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• W2076296145 abstract "Bcl-rambo binds to ANT1 by pull down (View interaction) Bcl-2 family members are key protein regulators of cell death. This family of proteins contains both pro-apoptotic and anti-apoptotic members that decide the fates of cells at the mitochondrial level [1, 2]. Bcl-rambo is Bcl-2 protein family member with a pro-apoptotic function [3, 4]. However, unlike other Bcl-2 family protein members, Bcl-rambo possesses a unique c-terminal extension (BHNo domain) with 2 repeated tandem domains A and B (RTA and RTB) [3]. In addition, the conserved BH domains of Bcl-rambo are not essential for triggering cell death [3], obscuring the mechanism by which this protein causes cell death. Mitochondria, and the mitochondrial permeability transition (MPT) in particular, are known to play a central role in apoptotic cell death [4, 5]. The cause of the MPT is the opening of a non-specific pore, known as the mitochondrial permeability transition pore (PTP), which is a protein aggregate composed of Cyclophilin D (Cyp-D), a voltage-dependent anion channel (VDAC), and the adenine nucleotide translocator (ANT) [6]. Members of the Bcl-2 family of proteins interact with VDAC or ANT to regulate PT. For examples, Bax and Bcl-2 physically bind to ANT to modulate its activity [7], and Bcl-XL prevents VDAC from forming large non-specific channels [8, 9]. However, the precise molecular mechanisms underlying the pro-apoptotic or anti-apoptotic activity of Bcl-2 family proteins with regard to PTP regulation are largely unknown. In this work, we report how Bcl-rambo facilitates apoptotic cell death and identify signaling molecules involved in the MPT using site-directed mutant plasmids and recombinant proteins. PC-3 prostatic cancer cells, purchased from the American Type Culture Collection (Rockville, MD), were cultured in RPMI 1640 medium. Anti-VDAC and -ANT antibodies were obtained from Calbiochem (La Jolla, CA). Anti-Cyclophilin D and anti-V5 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Invitrogen (Carlsbad, CA), respectively. Cyclosporin A (CsA) and bonkrekic acid (BA) were obtained from Calbiochem. Mitotracker was from Molecular Probes, Inc. (Eugene, OR). All other chemicals or reagents were obtained from Sigma–Aldrich (St. Louis, MO) unless otherwise specified. DNA encoding Bcl-2 or Bcl-rambo cloned into pcDNA3.1/V5 (Invitrogen, Carlsbad, CA). Deletion mutants of Bcl-rambo or chimeric TM mutants were generated by a splice overlap extension method as described previously [10]. After DAPI (1 μg/ml in PBS) staining, apoptotic cells with fragmented nuclei or chromatin condensation were counted manually under a immunofluorescence microscope. Caspase-3 activity was measured using the Colorimetric Caspase-3 Assay Kit (Calbiochem, CA) as described previously [11]. Absorbance at 405 nm was determined ∼6 h after initiation of the reaction. PC-3 cells (3 × 105/10 cm culture dish) were transiently transfected with 2 μg of each plasmid using Lipofectamine 2000 (Invitrogen). For immunoprecipitation, cells were lysed in ice cold NP40 lysis buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% NP-40 and complete protease inhibitor cocktail], incubated with the indicated antibodies, and immunoprecipitated with protein A (Sigma–Aldrich). The precipitates were washed four times, subjected to SDS–PAGE, and analyzed by western blotting. Bcl-rambo, Bcl-ramboΔTM, Rambo-Bcl2-TM, Rambo-BNip3-TM, and Rambo-Cyb5-TM cDNA were subcloned into the pGEX-2TK vector (Amersham Pharmacia Biotechnology, NJ) tagged with glutathione S-transferase (GST). Bound GST-fusion proteins were eluted by thrombin protease treatment. The amount of purified recombinant protein was estimated by the Biuret method. PC-3 cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. The cells were incubated with primary antibody for 2 h at room temperature, and then stained with secondary antibody conjugated with FITC or Texas-Red and viewed using a confocal microscope (META 510, Zeiss, Germany). Mitochondria were freshly isolated from the liver of a 1-month-old rat by differential centrifugation as described previously [12]. Mitochondrial swelling was monitored by measuring the spectrophotometric refractory index at 540 nm at 10 min intervals. To assess mitochondrial membrane potential, PC-3 cells were incubated with 400 nm MitoTracker Red CMXRos (Molecular Probes Inc.). The fluorescent intensity was measured by flow cytometry (FACScaliver, Beckton Dickinson, CA) using the CellQuest program. The effect of Bcl-rambo on ADP transport was determined as described previously [13]. Mitochondria (1 mg/ml) were pre-treated with various concentrations of recombinant Bcl-rambo in assay buffer for 10 min, and then further incubated with 20 μM [14C] ADP for 20 s at 0 °C. The radioactivity of the mitochondrial lysate was determined using a liquid scintillation counter (Wallac 1409, Perkin-Elmer). Liposomes were prepared by solubilization of 400 mg of phosphatidylcholine, 400 mg of phosphatidylserine, and 230 mg cholesterol in PBS, pH 7.2, as described previously [14, 15]. Purified ANT protein were mixed with the liposome and then loaded with ATP. Unincorporated ATP was removed by chromatography on Sephadex G50. These liposome were incubated for 30 min at room temperature with 100 μM ADP and purified Bcl-rambo recombinant proteins or GST control protein were added. Analysied for the released ATP in the supernatant was quantified by a luciferine-luciferase assay as described in Ref. [16]. Unlike the other Bcl-2 family proteins, Bcl-rambo contains a unique BHNo domain with 2 repeated tandem domains, RTA and RTB [3], as well as conserved BH domains and carboxyl-terminal membrane anchor domain (TM). To understand the contribution of the BH, BHNo, and TM domains of Bcl-rambo in its proapoptotic activity, we constructed deletion mutant plasmids (Fig. 1 A). After 48 h of transient transfection with wild-type or mutant constructs, cell death rates were determined by DAPI staining and caspase-3 activity assay. Deletion of the BH1, BH2, BH3, or BH4 domains, and RTA, or RTB did not affect the pro-apoptotic activity of Bcl-rambo (Fig. 1B). However, consistent with a previous report [3], deletion of the TM domain abolished the protein's death-inducing property (Fig. 1, B, and C), indicating that the TM domain plays a pivotal role in the cell death pathway. The TM domain of Bcl-rambo probably is essential for mitochondrial targeting and pro-apoptotic activity [3]. This raises the question of whether the unique sequence identity of the TM domain or its mitochondrial localization ability is crucial for the pro-apoptotic activity of Bcl-rambo. To analyze this, we constructed chimeric mutant plasmids by deleting TM domain of Bcl-rambo (Bcl-ramboΔTM) or mutants with the TM domain replaced with heterologous TM domain sequences from Bcl-2 (Rambo-Bcl2-TM), BNip3 (Rambo-BNip3-TM), or rat cytochrome b5 (Rambo-Cyb5-TM). We transiently transfected PC-3 cells with wild-type Bcl-rambo or chimeric mutant plasmids. The staining patterns obtained for Bcl-rambo, Rambo-Bcl2-TM, and Rambo-BNip3-TM resembled the punctuate mitochondrial distribution of heat shock protein 60 (HSP60) (Fig. 2 A) [17]. Rambo-Cyb5-TM and Bcl-ramboΔTM showed a globular or diffuse staining pattern (Fig. 2A). To determine whether Bcl-rambo mutants targeted to mitochondrial or non-mitochondrial sites were able to promote cell death, we determined the cell death rates with DAPI staining (Fig. 2B) and caspase-3 assay (Fig. 2C) after 48 h of transfection. Cell death rates were significantly increased in cells transfected with Bcl-rambo, Rambo-Bcl2-TM, or Rambo-BNip3-TM. In contrast, transfection with Rambo-Cyb5-TM and Bcl-ramboΔTM did not increase the cell death rates. These data indicate that the pro-apoptotic activity of Bcl-rambo depends on mitochondrial localization, but not on the unique amino acid sequence of the TM domain. Because mitochondrial localization is crucial for the pro-apoptotic activity of Bcl-rambo, we investigated whether Bcl-rambo induces the loss of mitochondrial membrane potential (MMP, ΔΨ m). After 10 h of transfection with Bcl-rambo or the mock control, MMP was determined at 2-h intervals with MitoTracker Red. Decrease in MMP was observed 12 h after transfection of the cells with Bcl-rambo, whereas no decrease was observed in the mock-transfected cells (Fig. 3 A). Bax and BNip3 are known to induce MMP loss; thus, Bax and BNip3 were used as a positive control for determining MMP loss. Cytochrome c was translocated into the cytosol by Bcl-rambo and mitochondria-targeted molecules, Rambo-Bcl2-TM and Rambo-BNip3-TM. (Fig. 3B), suggesting that mitochondrial damage occurs during Bcl-rambo-induced cell death. To further characterize the pathways by which Bcl-rambo induced mitochondrial damage, we transiently transfected PC-3 cells with Bcl-rambo in the presence or absence of specific PT inhibitors such as CsA and BA, or co-transfected the cells with pcDNA-Bcl-2. BA inhibits ANT and CsA attach to CyP-D, and thereby prevents their interaction with other PTP components and effectively blocks PT [18]. BA suppressed Bcl-rambo-induced membrane perturbation (Fig. 3C), whereas, CsA did not block MMP loss. Bcl-2 overexpression partly inhibited MMP loss. Consistent with the MMP alterations, BA protected against Bcl-rambo-induced apoptotic cell death, whereas CsA failed to inhibit Bcl-rambo-induced apoptotic cell death. Bcl-2 overexpression did not rescue cells from apoptosis (Fig. 3C and D). Our data indicate that Bcl-rambo facilitates cell death by inducing BA-sensitive MMP loss. MPT plays a central role in apoptotic cell death [4, 5]. MPT is caused by the opening of PTP, which supposedly leads to the MMP loss and cytochrome c release. To confirm our results and identify what other components are involved in PT induction, we tested whether recombinant Bcl-rambo (rBcl-rambo) or its chimeric mutants were able to dissipate MMP in isolated mitochondria. We incubated freshly isolated mitochondria from rat liver with 1 μg of purified rBcl-rambo, rRamboΔTM, rRambo-BNip3-TM, rRambo-Bcl2-TM, or rRambo-Cyb5-TM in respiration buffer, and assessed mitochondrial swelling indicative of PT induction by measuring the optical density at 540 nm. In agreement with the in vivo data, the mitochondria were swollen in the presence of rBcl-rambo, rRambo-BNip3-TM, or rRambo-Bcl2-TM, but not in the presence of rRamboΔTM or rRambo-Cyb5-TM (Fig. 4 A). Mitochondrial swelling was suppressed by the PT inhibitor BA, but not by CsA or rBcl-2 (Fig. 4B). These in vitro data indicate that Bcl-rambo directly induces PT through a BA-sensitive pathway. The in vivo and in vitro data suggest that Bcl-rambo induces MMP loss through a BA-sensitive pathway. We therefore investigated the interactions between Bcl-rambo and the components of PTP. Proteins extracted from PC-3 cells transfected with Bcl-rambo were immunoprecipitated with anti-V5 antibody and then blotted with anti-ANT, anti-VDAC, or anti-Cyp-D antibody. ANT co-precipitated with Bcl-rambo, whereas VDAC or Cyp-D did not (Fig. 5 A). We then investigated whether the physical interaction between Bcl-rambo and ANT is crucial for cell death. PC-3 cells were transiently transfected with Bcl-rambo or its chimeric mutants. The protein lysates were immunoprecipitated with anti-V5 antibody and blotted with anti-ANT antibody. Rambo-Bcl2-TM and Rambo-BNip3-TM, both of which induce cell death, were co-precipitated with ANT, whereas Rambo-Cyb5-TM and Bcl-ramboΔTM, neither of which induces cell death, were not co-precipitated with ANT (Fig. 5B). Interaction study with purified Bcl-rambo mutants and purified ANT in GST full-down assay and co-localization study by confocal microscopy showed that Bcl-rambo directly interacts with ANT (Fig. 5C and D). To further demonstrate the effect of ANT in Bcl-rambo-induced cell death, we transfected the cells with siRNA targeting ANT1 and ANT2 (siANT1, siANT2) or scrambled siRNA (siSCR) to downregulate endogenous ANT expression; immunoblotting confirmed that the expression of ANT was significantly reduced by more than 80% in the PC-3 cells (Fig. 5E). After a 48-h transfection of ANT-knockdown cells with Bcl-rambo wild-type and mutants (Fig. 5E), Bcl-rambo-induced cell death reduced. These data indicate that ANT is crucial for Bcl-rambo induced cell death. To determine whether Bcl-rambo affects ANT activity, we investigated the effects of Bcl-rambo and its chimeric mutants on mitochondrial [14C]ADP transport. Isolated mitochondria were incubated with 20 μM [14C]ADP in the presence of rBcl-rambo, rBcl-RamboΔTM, rRambo-Bcl2-TM, rRambo-BNip3-TM, or rRambo-Cyb5-TM. rBcl-rambo, rRambo-Bcl2-TM, and rRambo-BNip3-TM significantly inhibited ADP uptake (P < 0.05), whereas rRambo-Cyb5-TM or rBcl-ramboΔTM did not (Fig. 6 A). To show that the decrease in ADP uptake was not due to Bcl-rambo-induced mitochondrial swelling/dysfunction, we assessed ANT activity in liposomes by analyzing the released ATP in the supernatant. The ADP-induced release of ATP was significantly inhibited by Bcl-rambo, suggesting that ADP uptake is not due to Bcl-rambo-induced mitochondrial swelling/dysfunction (Fig. 6B). Bcl-2 family proteins can be subdivided into pro-apoptotic or anti-apoptotic members on the basis of their ability to induce or inhibit cell death, respectively [19]. Bcl-rambo is a pro-apoptotic member of the Bcl-2 family of proteins. Although the TM domain is known to be important for the optimal activity of Bcl-rambo, the molecular mechanisms of Bcl-rambo-induced cell death remain largely unknown. We initially characterized which domains of Bcl-rambo are essential for its pro-apoptotic activity, using deletion mutants (Fig. 1A). Deletion of the BH domain, RTA, or RTB did not significantly affect Bcl-rambo's cell-killing activity, suggesting that these domains participate in other biological functions such as signal transduction. Consistent with a previous report [3], deletion of the TM domain abolished the pro-apoptotic ability (Fig. 1B and C). These results indicate 2 possibilities: First, mitochondrial localization per se is essential for the activity of this protein. If this were the case, substitution of the TM domain of Bcl-rambo with the TM domain of other members that efficiently target molecules to mitochondria would yield chimeric proteins with cell-killing ability. Second, mitochondrial localization is important, but not essential, for the optimal activity of these proteins. For example, substitution of the TM domain of BNip3 with the corresponding sequence of cytochrome b5, which targets BNip3 to non-mitochondrial sites, diminished the proapoptotic activity of BNip3 to half that of wild-type BNip3 [10]. This suggests that BNip3 activates different pathways for cell-death induction other than the mitochondrial pathway. To date, Bcl-rambo and BNip3 have several cell death pathway characteristics in common. First, both of them induce BH3-independent cell death [20]. Second, the TM domains of Bcl-rambo and BNip3 are crucial for their pro-apoptotic activity, as shown by other researchers and us previously [21, 22]. In addition, substitution of the TM domain of Bcl-rambo or BNip3 with the corresponding domain of Bcl-2 does not affect the pro-apoptotic activity of Bcl2-rambo or BNip3. However, there probably are differences in the cell death pathways activated by Bcl-rambo and BNip3: for instance, targeting of Bcl-rambo to non-mitochondrial sites resulted in a loss of its pro-apoptotic activity, whereas this was not true for BNip3 [10]. Therefore, we propose that mitochondrial localization, rather than a unique TM domain sequence, is essential for the pro-apoptotic activity of Bcl-rambo, which is consistent with our findings on the involvement of MMP loss in Bcl-rambo-induced cell death (Fig. 3). Although the detailed mechanisms of PT induction remains controversial, pro-apoptotic or anti-apoptotic Bcl-2 family proteins regulate PT by interacting with the component(s) of the PTP [23]. Our finding that Bcl-rambo induced a BA-inhibitable PT led us to identify critical proteins involved in the Bcl-rambo-induced PT. We performed immunoprecipitation to understand the interaction between the components of PTP and Bcl-rambo. We found that Bcl-rambo interacts with ANT to induce PT. In addition, the mitochondria-targeted molecules, Rambo-Bcl2-TM and Rambo-BNip3-TM, interact with ANT, whereas the non-mitochondria-targeted proteins, Rambo-Cyb5-TM and Rambo-ΔTM, do not. These results strongly suggest that cell death signaling by Bcl-rambo is linked to ANT. Our results suggest that the TM domain is not responsible for the interaction with ANT because, as shown Fig. 5A, wild-type BNip3 didn't interact with ANT, suggesting TM domain is only responsible for only mitochondrial localization. ANT catalyzes the import of cytosolic ADP and the export of matrix ATP [24]. Our results led us to study the effects of Bcl-rambo on the ADP translocation ability of ANT. Recombinant Bcl-rambo, Rambo-Bcl2-TM, and Rambo-BNip3-TM suppressed ADP/ATP translocation of ANT, whereas Rambo-ΔTM and Rambo-Cyb5-TM (non-mitochondrial proteins) failed to suppress the ADP/ATP translocation of ANT. Collectively, our data demonstrated that Bcl-rambo interacts with the PTP composite protein ANT to induce the loss of MMP, resulting in caspase activation and apoptotic cell death. This work was supported by a Grant from Kyung Hee University in 2011 (KHU-20100136)." @default.
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• W2076296145 title "Bcl-rambo induces apoptosis via interaction with the adenine nucleotide translocator" @default.
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• W2076296145 cites W2004139620 @default.
• W2076296145 cites W2024505421 @default.
• W2076296145 cites W2032405173 @default.
• W2076296145 cites W2032580133 @default.
• W2076296145 cites W2035028095 @default.
• W2076296145 cites W2035549170 @default.
• W2076296145 cites W2042584010 @default.
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• W2076296145 cites W2069635784 @default.
• W2076296145 cites W2161641464 @default.
• W2076296145 cites W2167293805 @default.
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