All DNA manipulations were carried out using standard subcloning techniques, and plasmids were propagated in E. coli DH5α. All newly created constructs were verified by DNA sequencing. The full-length and spliced forms of the genes encoding human BCA3 and BAX were obtained by reverse transcription of total RNA from HeLa cells and cloned into pCMV-HA/c-myc (Clontech) or pET22b, resulting in HA-BCA3, c-myc-BCA3 or BAX vectors, respectively. PCR fragments encoding M-PMV or HIV-1 protease (PR), their inactive forms (D26N, D25N) or HIV-1 protease precursor (PRO) were subcloned into plasmids for mammalian (pCMV-HA/c-myc) or bacterial (pET22b) expression, resulting in vectors c-myc-M-PMV PR17 (D26N), c-myc-M-PMV PR12 (D26N), c-myc-HIV-1 PR, HA-HIV-1 PR, c-myc-HIV-1 PR (D25N), HA-HIV-1 PR (D25N)c-myc-HIV-1 PRO, and HIV-1 PR pET22b. Point mutations and deletions within BCA3, HIV-1 PR, and BAX genes were introduced by two-step PCR mutagenesis using primers carrying the desired mutations and suitable restriction sites. The PCR fragments were digested with appropriate restriction enzymes and ligated into HA/c-myc-pCMV or pET22b.
The pSAX2 vector, which encodes HIV-1 proviral DNA with deletion of the env gene, was kindly provided by Dr. J. Luban. The pGL3-bax-luc luciferase reporter vector was kindly provided by Prof. L. Tuosto. The pDsRed2-Mito mammalian expression vector, which encodes a fusion of Discosoma sp. red fluorescent protein (DsRed2) and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase, was purchased from Clontech. Further details of the cloning strategy and full sequences of all PCR primers can be obtained from the authors upon request.
Cell lines and protein expression
HEK-293 and HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1% L-glutamine (PAA) at 37°C under 5% CO2. Typically, cells were plated at a density of 2–5 × 105 cells/ml one day before transfection. The following day, cells were transfected with the appropriate plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions. The cells were grown for 6–48 h post-transfection and washed with phosphate-buffered saline (PBS). Further processing depended on the type of experiment. A stable HEK-293 cell line expressing HA-BCA3 was prepared as described in .
Expression and purification of HIV-1 PR
HIV-1 PR was purified as previously described . Briefly, the vector HIV-1 PR pET22b was introduced in E.coli BL21 (DE3) RIL cells (Invitrogen) and HIV-1 PR was produced in the form of inclusion bodies. The inclusion bodies were isolated and resuspended in 60% acetic acid. The suspension was diluted with water and dialyzed against a chromatographic buffer (50 mM MES, 10% glycerol, 1 mM EDTA, 0.05% ME, pH 5.8). The refolded protein was then purified by ion-exchange chromatography (Mono S 5/50 GL, GE Healthcare). The appropriate fractions were aliquoted and frozen at −20°C.
Proteins were resolved by reducing SDS-PAGE and blotted onto a nitrocellulose membrane. To prevent nonspecific interactions, the membrane was blocked with Blocking Solution (Thermo Scientific) and then incubated with primary antibody for 2 h to overnight. After washing with PBS (3×), the membrane was incubated with HRP-conjugated secondary antibody for 1–2 h at 4°C. The antigen-antibody complexes were detected by West Femto Chemiluminescent Substrate (Thermo Scientific) using a LAS-2000 imager. The following antibodies were purchased from Sigma-Aldrich: monoclonal anti-mitochondrial protein (MTC02) and rabbit anti- COX IV antibodies and anti-VDAC1/Porin from Abcam; monoclonal anti-HA peroxidase conjugate clone HA-7, EZview Red Anti-HA Affinity Gel, anti-alpha-tubulin (B512), anti-C11orf17(BCA3), anti-HA-TRITC, anti-HA clone HA-7 FITC, anti-c-myc FITC, and anti-p53 (PAb1801) antibodies and rabbit polyclonal anti-HSPD1, anti-AIF, anti-Tomm22, anti-c-myc, anti-VDAC/Porin, anti-CANX, and anti-BCA3 antibodies. The monoclonal anti-c-myc HRP, anti-c-myc (9E10), anti-Bax (2D2), and anti-HA (F-7) antibodies and goat anti-caspase 8p18(C-20) and anti-BCA3 (N-12) antibodies were purchased from Santa Cruz. The following antibodies were purchased from Cell Signaling; rabbit anti-Bcl2, anti-PARP, anti-Bid, anti-caspase 9, anti-Bax, and anti–acetyl-p53 (Lys382) antibodies and rabbit monoclonal anti-Bak (D2D3), anti-Bax (D2E11), anti-COXIV (3E11), and anti-cleaved caspase 8 (Asp391) antibodies and monoclonal anti-caspase-8 (1C12) antibody. Monoclonal anti-cytochrome C and anti-PKA(C) antibodies were purchased from BD Pharmingen; and rabbit anti-ANT antibody from LSBio. All secondary antibodies (anti-rabbit IgG, anti-mouse IgG) were HRP- (Sigma) or gold- (British Biocell) conjugated.
Co-immunoprecipitation was carried out as previously described . Briefly, HEK-293 or HeLa cells were grown on 60 mm plates and co-transfected with HA-BCA3, c-myc-M-PMV PR17(D26N), c-myc M-PMV PR12(D26N) or c-myc-HIV-1 PR(D25N). After 48 h, the cells were washed 1× with PBS and lysed in 300 μl CO-IP buffer B (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2, 0.5% NP-40) containing Halt Protease Inhibitor Mix (Thermo Scientific) for 30 min on ice. A 1/5 of the total cell lysate sample was used for Western blot analysis. The rest of the cell lysate was cleared by centrifugation and diluted with 1 200 μl CO-IP A buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2). Primary antibody [monoclonal anti-c-myc (Santa-Cruz), rabbit anti-c-myc or EZview Red Anti-HA Affinity Gel (Sigma)] was added to the cell extract and incubated overnight at 4°C. Optionally, 20 μl Protein-A/G-Sepharose beads were added, and after 2 h incubation at 4°C, the immunoprecipitates were collected by centrifugation. The pellets were washed 3× with CO-IP buffer and then 1× with PBS. Proteins were resolved by SDS-PAGE and analyzed by Western blotting.
Immunoprecipitation from mitochondrial and cytosolic fractions
HEK-293 cells (4×105cells/ml) were seeded on 2×100 mm plates and co-transfected with HA-BCA3pCMV and c-myc-HIV-1 PR(D25N). After 48 h, the cells were washed 1× with PBS and 1/20 of the cells was resuspended in PLB and used as total cell lysate sample for Western blot analysis. The rest of the cells was aliquoted: 1/4 was resuspended in 300 μl CO-IP buffer B, and immunoprecipitation was carried out as described above; 3/4 were resuspended in 0.5 ml homogenization medium HB (250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES-NaOH, 10 mM KCl, 1.5 mM MgCl2, pH 7.4) supplemented with Halt Protease Inhibitor Mix (Thermo Scientific) and homogenized by 50 strokes in a Dounce homogenizer. The cell lysate was centrifuged for 10 min at 1000 × g. The pellet was resuspended in 0.5 ml HB, re-homogenized by 30 strokes in a Dounce homogenizer, and centrifuged for 10 min at 1 000 × g. Both supernatants were combined and centrifuged at 17 000 × g for 15 min. NP-40 and NaCl were added to the supernatant (cytosolic fraction) to final concentrations of 0.1% and 150 mM, respectively. The mitochondrial pellet was washed 1× with 400 μl HB and resuspended in 1 ml of CO-IP buffer A supplemented with 0.1% NP-40. 100 μl aliquots of both cytosolic and mitochondrial fractions were mixed with PLB and used as total cytosolic and mitochondrial lysate samples for Western blot analysis. 900 μl aliquots of both samples were used for immunoprecipitation using anti-c-myc and anti-HA antibodies, as described above.
Subcellular fractionation and mitochondria isolation
To obtain a crude mitochondrial fraction, cells (1 × 108 cells/ml) were washed with PBS, pelleted by centrifugation at 300 × g for 5 min and frozen at −70°C. The cell pellet was resuspended in 1 ml homogenization medium HB supplemented with Halt Protease Inhibitor Mix (Thermo Scientific) and homogenized by 50 strokes in a Dounce homogenizer. The cell lysate was centrifuged for 10 min at 1 000 × g. The pellet was resuspended in 1 ml HB, re-homogenized by 30 strokes in a Dounce homogenizer, and centrifuged for 10 min at 1 000 × g. Both supernatants were combined, and a crude mitochondrial fraction was obtained by centrifugation at 17 000 × g for 15 min. The pellet containing crude mitochondria was washed with HB (1×), resuspended in the same buffer and used for subsequent experiments. The remaining supernatant was subjected to ultracentrifugation at 100 000 × g for 1 h at 4°C, and the microsome-containing pellet was analyzed by Western blotting . Mitochondria were further purified by centrifugation in a discontinuous gradient of Optiprep according to the manufacturer’s instructions (http://www.axis-shield-density-gradient-media.com). Briefly, the crude mitochondrial fraction was resuspended in 1.4 ml HB and 3.6 ml solution D [5 volumes of 50% iodixanol and 1 volume of solution C (0.25 M sucrose, 6 mM EDTA, 120 mM HEPES-NaOH, pH 7.4)]. This suspension was overlaid with 3.6 ml of each of two gradient solutions (with densities of 1.175 g/ml and 1.079 g/ml) and centrifuged at 50 000 × g for 3 h in a Beckman SW41 rotor. The gradient was collected in 1 ml fractions and analyzed by Western blotting. To recover purified mitochondria, the appropriate gradient fraction(s) was diluted by HB (1:3) and centrifuged at 17 000 × g for 15 min. Pelleted mitochondria were washed with HB and resuspended in HB solution. In experiments in which nuclear and mitochondrial fractions were isolated, the NE-PER Nuclear Extraction Kit and Mitochondrial Isolation Kit (Thermo Scientific), respectively, were used.
HIV-1 PR treatment of cell lysates
HEK-293 or HeLa cells, optionally transfected with HIV-1 Gag, c-myc-HIV-1 PR and HA-BCA3, were washed with PBS, pelleted by centrifugation at 300 × g for 5 min and frozen at −70°C. The cell pellet was resuspended in 500 μl solution B (60 mM MES, pH 6.5, 120 mM NaCl, 0.1% NP-40) and homogenized by 50 strokes in a Dounce homogenizer. The cell lysate was centrifuged for 10 min at 1 000 × g. The pellet was resuspended in 500 μl 60 mM MES, pH 6.5, containing 120 mM NaCl, homogenized by 30 strokes in a Dounce homogenizer and centrifuged for 10 min at 1 000 × g. Both supernatants were combined and incubated with recombinant HIV-1 PR (1.25 μM final concentration), optionally in the presence of ritonavir (Sigma) (4 μM final concentration) or calpain inhibitor I (Sigma) (33–100 μM final concentration) at 37°C. The reaction was stopped by addition of 2× protein loading buffer (PLB). Samples were separated on 12 or 15% SDS-PAGE and analyzed by Western blotting.
HIV-1 PR treatment of purified mitochondria
Freshly isolated mitochondria were diluted in SB buffer (200 mM sucrose, 10 mM HEPES-NaOH, pH 7.4, 2 mM NaCl, 5 mM KH2PO4, 2 mM MgCl2) to a final protein concentration of 1 mg/ml. Various amounts of recombinant HIV-1 PR (4 μM storage concentration) in the presence or absence of ritonavir (4 μM final concentration) were added, and incubation proceeded overnight at room temperature. The reaction was stopped by addition of 2× PLB. Proteins were separated by 12-15% SDS-PAGE and analyzed by Western blotting. In the experiments studying the release of the mitochondrial protein, the mitochondria were after incubation with HIV-1 PR centrifuged for 16 000 × g for 10 min at 4°C. The selected proteins from the supernatant were analyzed by Western blot.
In vitro preparation and cleavage of Bax protein
The p21 and p18 forms of Bax protein were prepared by TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer’s instructions. Briefly, 40 μl TNT Quick Master Mix was mixed with 1 μg plasmid DNA together with 2 μl 35S methionine (10 mCi/ml) in nuclease-free water to a final volume of 50 μl. The mixture was incubated for 3 h at 30°C. Buffer containing 60 mM MES, 120 mM NaCl, pH 5.5, (50 μl) was added to each sample, followed by treatment with HIV-1 PR (1.25 μM final concentration) in the presence or absence of ritonavir (8 μM final concentration) for various time at room temperature. The reaction was stopped by addition of 2× PLB. HIV-1 PR activity was monitored by cleavage of purified HIV-1 CANC protein. Samples were resolved by 15% SDS-PAGE and visualized by autoradiography.
Proteinase K digestion of isolated mitochondria
The basic experimental procedure was adapted from previously described methods [56, 57]. Freshly isolated mitochondria from 0.5 × 108 HEK-293 cells co-transfected with c-myc-HIV-1 PR(D25N) and HA-BCA3 were resuspended in 350 μl homogenization buffer without protease inhibitors, and protein concentration was assessed by Bradford assay. Fresh mitochondrial samples (300 μg/100 μl) were incubated (i) alone, (ii) in the presence of proteinase K (Sigma) (0.14, 1.4 or 14 μM final concentration) or (iii) in the presence of Triton X-100 (1% final concentration) and proteinase K (0.14, 1.4 or 14 μM final concentration) for 10 or 60 min on ice. The reaction was stopped by the addition of 6× PLB, and samples were analyzed by Western blotting.
HEK-293 cells were grown in a 24-well plate and were transfected with the appropriate vectors. Cells were lysed in Glo™ Lysis Buffer (Promega) 48 h post-transfection, and luciferase activity was determined by One-Glo™ Luciferase Assay System (Promega) using a fluorescence reader. Relative luciferase activity was corrected for differences in transfection efficiency by co-transfection with a GFPpCMV construct followed by fluorescence measurement. Each transfection was performed in triplicate; at least three independent experiments were performed.
Flow cytometry analysis of dead and apoptotic cells
Determination of dead cells was adapted from the procedure described by Vermes et al.. HeLa cells growing in six-well plates were transfected with c-myc-HIV-1 PR at various times, and the cells were harvested at 12, 18, 24 and 48 h post-transfection by standard trypsinization . The cells were gently washed twice in PBS (300 × g, 5 min), resuspended in annexin-binding buffer and labeled with FITC Alexa Fluor® 488 annexin V and propidium iodide (PI) according to the manufacturer’s protocol for Dead Cell Apoptosis Kit (Invitrogen). Analyses were performed by flow cytometry using BD LSR Fortessa with BD FACSDiva software. Ten thousand events were collected for each sample.
Flow cytometry analysis of mitochondrial membrane potential (ΔΨm)
The protocol was adapted from previous reports [60, 61]. HEK-293 cells were seeded in 12-well plates overnight and transfected with 0.5, 1 or 2 μg/ml c-myc-HIV-1PR. Changes in ΔΨm were determined by JC-1 fluorochrome (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide) stored in DMSO (Sigma) 24 h post-transfection. The cells were incubated in DMEM containing 12.5 μg/ml JC-1 at 37°C under 5% CO2 for 45 min. Cells incubated with 400 μM CCCP (carbonyl cyanide 3-chlorophenylhydrazone) stored in DMSO (Sigma) for 2 hours were used as a positive control. After incubation, the samples were washed twice with cold PBS, and cell pellets were resuspended in 500 μl PBS and immediately analyzed by flow cytometry using BD LSR Fortessa. Healthy cells containing red JC-1 aggregates were detected in the FL 2 channel, while green JC-1 monomers in apoptotic cells were monitored in the FL1 channel (FITC). Ten thousand events were collected for each sample.
HEK-293 or HeLa cells at a density of 1 × 105 cells/ml were plated on a glass coverslip placed in a 6-well plate and incubated in DMEM medium supplemented with 10% FBS (Sigma) and 1% L-glutamine (PAA) at 37°C and 5% CO2. The following day, the cells were transfected with appropriate plasmid DNA using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol. 48 h post transfection, the cells were washed with PBS, fixed with 4% formaldehyde and incubated for 30 min at room temperature. The cells were washed 2× with PBS and incubated 10 min in 0.4% Triton X-100, followed by 2 × 5 min incubation with 0.1% Triton X-100. The cells were then incubated with primary fluorescently labeled antibodies for 60 min at room temperature, washed 2× with PBS and mounted using mounting media (Vectashield, Vector Laboratories), containing or not DAPI (4′,6-diamidine-2-phenyl indole) at a final concentration of 1.5 μg/ml. The samples were then imaged using a laser scanning confocal microscope (Leica) or Olympus IX 81 microscope.
HEK-293 cells were fixed for 30 min at 4°C in 0.1 M PIPES, pH 7.5, containing 2% formaldehyde and 0.1% glutaraldehyde. After washing, the samples were gradually dehydrated at 4°C in 30%, 50%, 70%, 90% aqueous ethanol (10 min in each bath) and finally twice in anhydrous ethanol for 10 min. After dehydration, the samples were embedded in LR White. The cells were infiltrated at 4°C in a 1:2 (v/v) mixture of LR White and ethanol for 15 min, a 1:1 (v/v) mixture for 30 min and a 2:1 (v/v) mixture for 30 min, followed by overnight incubation in pure resin at 4°C. Polymerization was carried out at 18°C under UV light for 72 h.
Ultrathin sections (90 nm) were mounted on parlodion-coated nickel grids. The grids were washed briefly in water and incubated in blocking buffer [1% BSA (w/v) in PBS, pH 7.4] containing 10% normal goat serum (v/v) for 25 min. The grids were then incubated in buffer A [0.5% BSA (w/v), 0.05% (v/v) TWEEN 20 in PBS, pH 7.4] containing monoclonal antibody against HA (Santa Cruz) or VDAC (Sigma), diluted 1:100, for 2 h at room temperature. The grids were washed three times in buffer A and then transferred to a droplet of goat anti-mouse IgG conjugated to 10 nm gold particles (British Biocell), diluted 1:25 in buffer A, and incubated 1 h at room temperature. After washing in buffer A, the grids were stained with uranyl acetate and examined with a JEOL JEM 1200EX electron microscope.