane space of these organelles. Among these factors is cytochrome c, an important component of the mitochondrial respiratory chain. Once released into the cytosol, cytochrome c activates caspase-9, which in turn activates caspase-3 and caspase-7. These activated caspases kill the cell by proteolysis, leading to biochemical and morphological features characteristic of apoptosis (Li et al. 1997). The release of cytochrome c has been widely believed to be mediated by the opening of mitochondrial permeability transition (PT) pores; these pores include the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), and cyclophilin D (Tsujimoto & Shimizu 2007). Opening of the PT pore is strictly regulated by proteins of the Bcl-2 family. Bax, one of these proteins, physically interacts with VDAC in the outer MM, leading to opening of the PT pore and induction of cytochrome c release. Conversely, the anti-apoptotic factors Bcl-2 and Bcl-xL induce PT pore closing and the blocking of cytochrome c release through their association with VDAC (Shimizu et al. 1999, 2000a).
In the present study, we show that binding of polyalanine tracts to mitochondria induces the release of cytochrome c into the cytoplasm, independently of the PT pore and the subsequent activation of caspase-3. Our results suggest a novel mechanism by which polyalanine tracts bring about cytotoxicity through the apoptotic process. The investigation of this mechanism may provide insights into the pathogenesis of, and possible therapeutic strategies for, polyalanine diseases.
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Results |
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In a previous study, we detected polyalanine tracts in a mitochondrial fraction, suggesting that polyalanine tracts associate with mitochondria (Toriumi et al. 2008). To confirm this association in vitro, we performed a mitochondrial-binding assay using isolated mouse liver mitochondria. The mitochondria were incubated with GST or GST-Ala29 purified from Escherichia coli and then collected by discontinuous sucrose gradient centrifugation. Immunoblot analysis with an anti-GST antibody indicated that GST-Ala29, but not GST, was tightly associated with the recovered mitochondrial pellet (Fig. 1A).
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Expression of a long polyalanine tract induces cytochrome c release from mitochondria
In our previous study, we showed that the expression of polyalanine tracts decreases the MM potential, suggesting that an initial step of apoptosis is mediated by the mitochondria. We hypothesized that by directly binding to the mitochondria, polyalanine tracts induce the release of apoptogenic mitochondrial factors leading to an activation of an apoptotic cascade. Here, we evaluated this hypothesis by quantifying the amount of cytochrome c in the cytosol of COS-7 cells expressing YFP, YFP-Ala29, or YFP-Ala70 (Fig. 2). Staurosporine, an inducer of apoptosis, was used as a positive control. We found that the amount of cytosolic cytochrome c increased as the length of the expressed polyalanine tracts increased, suggesting that apoptosis induced by expression of polyalanine tracts is mediated by the mitochondria.
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Next, we examined the more direct effects of abnormally expanded polyalanine tracts on the release of cytochrome c using an in vitro cytochrome c-release assay. Mitochondria isolated from mouse liver were incubated in the absence or presence of GST or GST-Ala29 (2 µM), and the organelles were centrifuged. The supernatant fractions were then analyzed for their cytochrome c content by immunoblotting (Fig. 3A), which demonstrated that incubation of the mitochondria with GST-Ala29 resulted in a significant release of cytochrome c to the cytoplasm. CaCl2 was used as a positive control because Ca2+ is a known permeabilizer and PT pore opener.
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Polyalanine tracts induce cytochrome c release, independently of the PT pore
Although regulatory release of cytochrome c has been reported to occur through the PT pore, the GST-Ala29-induced release of cytochrome c seen in our study was probably not a result of regulatory opening of the PT pore; rather, it probably leaked through a rupture in the outer MM. To explore this possibility, we performed the in vitro cytochrome c-release assay in the presence of an inhibitor of PT pore opening. As the inhibitor, we used the conserved N-terminal homology domain (BH4) of Bcl-xL (amino acids 4–23), which inhibits VDAC activity in liposomes and isolated mitochondria (Shimizu et al. 2000b), thereby inhibiting cytochrome c release and loss of the MM potential. Although addition of the PT pore inhibitor prevented the release of cytochrome c normally caused by calcium overload, it did not prevent the GST-Ala29-induced release of cytochrome c (Fig. 4). This result is consistent with the proposal that polyalanine tracts disrupt the MM, allowing release of cytochrome c into the cytoplasm.
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Release of cytochrome c into the cytoplasm has been reported to activate a sequential apoptotic cascade. To confirm that the polyalanine-induced release of cytochrome c leads to apoptosis, we examined caspase-3 activity, which is known to rise at the point in the cascade immediately downstream of cytochrome c release. Forty-eight hours after transfection of COS-7 cells with plasmids expressing YFP, YFP-Ala29, or YFP-Ala70, we assessed caspase-3 activity by measuring cleavage of Ac-Asp-Glu-Val-Asp-MCA, a specific peptide substrate of caspase-3. The caspase-3 activity in cells expressing YFP-polyalanine fusion proteins increased in a polyalanine-length-dependent manner and was greater than that in cells expressing YFP alone (Fig. 5), suggesting that the apoptosis induced by expression of polyalanine tracts is mediated by the mitochondria.
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Discussion |
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In a previous study of cell lysates from COS-7 cells transfected with YFP–polyalanine constructs (Oma et al. 2007), immunoblotting showed that the constructs with more than 23 alanine repeats were retained in the stacking gel after native PAGE, suggesting that they formed oligomers. After SDS-PAGE, however, only the construct with 70 repeats was retained in the stacking gel; those constructs with 23 to 35 alanine repeats were not retained. These results suggest that moderately long polyalanine tracts (23–35 repeats) might form SDS-sensitive oligomers in the cell. In short, our study shows that the oligomerization of polyalanine tracts is sufficient to induce their cytotoxicity.
Since we were able to detect an association between polyalanine tracts and mitochondria in vitro in the absence of a cytosolic factor for mitochondrial import, such as MSF or Hsp70 (Hachiya et al. 1994; Komiya et al. 1996), we conclude that the polyalanine tracts were probably bound directly to the outer MM. Furthermore, we found that the amount of polyalanine tract detected in association with the mitochondria decreased gradually with a high-pH wash (data not shown), consistent with their binding to the outer MM.
Next, using an in vitro cytochrome c-release assay, we found that polyalanine tracts at concentrations greater than 1.5 µM directly induced the release of cytochrome c. This release was not blocked by BH4 peptides, suggesting that the release was not mediated by the PT pore (Figs 3 and 4). The addition of polyalanine tracts at 4.0 or 8.0 µM also promoted an efflux of COX IV, which is normally localized to the inner MM. These results suggest that polyalanine tracts cause the MM to rupture, leading to the release of cytochrome c. This possibility is also supported by the observation that the amount of cytochrome c released from mitochondria was independent of the polyalanine tract concentration in an "all-or-none" fashion, and the release occurred in the absence of other apoptotic signals, such as Ca2+. In the affected cells, oligomerization causes a local increase in the polyalanine tract concentration. When these tracts attack mitochondria, they may in turn induce MM rupture, leading to cytochrome c release.
We demonstrated the release of cytochrome c and the activation of caspase-3 in COS-7 cells expressing YFP-A70 (Figs 2 and 5). These findings were consistent with a report that expression of PABPN1 containing a polyalanine tract long enough to cause OPMD induced the release of cytochrome c, followed by apoptosis (Davies et al. 2008). In addition, polyalanine tracts have been reported to form a β-sheet and then to activate an apoptotic pathway via caspase-8 (Giri et al. 2003). Thus, the cytotoxicity induced by polyalanine tracts might be related to apoptotic cell death. However, YFP-A29 didn't induce the release of cytochrome c and the activation of caspase-3 (Figs 2 and 5) although it can induce its release in in vitro experiment (Fig. 3). We think the inconsistency might be produced by the expression level of YFP-A29 in COS-7 cells. Since it is unlikely that the concentration of YFP-Ala29 in cells is the same as used in in vitro experiment, YFP-A29 couldn't induce the release of cyctochrome c from mitochondria and subsequent activation of caspase-3. However, it is possible that the local concentration increased due to the oligomerizing property of YFP-A29, so that the increasing tendencies were observed in YFP-A29 expressing cells in Figs 2 and 5. We think that increase in local concentration of polyalanine stretch is important to attack mitochondria. If we consider this idea as a pharmacological target, we can suggest some potentially effective therapeutics against polyalanine diseases. In fact, some reagent, inhibitors of oligomerization such as trehalose and geldanamycine, have been reported to be effective against polyalanine disease model (Albrecht et al. 2004; Davies et al. 2006).
In conclusion, we propose a novel mechanism by which the polyalanine tracts cause apoptosis (Fig. 6). This mechanism for polyalanine tract cytotoxicity might be common to the pathogenesis of all polyalanine diseases. These findings in this report point to the mitochondria as good therapeutic targets for polyalanine diseases. We hope that these findings will benefit patients suffering from these diseases.
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Experimental procedures |
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Polyalanine repeat sequences [(Ala-)n] were synthesized by annealing of double-stranded oligonucleotides. These sequences were inserted into the EcoRV site of the pBlueScript KS(–) vector (TaKaRa), and the resulting constructs were digested with BamHI and ApaI. The excised repeat sequences were then inserted into the BglII and ApaI sites of the pEYFP-C1 vector (Clontech) to allow the expression of the alanine repeats fused to the C-terminus of YFP. The resulting constructs were named YFP-Ala(n) according to the number of alanine repeats (n).
For in vitro assays of cytochrome c release, we prepared a construct encoding an (Ala)29 tract fused to the glutathione-S-transferase (GST) protein. This construct, GST-Ala29, was prepared by excision of the (Ala)29 sequence from YFP-Ala29 by digestion with XhoI and BamHI and insertion of the fragment into the XhoI–BamHI site of pGEX 4T-2 (Amersham Bioscience), a vector for expression of GST-fusion proteins in Escherichia coli.
Isolation of mitochondrial and cytosolic fractions
Transfected COS-7 cells were washed twice with ice-cold phosphate-buffered saline and scraped into mitochondrial buffer [20 mM Tris–HCl, pH 7.2, 250 mM sucrose, 2 mM EGTA, 40 mM KCl, 1 mg/mL bovine serum albumin (BSA)] containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 : 1000 protease inhibitor cocktail (Sigma). The cells were fractured on ice using a 27-gauge needle. Unlysed cells, large debris, and nuclei were removed by centrifugation at low speed (50 x g, 10 min, 4 °C). The supernatant fraction was centrifuged at 1000 x g (10 min, 4 °C), and the resulting supernatant fraction was removed and centrifuged at 20 000 x g (10 min, 4 °C) to obtain the cytosolic fraction.
To purify mitochondria, the 1000 x g pellet was resuspended in mitochondrial buffer containing protease inhibitors, layered over a discontinuous 1.0/1.5 M sucrose gradient, and separated ("floated") by centrifugation at 100 000 x g in a swinging-bucket rotor (2 h, 4 °C). The hazy ring corresponding to the mitochondrial fraction was recovered carefully from the interface between the two sucrose solutions, diluted 1:2 in a buffer consisting of 20 mM Tris–HCl, pH 7.2, 2 mM EGTA, 40 mM KCl, and 1 mg/mL BSA, and centrifuged at 20 000 x g (20 min). The resulting pellet was resuspended in mitochondrial buffer containing protease inhibitors and washed twice by centrifugation (6800 x g for 5 min followed by 3800 x g for 5 min). The identity and purity of the mitochondrial fraction were determined by Western blot analysis using various cellular markers. Mitochondria were identified with antibodies against the nucleusencoded cytochrome c oxidase subunit IV (COX IV; BD Biosciences); the cytosolic fraction was identified with antibodies against 
-tubulin (Sigma).
Isolation of mitochondria from mouse liver
Mice were fasted overnight, and their liver mitochondria were isolated by the method of Hogeboom (Hogeboom 1955) using a medium containing 0.25 M sucrose, 10 mM Tris–HCl, pH 7.4, and 0.1 mM EDTA. EDTA was omitted in the final wash, and the mitochondrial preparation was suspended in 0.25 M sucrose containing 10 mM Tris–HCl, pH 7.4.
Preparation of GST-fusion proteins
Escherichia coli cells were transformed with pGEX 4T-2 or the GST-Ala29-encoding plasmid construct and grown to an OD600 of 1.0. Expression was then induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the cells were grown at 27 °C for 1 h. After centrifugation of the cells (1600 x g, 15 min), the pellet was placed on ice and resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA) containing 1 mM PMSF and 1:1000 protease inhibitor cocktail. The cells were fractured by sonication, and Triton X-100 was added to a final concentration of 1%. The resulting lysates were chilled on ice for 30 min and clarified by centrifugation (16 000 x g, 15 min). The supernatant fractions were incubated for 2 h at 4 °C with glutathione-Sepharose beads (Amersham Bioscience) that had been washed three times and resuspended in lysis buffer containing 1% Triton X-100.
Mitochondrial binding assay
Purified GST or GST-Ala29 (0.8 µM) were added to isolated mouse liver mitochondria (1 µg/µL) in binding buffer (20 mM HEPES, pH 6.8, 250 mM sucrose, 150 mM potassium acetate, 5 mM magnesium acetate), and the mixture was incubated at room temperature for 10 min. After centrifugation (1600 x g, 10 min), the pellet containing mitochondria was washed with binding buffer and resuspended in 50 µL of binding buffer. The mitochondrial suspension was added to 350 µL of 2.5 M sucrose buffer (20 mM HEPES, pH 6.8, 2.5 M sucrose, 150 mM potassium acetate, 5 mM magnesium acetate), and then layered under a discontinuous 1.0/1.5 M sucrose gradient. The mitochondria were floated by centrifugation at 100 000 x g in a swinging-bucket rotor (2 h, 4 °C). The hazy ring between the two sucrose solutions was recovered, diluted 1:2 in binding buffer, and centrifuged at 16 000 x g for 20 min. The resulting pellet was resuspended in binding buffer and washed by centrifugation at 16 000 x g for 10 min. GST-Ala29 protein associated with mitochondria was detected by Western blot analysis using anti-GST antibody. Mitochondria were identified with antibodies against the nucleus-encoded COX IV protein.
In vitro cytochrome c-release assay
To determine the effect of polyalanine tracts on the release of cytochrome c, isolated mouse liver mitochondria (0.6 µg/µL) were incubated in the presence or absence of GST or GST-Ala29 (various concentrations) for 30 min at 30 °C in a KCl-based medium (125 mM KCl, 15 mM HEPES, pH 7.4, 0.5 mM EGTA, 4 mM MgCl2, 5 mM Na2HPO4) containing glutamate and malate (1 mM each) as respiratory substrates. At the end of the incubation period, the mitochondrial suspensions were centrifuged at 1600 x g for 15 min. The supernatant fractions were removed and mixed with 5x SDS sample buffer, and the mitochondrial pellets were resuspended in 1x SDS sample buffer. Samples were boiled for 5 min and electrophoresed on a 12.5% SDS–polyacrylamide gel. The separated proteins were transferred to nitrocellulose and immunoblotted with antibodies specific for cytochrome c or COX IV.
For assays of cytochrome c release in the presence of PT pore inhibitor, the mitochondria were incubated with BH4 peptides (20 µg/mL, Calbiochem), which inhibit induction of the PT pore, for 15 min at 30 °C before polyalanine tracts were added.
Measurement of caspase-3 activity
COS-7 cells were transiently transfected with plasmids encoding YFP, YFP-Ala29, or YFP-Ala70, and the cells were harvested 48 h after transfection. The harvested cells were dissolved in extraction buffer (50 mM Tris–HCl, pH 7.5, 10 mM 2-mercaptoethanol, 1 mM EDTA) and subjected to three freeze-thaw rounds consisting of 60 s in liquid nitrogen followed by 90 s in a 30 °C water bath. The samples were centrifuged at 10 000 x g for 5 min, and the total protein (7.5 µg) in the supernatant fraction was dissolved in 200 µL of assay buffer (25 mM Tris–HCl, pH 7.5, 10 mM 2-mercaptoethanol, 1 mM EDTA). The fluorescent caspase-3 substrate Ac-Asp-Glu-Val-Asp-MCA (Peptide Institute) was added to a final concentration of 5 µM, and the mixtures were incubated at 37 °C for 30 min. The reactions were stopped by the addition of 100 µL of 10% SDS and 1 ml of 0.1 M sodium acetate, and the fluorescence was measured with a JASCO FP-777 fluorescence spectrometer with excitation at 380 nm and emission at 460 nm.
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Acknowledgements |
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Footnotes |
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* Correspondence: cishiura{at}mail.ecc.u-tokyo.ac.jp
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References |
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Accepted: 22 March 2009
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