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A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax
Analytical Biochemistry 497 (2016) 90e94
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A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax Oscar Teijido a, Yogesh Tengarai Ganesan b, Raul Llanos c, Ashley Peton c, Jean-Baptiste Urtecho c, Adauri Soprani d, Aimee Villamayor d, Bruno Antonsson e, phen Manon f, Laurent Dejean c, * Ste a
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Care and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY 10065, USA c Department of Chemistry, California State University, Fresno, CA 93740, USA d Department of Basic Sciences, New York University, College of Dentistry, New York, NY 10010, USA e Merck Serono S.A. Geneva Research Center, Geneva, Switzerland f CNRS, Universit e de Bordeaux, UMR5095, 1 Rue Camille Saint-Sa€ ens, 33077 Bordeaux, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2015 Received in revised form 25 November 2015 Accepted 22 December 2015 Available online 31 December 2015
Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. © 2016 Elsevier Inc. All rights reserved.
Keywords: ELISA Bax Conformation specificity Apoptosis Mitochondria
Introduction Apoptosis is a programmed cell death mechanism that is finely regulated by proteineprotein interaction at the mitochondria between pro- and anti-apoptotic members of the Bcl-2 family proteins [1e5]. Defects in this regulation are associated with several diseases, such as neurodegenerative disorders and cancer [6e10]. Cytochrome c release is the point of no return of apoptosis, which promotes and amplifies the cell death cascade [1,2,5,11]. The permeabilization of the mitochondrial outer membrane involves the translocation of Bax to the mitochondria, where it undergoes a change of conformation (Bax activation) that leads to the exposure of the Bax N-terminal region [1,2,4,5,8,12]. Activated Bax forms
* Corresponding author. E-mail address: [email protected] (L. Dejean). http://dx.doi.org/10.1016/j.ab.2015.12.016 0003-2697/© 2016 Elsevier Inc. All rights reserved.
oligomers that are the principal components of the mitochondrial apoptosis-induced channel (MAC), through which cytochrome c is released to the cytosol [5,8,9,11,13e16]. Activated Bax is extensively used as an early marker of apoptosis [1e5,8,11,12]. Quantification of the accumulation of activated Bax in mitochondria is a valuable tool to clinically diagnose pathologies associated with cell degeneration or tumorigenesis [7,10,22,23]. Antibodies that recognize the Bax N-terminus allow the discrimination of activated/oligomerized from native cytosolic Bax [17,18]. Current methods used to detect activated Bax are based on immunoprecipitation followed by immunodetection by Western blot. These time-consuming techniques provide only qualitative detection or, at best, semiquantification of activated Bax, which in many cases is not reproducible enough to report small but significant differences between control and apoptotic conditions. This article describes a rapid method based on a sandwich ELISA that allows an accurate and reproducible quantification of recombinant
O. Teijido et al. / Analytical Biochemistry 497 (2016) 90e94
human activated/oligomerized Bax in vitro. In addition, this ELISA also permits reproducible quantification of activated Bax in mitochondrial or whole cell protein extracts from normal and apoptotic HeLa cells. This sandwich ELISA allows rapid and convenient quantification of recombinant and native activated Bax, a wellrecognized early apoptotic marker with potential prognostic value in degenerative disorders and/or cancer therapeutics. Materials and methods Cell culture and apoptosis induction HeLa cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 50 mg/ml streptomycin, and 50 IU/ml penicillin at 37 C in 5% CO2. Cells were split 1/2 (v/v) in 175 cm2 flasks 24 h before the treatment, and then untreated or treated with 0.5 mM staurosporine for 16 h to induce apoptosis [19]. Recombinant proteins Recombinant monomeric/nonactivated (monomeric) and oligomeric/activated (activated) human Bax of full length, with six histidines at the N-terminus, were expressed in Escherichia coli and purified as previously described [19,20]. Activated Bax was obtained by purifying the oligomers after disrupting bacteria in a buffer containing 1% Triton X-100 (v/v), a detergent that artificially induces Bax activation/oligomerization [21]. Once the oligomers were obtained, Triton X-100 was exchanged for 1% octyl glucoside by dialysis. The purified protein was stored in 25 mM Hepes-NaOH, 0.2 mM DTT, 1% octyl glucoside, 30% glycerol, pH 7.5, at 80 C [19]. Total protein extraction from whole cells and mitochondria Total proteins were extracted from control (untreated) or staurosporine-treated (apoptotic) HeLa cells (4 175 cm2 flasks each). At the end of the treatment, cells were harvested in phosphate-buffered saline (PBS) containing 1 mM EDTA and recovered by centrifugation at 750g for 10 min. Pellets containing cells were washed with 1X PBS and suspended in lysis buffer consisting of a 1x PBS supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals) and 2% CHAPS (w/ v) (Sigma-Aldrich), in order to prevent artificial Bax oligomerization [21]. Cells were incubated on ice for 60 min, sonicated for 4 30 s, incubated on ice for 90 min, sonicated again, and centrifuged at 100,000g for 30 min. Supernatants contained the extracted proteins. Mitochondria were isolated from untreated or apoptotic HeLa cells (10 175 cm2 flasks each), as previously described [19]. Pellets containing mitochondria were lyzed in mitochondrial buffer (230 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HepesNaOH, pH 7.5) supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals) and 2% CHAPS (v/v). Mitochondria were then incubated on ice for 1 h, sonicated for 4 30 s, and finally centrifuged for 30 min at 100,000g. Supernatants contained the mitochondrial extracted proteins. The total protein content in both whole cells and mitochondria extracts was measured using a BCA assay (Pierce). Estimation of total Bax content by Western blot and densitometry Total protein extracts (10 mg) from untreated and apoptotic cells and mitochondria were run in a SDS-PAGE gel, along with different concentrations of oligomeric recombinant Bax. Proteins were then transferred onto a PVDF membrane (BioRad). Membrane was blocked in 1x PBS, 0.2% (v/v) Tween-20, 5% (w/v) milk for 30 min,
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followed by incubation with 0.2 ng/mL polyclonal HRP-linked antiBax antibody (N20-HRP, Santa Cruz Biotechnology, Inc.) for 1 h at room temperature or overnight at 4 C. Bax signal was detecting using HRP-dependent luminescence (ECL, Pierce), which was collected on an X-ray film after different exposure times (GE Healthcare). Nonsaturated Western blot signals were scanned, and the pixel density of the Bax bands was quantified using the software Image J (NIH Open Source). Pixel quantification of the bands corresponding to increasing amounts of recombinant oligomeric Bax were used to build a standard curve, which allowed estimation of the Bax content in the different protein extracts tested [22,23]. Immunoprecipitation of activated/oligomeric BAX Mitochondrial protein lysates (35e55 mg), HeLa total cell protein lysates (60e140 mg), or recombinant human Bax monomers and oligomers (10 ng total Bax) were resuspended in mitochondrial buffer or in 1x PBS supplemented with complete protease inhibitor mixture and 2% CHAPS (w/v) (Sigma). Samples were incubated overnight at 4 C with 1 mg of monoclonal (6A7, Sigma-Aldrich) or polyclonal anti-Bax antibody (N20, Santa Cruz Biotechnology, Inc.), or alternatively with control mouse or rabbit total IgGs (Santa Cruz Biotechnology, Inc.). The different samples were then incubated with 1/1 (v/v) protein G-coupled magnetic beads (Invitrogen) at room temperature for 45 min, and the immunocomplexes were precipitated by placing the assay tubes in a holder magnet for 10e30 s. Proteins contained in the pellets were separated from the magnetic beads by washing them twice with 1x PBS, resuspension in sample buffer, incubation at 60 C for 5 min, and magnetic precipitation of the denatured protein G magnetic beads. Supernatants were directly supplemented with sample buffer (BioRad) and heated at 60 C for 5 min. Bax content in supernatants and pellets was estimated by Western blot and densitometry using Image J as described in Refs. [22,23]. Sandwich ELISA for the quantification of activated Bax Activated Bax ELISA was performed in a 96-well plate (Costar). Each well used for the assay was coated overnight with 100 mL of capture antibody solution containing 0.25 mg/mL of the monoclonal anti-Bax 6A7 antibody (Sigma-Aldrich) in DPBS (Fisher) at 4 C. All the following incubations were performed at room temperature and with continuous plate shaking. Coated wells were washed four times with 100 ml DPBS, 0.05% Tween-20 (Sigma-Aldrich), and incubated for 1 h with 200 mL blocking buffer containing 1% BSA (w/ v) (Sigma-Aldrich) in DPBS, followed by four washes with regular DPBS. Both standards and samples were diluted in DPBS 2% (w/v) CHAPS and analyzed in duplicate or triplicate. Activated Bax standards contained 0, 0.3, 0.6, 1.2, 2.5, 5.0, 10, 20, 40, or 80 ng/mL of recombinant activated Bax. Total protein extracts from isolated mitochondria of control and apoptotic HeLa cells were resuspended in DPBS 2% (w/v) CHAPS at a final concentration of 0.2e0.45 mg/ mL, whereas proteins extracted from whole HeLa cells were resuspended at a final concentration of 3.5e10 mg/mL in the same buffer. Activated Bax standards and samples were incubated for 2 h at room temperature in the wells precoated with the 6A7 antibody. The plate was then washed four times with DPBS. Each sample was incubated for 1 h with 100 mL of detection antibody solution containing 0.1 mg/mL of the polyclonal anti-Bax N20 antibody (Santa Cruz Biotechnology, Inc.) in blocking buffer. After incubation, the plate was washed four times with DPBS, and then wells were incubated for 1 h with 100 ml of a solution containing 0.12 mg/mL goat anti-rabbit biotinylated third antibody (Abcam) in blocking buffer. Each well was washed four times with DPBS and incubated
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30 min with 100 mL of a solution of 1x streptavidin-HRP (R&D Systems). The plate was finally submitted to five rapid washings followed by four longer washings (30 se1 min each) in order to maximally reduce the background signals. Immunodetection was performed by incubating the samples for 10 min in the dark with 100 mL of freshly mixed TMB substrate solution (R&D Systems). The developing reaction was finally stopped by adding 100 mL of 2 N H2SO4: the intensity of the yellow color was proportional to the amount of immunocomplexes contained in each well. Absorbance was read at 450 nm and 540 nm within 30 min after the developing reaction was stopped. Final sample concentration values were calculated by the Delta OD (450e540 nm) using a standard curve made with increasing concentrations of recombinant Bax oligomers (0.5e80 ng/mL).
A
Bax monomers Control Ab Bax Ab
B
Results Activated Bax ELISA allows the specific detection and quantification of recombinant human Bax oligomers
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
+ _
+ _
S
P
_ +
S
Bax oligomers
_ +
+ _
+ _
_ +
_ +
P
S
P
S
P
y = 0.0071x + 0.1359 2 R = 0.9966
Bax oligomers
Bax monomers 0
20
40
60
80
100
[BAX] (ng/mL)
C Back-calculated Bax ratio (%)
The binding specificity of the antibodies for activated Bax was first tested by immunoprecipitation of recombinant human Bax monomers and oligomers. As shown in Fig. 1A, Bax oligomers, but not monomers, were immunoprecipitated by the monoclonal antiactivated Bax antibody 6A7 (Bax ab). Similar results were previously obtained with the polyclonal anti-activated Bax antibody N20 [24]. Total mouse or rabbit IgGs (control ab), however, did not immunprecipitate either Bax monomers or oligomers (Fig. 1A). Specificity of these antibodies for the activated form of Bax was then assessed using a sandwich-based ELISA on increasing concentrations of human Bax oligomers or monomers (Fig. 1B). During these preliminary experiments, monoclonal 6A7 and polyclonal N20 were used as “capture” and “detection” antibodies, respectively. Once again, the increase of the ELISA signals (delta OD) was proportional to the concentrations of Bax oligomers tested, whereas Bax monomers produced a low-intensity signal independent of Bax concentrations (Fig. 1B). The linear range of the ELISA using Bax oligomer standards was between 0.5 and 80 ng/mL, which allowed a linear regression analysis of the data, and thus the calculation of Bax oligomer concentrations in a sample. The coefficient of determination (R2) using Bax oligomer standards was above 0.99 in the experiment reported in Fig. 1B, which is representative of 35 independent ELISAs performed so far. Finally, the limit of detection of Bax oligomers with this ELISA was 5 ng/mL and was defined as the lowest concentration for which the signal is statistically different from the signal generated by the same concentration of Bax monomers (Fig. 1B). Biological samples contain a mix of monomeric and activated/ oligomeric Bax. The ratio between the two conformations of Bax is modified during apoptosis or under pathological conditions. To mimic these conditions, the activated-Bax ELISA was tested with samples containing the same amount of total Bax, but different proportions of Bax monomers vs. oligomers (Fig. 1C). Thus, samples were prepared with mixes of 0/100, 25/75, 50/50, 75/25, and 100/ 0% Bax oligomers/monomers, in which 100% corresponded to 80 ng/mL of total Bax. As expected, the delta ODs were higher as the proportion of oligomeric Bax increased (Fig. 1C). Bax concentration back-calculated from the delta ODs corresponded strictly to the amount of Bax oligomers in each sample tested, demonstrating that the presence of monomeric Bax did not interfere with the ELISA signal (Fig. 1C). Activated Bax ELISA allows the specific quantification of activated/oligomeric Bax in mitochondrial and whole cell proteins extracted from human cells.
Recombinant human Bax
Delta OD (450-540 nm)
92
y= 1.0639x R2 = 0.9879
100 75 50 25 0
0/100
25/75
50/50
75/25
100/0
Bax oligomers/monomers ratio (%) Fig. 1. Activated Bax ELISA allows the specific detection and quantification of recombinant human Bax oligomers. (A) Recombinant human Bax oligomers are specifically immunoprecipitated by the 6A7 monoclonal antibody. Equal total amounts of Bax monomers or oligomers (i.e., 10 ng) were submitted to immunoprecipitation using 6A7 anti-Bax monoclonal antibody (Bax Ab) or total mouse IgGs (Control Ab) as described in the Materials and methods section. S: supernatants, P: pellets. (B) A 6A7/N20 antibodies-based sandwich ELISA allows the specific detection and quantification of recombinant human Bax oligomers. Sandwich ELISA was performed on samples containing increasing concentrations of recombinant human Bax monomers and oligomers (0.5e80 ng/mL). During the assay, mouse monoclonal 6A7 and rabbit polyclonal N20 Bax antibodies were used as capture and detection antibodies, respectively. The detection of the immunocomplexes was realized using a biotinylated anti-rabbit tertiary antibody and streptavidin-HRP (see the Materials and methods section for details). Results are representative of 35 independent ELISAs. The detection limit was defined as the lowest concentration at which the signal is statistically different from the signal generated by the same concentration of Bax monomers using a Student's t-test (i.e., p < 0.05). (C) Recombinant human Bax monomers do not interfere with the activated Bax ELISA signals. Sandwich ELISA of sample mixtures of recombinant Bax monomers and oligomers. Samples contained increasing amounts of Bax oligomers (0e100%) and proportional decreasing amounts of Bax monomers (100e0%), to have a final total Bax concentration of 80 ng/mL (Bax oligomers/monomers ratio (%)). The Bax ratio was then recalculated for each sample using the ELISA data, making the assumption that only Bax oligomers contributed to the signal (back-calculated Bax ratio (%)). Results are shown as mean ± s.e.m. (n ¼ 3).
We finally tested the ability to detect and quantify activated Bax in human biological samples comparing the classical immunoprecipitation technique with our new ELISA assay (Fig. 2). Fig. 2A shows the immunoprecipitation of activated Bax from total (top) and mitochondrial (bottom) proteins extracted from HeLa cells treated with the classic apoptotic inducer staurosporine [13]. Immunoprecipitation with the monoclonal anti-Bax 6A7 (Bax
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Fig. 2. Activated Bax ELISA allows the quantification of activated human Bax in biological samples. Apoptosis was induced by incubating HeLa cells in the presence of 0.5 mM staurosporine (STS) for 16 h. Total proteins were extracted from control (untreated) or staurosporine-treated (apoptotic) cells in the presence of 2% CHAPS. Mitochondria were isolated from untreated or apoptotic HeLa cells and mitochondrial proteins extracted in the presence of CHAPS 2% as described in the Materials and Methods section. (A) Endogenous human Bax oligomers are specifically immunoprecipitated by the 6A7 monoclonal antibody in mitochondrial or cell protein extracts. Immunoprecipitation of total protein extracts from untreated and apoptotic whole cells (top) or isolated mitochondria (bottom) using 6A7 anti-Bax antibody (Bax Ab) or total mouse IgGs (Control Ab). S: supernatants, P: pellets. (B) The 6A7/N20 Bax ELISA allows sensitive detection and quantification of Bax activation in protein extracts from whole cells or mitochondrial samples. Cell and mitochondrial protein extracts from HeLa cells were obtained and assayed as described in the Materials and methods section. The amounts of activated oligomeric Bax concentrations (ng Bax/mg total protein) from untreated and apoptotic protein extracts from whole cells (left) or isolated mitochondria (right) were calculated with reference to a standard curve built with increasing concentrations of recombinant human Bax (1e80 ng/mL). Results are shown as mean ± s.e.m. (n ¼ 12e18). Statistical differences were determined by Student's t-test and * corresponds to p < 0.05.
ab) showed a barely visible band in the pellet, corresponding to the activated Bax population, in untreated cells or mitochondria compared with an intense signal in apoptotic samples. Total mouse IgGs (control ab) did not immunoprecipitate any Bax conformation. Similar results were obtained using the polyclonal anti-Bax N20 (Bax antibody) on total and mitochondrial protein extracts from apoptotic HeLa cells, as previously shown in Ref. [13]. These same total and mitochondrial protein extracts were then submitted to the activated-Bax ELISA assay (Fig. 2B). The standard curve was performed with increasing concentrations of recombinant Bax oligomers (1.25e80 ng/mL range, as shown in Fig. 1C). As expected, the activated Bax content was significantly higher in apoptotic whole cells, i.e., 2.7 ± 0.4 vs. 1.0 ± 0.1 ng/mg of total protein (Fig. 2B, left panel), and in apoptotic mitochondria, i.e., 34 ± 6 vs. 13 ± 1 ng/mg of total protein (Fig. 2B, right panel). Interestingly, the high sensitivity of the ELISA allowed a clear and reproducible quantification of activated Bax from untreated HeLa cells, which was, at best, barely detected by the immunoprecipitation assay (compare Fig. 1B with Fig. 1A). The concentration of activated Bax detected by ELISA was also found to be about 10 times higher in mitochondria than in whole cells, in agreement with Bax translocation and activation at the mitochondria during staurosporine-induced apoptosis (Fig. 2B and [25]). However, the relative increase of activated Bax during apoptosis was found to be very similar in both cases (i.e., 2.5-fold increase of activated Bax in
apoptotic whole cells vs. untreated, compared with the 3-fold increase in apoptotic vs. normal in mitochondria), thus allowing clear discrimination between normal and apoptotic situations in both types of samples (Fig. 2B). Discussion Bax activation/oligomerization in mitochondria is a very early event in the apoptotic cascade [1e5,8,11,12]. In this respect, activated Bax content is an early marker for apoptosis, but also a potential biomarker for the clinical diagnosis of diseases associated with defects in apoptosis regulation (e.g., neurodegenerative diseases or cancer). Activated/oligomerized Bax is discriminated from cytosolic Bax by antibodies that recognize the conformational change that leads to the exposure of the Bax N-terminal region. The antibodies used during this study include monoclonal (6A7) and polyclonal (N20) Bax antibodies. Techniques used nowadays to detect activated Bax are based on immunocytochemistry or immunoprecipitation assays using those same antibodies or antibodies directed against similar epitopes [1e5,8,11,12]. However, these time-consuming and multistep methods allow only a few samples to be processed at a time and provide a qualitative, or at best semiquantitative type of detection for activated Bax. Our study describes an ELISA method that allows (i) an absolute quantification of activated Bax, (ii) a higher sensitivity than immunoblots, (iii)
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multiple determinations by performing the experiments on a 96well plate, and (iv) a short processing time. The activated Bax ELISA assay uses monoclonal antibodies (6A7, Sigma-Aldrich), which provide high specificity, and polyclonal antibodies (N20, Santa Cruz Biotechnology, Inc.), which increase the sensitivity of the detection. This method allows the specific detection and quantification of recombinant Bax oligomers, but not monomers, either sampled standalone or in a mix (Fig. 1B and C). The reliability of the ELISA was further tested on human biological samples. In order to specifically determine and quantify activated Bax, total proteins were extracted from HeLa cells or from mitochondria in buffer containing 2% CHAPS, as this detergent does not induce artificial change of the conformation of Bax [21]. The high sensitivity of the ELISA assay allows detection and quantification of activated Bax traces in untreated cells, which are undetectable in the classic Western blot assays. As expected, the amount of activated/oligomeric Bax (ng/mg total protein) was significantly higher in samples from apoptotic cells than from untreated cells (Fig. 2B). Interestingly, the increase of activated Bax in apoptotic extracts (2.5e3 fold) was very similar in whole cells and in isolated mitochondria, confirming that Bax activation and oligomerization occur in the mitochondria during staurosporine-induced apoptosis [25]. Immunoassays such as ELISA are prone to matrix effects when used to quantify membrane proteins [26]. In this study, we observed a decrease of signal linearity vs. Bax oligomer concentrations in protein samples extracted from isolated mitochondria, more specifically when these samples were diluted less than 10 times in PBS 2% CHAPS (not shown). We did not observe this phenomenon in protein samples from whole cells that are generated in the same buffer further used during the ELISA (i.e., PBS 2% CHAPS). Taken together, these results indicate that high concentrations of mitochondrial buffer components such as mannitol, sucrose, or EDTA may generate negative matrix effects on this activated Bax ELISA. We believe that the combination of the results presented in this study validates an ELISA protocol allowing a rapid, specific, and sensitive quantification of activated Bax in human biological samples. Therefore, the application of this protocol to different cell models, including tissue samples, may prove useful in the diagnosis of various pathologies related to defective regulation of apoptosis, such as neurodegenerative diseases or cancer. Acknowledgments We are grateful to Kathleen Kinnally, Pablo Peixoto, and ShinYoung Ryu (NYU CD) for helpful discussions. This study has been supported by NYU Research Challenge Funds and CSU Fresno startup funds to LD. References [1] J.E. Chipuk, D.R. Green, How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol. 18 (2008) 157e164. [2] N.N. Danial, BCL-2 family proteins: critical checkpoints of apoptotic cell death, Clin. Cancer Res. 13 (2007) 7254e7263.
[3] B. Leber, J. Lin, D.W. Andrews, Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes, Apoptosis 12 (2007) 897e911. [4] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99e163. [5] L.M. Dejean, S. Martinez-Caballero, K.W. Kinnally, Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? Cell Death Differ. 13 (2006) 1387e1395. [6] J.P. Meijerink, E.M. Van Lieshout, H.B. Beverloo, E. Van Drunen, E.J. Mensink, M. Macville, R. Pieters, Novel murine B-cell lymphoma/leukemia model to study BCL2-driven oncogenesis, Int. J. Cancer 114 (2005) 917e925. [7] M. Certo, V. Del Gaizo Moore, M. Nishino, G. Wei, S. Korsmeyer, S.A. Armstrong, A. Letai, Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members, Cancer Cell 9 (2006) 351e365. [8] L.M. Dejean, S.Y. Ryu, S. Martinez-Caballero, O. Teijido, P.M. Peixoto, K.W. Kinnally, MAC and Bcl-2 family proteins conspire in a deadly plot, Biochim. Biophys. Acta 1797 (2010) 1231e1238. [9] K.W. Kinnally, P.M. Peixoto, S.Y. Ryu, L.M. Dejean, Is mPTP the gatekeeper for necrosis, apoptosis, or both? Biochim. Biophys. Acta 1813 (2011) 616e622. [10] T.T. Renault, O. Teijido, B. Antonsson, L.M. Dejean, S. Manon, Regulation of Bax mitochondrial localization by Bcl-2 and Bcl-x(L): keep your friends close but your enemies closer, Int. J. Biochem. Cell Biol. 45 (2013) 64e67. [11] K.W. Kinnally, B. Antonsson, A tale of two mitochondrial channels, MAC and PTP, in apoptosis, Apoptosis 12 (2007) 857e868. [12] A. Antignani, R.J. Youle, How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr. Opin. Cell Biol. 18 (2006) 685e689. [13] C. Chinopoulos, A.A. Starkov, S. Grigoriev, L.M. Dejean, K.W. Kinnally, X. Liu, I.S. Ambudkar, G. Fiskum, Diacylglycerols activate mitochondrial cationic channel(s) and release sequestered Ca(2þ), J. Bioenergy Biomembr. 37 (2005) 237e247. [14] L. Guo, D. Pietkiewicz, E.V. Pavlov, S.M. Grigoriev, J.J. Kasianowicz, L.M. Dejean, S.J. Korsmeyer, B. Antonsson, K.W. Kinnally, Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC, Am. J. Physiol. Cell Physiol. 286 (2004) C1109eC1117. [15] S. Martinez-Caballero, L.M. Dejean, M.S. Kinnally, K.J. Oh, C.A. Mannella, K.W. Kinnally, Assembly of the mitochondrial apoptosis-induced channel, MAC, J. Biol. Chem. 284 (2009) 12235e12245. [16] E.V. Pavlov, M. Priault, D. Pietkiewicz, E.H. Cheng, B. Antonsson, S. Manon, S.J. Korsmeyer, C.A. Mannella, K.W. Kinnally, A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast, J. Cell Biol. 155 (2001) 725e731. [17] Y.T. Hsu, R.J. Youle, Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations, J. Biol. Chem. 273 (1998) 10777e10783. [18] Y. Takahashi, M. Karbowski, H. Yamaguchi, A. Kazi, J. Wu, S.M. Sebti, R.J. Youle, H.G. Wang, Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis, Mol. Cell Biol. 25 (2005) 9369e9382. [19] B. Antonsson, S. Montessuit, B. Sanchez, J.C. Martinou, Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells, J. Biol. Chem. 276 (2001) 11615e11623. [20] S. Montessuit, G. Mazzei, E. Magnenat, B. Antonsson, Expression and purification of full-length human Bax alpha, Protein Expr. Purif. 15 (1999) 202e206. [21] Y.T. Hsu, R.J. Youle, Nonionic detergents induce dimerization among members of the Bcl-2 family, J. Biol. Chem. 272 (1997) 13829e13834. [22] O. Teijido, L. Dejean, Upregulation of Bcl2 inhibits apoptosis-driven BAX insertion but favors BAX relocalization in mitochondria, FEBS Lett. 584 (2010) 3305e3310. [23] T.T. Renault, O. Teijido, F. Missire, Y.T. Ganesan, G. Velours, H. Arokium, F. Beaumatin, R. Llanos, A. Athane, N. Camougrand, M. Priault, B. Antonsson, L.M. Dejean, S. Manon, Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737, Int. J. Biochem. Cell Biol. 64 (2015) 136e146. [24] L.M. Dejean, S. Martinez-Caballero, L. Guo, C. Hughes, O. Teijido, T. Ducret, F. Ichas, S.J. Korsmeyer, B. Antonsson, E.A. Jonas, K.W. Kinnally, Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel, Mol. Biol. Cell 16 (2005) 2424e2432. [25] L. Dejean, Y. Camara, B. Sibille, G. Solanes, F. Villarroya, Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis, J. Cell Physiol. 201 (2004) 294e304. [26] A.Y. Kolosova, S. De Saeger, S.A. Eremin, C. Van Petegehem, Investigation of several parameters influencing signal generation in flow-through membranebased enzyme immunoassay, Anal. Bioanal. Chem. 387 (2007) 1095e1104.
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A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax Oscar Teijido a, Yogesh Tengarai Ganesan b, Raul Llanos c, Ashley Peton c, Jean-Baptiste Urtecho c, Adauri Soprani d, Aimee Villamayor d, Bruno Antonsson e, phen Manon f, Laurent Dejean c, * Ste a
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Care and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY 10065, USA c Department of Chemistry, California State University, Fresno, CA 93740, USA d Department of Basic Sciences, New York University, College of Dentistry, New York, NY 10010, USA e Merck Serono S.A. Geneva Research Center, Geneva, Switzerland f CNRS, Universit e de Bordeaux, UMR5095, 1 Rue Camille Saint-Sa€ ens, 33077 Bordeaux, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2015 Received in revised form 25 November 2015 Accepted 22 December 2015 Available online 31 December 2015
Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. © 2016 Elsevier Inc. All rights reserved.
Keywords: ELISA Bax Conformation specificity Apoptosis Mitochondria
Introduction Apoptosis is a programmed cell death mechanism that is finely regulated by proteineprotein interaction at the mitochondria between pro- and anti-apoptotic members of the Bcl-2 family proteins [1e5]. Defects in this regulation are associated with several diseases, such as neurodegenerative disorders and cancer [6e10]. Cytochrome c release is the point of no return of apoptosis, which promotes and amplifies the cell death cascade [1,2,5,11]. The permeabilization of the mitochondrial outer membrane involves the translocation of Bax to the mitochondria, where it undergoes a change of conformation (Bax activation) that leads to the exposure of the Bax N-terminal region [1,2,4,5,8,12]. Activated Bax forms
* Corresponding author. E-mail address: [email protected] (L. Dejean). http://dx.doi.org/10.1016/j.ab.2015.12.016 0003-2697/© 2016 Elsevier Inc. All rights reserved.
oligomers that are the principal components of the mitochondrial apoptosis-induced channel (MAC), through which cytochrome c is released to the cytosol [5,8,9,11,13e16]. Activated Bax is extensively used as an early marker of apoptosis [1e5,8,11,12]. Quantification of the accumulation of activated Bax in mitochondria is a valuable tool to clinically diagnose pathologies associated with cell degeneration or tumorigenesis [7,10,22,23]. Antibodies that recognize the Bax N-terminus allow the discrimination of activated/oligomerized from native cytosolic Bax [17,18]. Current methods used to detect activated Bax are based on immunoprecipitation followed by immunodetection by Western blot. These time-consuming techniques provide only qualitative detection or, at best, semiquantification of activated Bax, which in many cases is not reproducible enough to report small but significant differences between control and apoptotic conditions. This article describes a rapid method based on a sandwich ELISA that allows an accurate and reproducible quantification of recombinant
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human activated/oligomerized Bax in vitro. In addition, this ELISA also permits reproducible quantification of activated Bax in mitochondrial or whole cell protein extracts from normal and apoptotic HeLa cells. This sandwich ELISA allows rapid and convenient quantification of recombinant and native activated Bax, a wellrecognized early apoptotic marker with potential prognostic value in degenerative disorders and/or cancer therapeutics. Materials and methods Cell culture and apoptosis induction HeLa cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 50 mg/ml streptomycin, and 50 IU/ml penicillin at 37 C in 5% CO2. Cells were split 1/2 (v/v) in 175 cm2 flasks 24 h before the treatment, and then untreated or treated with 0.5 mM staurosporine for 16 h to induce apoptosis [19]. Recombinant proteins Recombinant monomeric/nonactivated (monomeric) and oligomeric/activated (activated) human Bax of full length, with six histidines at the N-terminus, were expressed in Escherichia coli and purified as previously described [19,20]. Activated Bax was obtained by purifying the oligomers after disrupting bacteria in a buffer containing 1% Triton X-100 (v/v), a detergent that artificially induces Bax activation/oligomerization [21]. Once the oligomers were obtained, Triton X-100 was exchanged for 1% octyl glucoside by dialysis. The purified protein was stored in 25 mM Hepes-NaOH, 0.2 mM DTT, 1% octyl glucoside, 30% glycerol, pH 7.5, at 80 C [19]. Total protein extraction from whole cells and mitochondria Total proteins were extracted from control (untreated) or staurosporine-treated (apoptotic) HeLa cells (4 175 cm2 flasks each). At the end of the treatment, cells were harvested in phosphate-buffered saline (PBS) containing 1 mM EDTA and recovered by centrifugation at 750g for 10 min. Pellets containing cells were washed with 1X PBS and suspended in lysis buffer consisting of a 1x PBS supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals) and 2% CHAPS (w/ v) (Sigma-Aldrich), in order to prevent artificial Bax oligomerization [21]. Cells were incubated on ice for 60 min, sonicated for 4 30 s, incubated on ice for 90 min, sonicated again, and centrifuged at 100,000g for 30 min. Supernatants contained the extracted proteins. Mitochondria were isolated from untreated or apoptotic HeLa cells (10 175 cm2 flasks each), as previously described [19]. Pellets containing mitochondria were lyzed in mitochondrial buffer (230 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HepesNaOH, pH 7.5) supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals) and 2% CHAPS (v/v). Mitochondria were then incubated on ice for 1 h, sonicated for 4 30 s, and finally centrifuged for 30 min at 100,000g. Supernatants contained the mitochondrial extracted proteins. The total protein content in both whole cells and mitochondria extracts was measured using a BCA assay (Pierce). Estimation of total Bax content by Western blot and densitometry Total protein extracts (10 mg) from untreated and apoptotic cells and mitochondria were run in a SDS-PAGE gel, along with different concentrations of oligomeric recombinant Bax. Proteins were then transferred onto a PVDF membrane (BioRad). Membrane was blocked in 1x PBS, 0.2% (v/v) Tween-20, 5% (w/v) milk for 30 min,
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followed by incubation with 0.2 ng/mL polyclonal HRP-linked antiBax antibody (N20-HRP, Santa Cruz Biotechnology, Inc.) for 1 h at room temperature or overnight at 4 C. Bax signal was detecting using HRP-dependent luminescence (ECL, Pierce), which was collected on an X-ray film after different exposure times (GE Healthcare). Nonsaturated Western blot signals were scanned, and the pixel density of the Bax bands was quantified using the software Image J (NIH Open Source). Pixel quantification of the bands corresponding to increasing amounts of recombinant oligomeric Bax were used to build a standard curve, which allowed estimation of the Bax content in the different protein extracts tested [22,23]. Immunoprecipitation of activated/oligomeric BAX Mitochondrial protein lysates (35e55 mg), HeLa total cell protein lysates (60e140 mg), or recombinant human Bax monomers and oligomers (10 ng total Bax) were resuspended in mitochondrial buffer or in 1x PBS supplemented with complete protease inhibitor mixture and 2% CHAPS (w/v) (Sigma). Samples were incubated overnight at 4 C with 1 mg of monoclonal (6A7, Sigma-Aldrich) or polyclonal anti-Bax antibody (N20, Santa Cruz Biotechnology, Inc.), or alternatively with control mouse or rabbit total IgGs (Santa Cruz Biotechnology, Inc.). The different samples were then incubated with 1/1 (v/v) protein G-coupled magnetic beads (Invitrogen) at room temperature for 45 min, and the immunocomplexes were precipitated by placing the assay tubes in a holder magnet for 10e30 s. Proteins contained in the pellets were separated from the magnetic beads by washing them twice with 1x PBS, resuspension in sample buffer, incubation at 60 C for 5 min, and magnetic precipitation of the denatured protein G magnetic beads. Supernatants were directly supplemented with sample buffer (BioRad) and heated at 60 C for 5 min. Bax content in supernatants and pellets was estimated by Western blot and densitometry using Image J as described in Refs. [22,23]. Sandwich ELISA for the quantification of activated Bax Activated Bax ELISA was performed in a 96-well plate (Costar). Each well used for the assay was coated overnight with 100 mL of capture antibody solution containing 0.25 mg/mL of the monoclonal anti-Bax 6A7 antibody (Sigma-Aldrich) in DPBS (Fisher) at 4 C. All the following incubations were performed at room temperature and with continuous plate shaking. Coated wells were washed four times with 100 ml DPBS, 0.05% Tween-20 (Sigma-Aldrich), and incubated for 1 h with 200 mL blocking buffer containing 1% BSA (w/ v) (Sigma-Aldrich) in DPBS, followed by four washes with regular DPBS. Both standards and samples were diluted in DPBS 2% (w/v) CHAPS and analyzed in duplicate or triplicate. Activated Bax standards contained 0, 0.3, 0.6, 1.2, 2.5, 5.0, 10, 20, 40, or 80 ng/mL of recombinant activated Bax. Total protein extracts from isolated mitochondria of control and apoptotic HeLa cells were resuspended in DPBS 2% (w/v) CHAPS at a final concentration of 0.2e0.45 mg/ mL, whereas proteins extracted from whole HeLa cells were resuspended at a final concentration of 3.5e10 mg/mL in the same buffer. Activated Bax standards and samples were incubated for 2 h at room temperature in the wells precoated with the 6A7 antibody. The plate was then washed four times with DPBS. Each sample was incubated for 1 h with 100 mL of detection antibody solution containing 0.1 mg/mL of the polyclonal anti-Bax N20 antibody (Santa Cruz Biotechnology, Inc.) in blocking buffer. After incubation, the plate was washed four times with DPBS, and then wells were incubated for 1 h with 100 ml of a solution containing 0.12 mg/mL goat anti-rabbit biotinylated third antibody (Abcam) in blocking buffer. Each well was washed four times with DPBS and incubated
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30 min with 100 mL of a solution of 1x streptavidin-HRP (R&D Systems). The plate was finally submitted to five rapid washings followed by four longer washings (30 se1 min each) in order to maximally reduce the background signals. Immunodetection was performed by incubating the samples for 10 min in the dark with 100 mL of freshly mixed TMB substrate solution (R&D Systems). The developing reaction was finally stopped by adding 100 mL of 2 N H2SO4: the intensity of the yellow color was proportional to the amount of immunocomplexes contained in each well. Absorbance was read at 450 nm and 540 nm within 30 min after the developing reaction was stopped. Final sample concentration values were calculated by the Delta OD (450e540 nm) using a standard curve made with increasing concentrations of recombinant Bax oligomers (0.5e80 ng/mL).
A
Bax monomers Control Ab Bax Ab
B
Results Activated Bax ELISA allows the specific detection and quantification of recombinant human Bax oligomers
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
+ _
+ _
S
P
_ +
S
Bax oligomers
_ +
+ _
+ _
_ +
_ +
P
S
P
S
P
y = 0.0071x + 0.1359 2 R = 0.9966
Bax oligomers
Bax monomers 0
20
40
60
80
100
[BAX] (ng/mL)
C Back-calculated Bax ratio (%)
The binding specificity of the antibodies for activated Bax was first tested by immunoprecipitation of recombinant human Bax monomers and oligomers. As shown in Fig. 1A, Bax oligomers, but not monomers, were immunoprecipitated by the monoclonal antiactivated Bax antibody 6A7 (Bax ab). Similar results were previously obtained with the polyclonal anti-activated Bax antibody N20 [24]. Total mouse or rabbit IgGs (control ab), however, did not immunprecipitate either Bax monomers or oligomers (Fig. 1A). Specificity of these antibodies for the activated form of Bax was then assessed using a sandwich-based ELISA on increasing concentrations of human Bax oligomers or monomers (Fig. 1B). During these preliminary experiments, monoclonal 6A7 and polyclonal N20 were used as “capture” and “detection” antibodies, respectively. Once again, the increase of the ELISA signals (delta OD) was proportional to the concentrations of Bax oligomers tested, whereas Bax monomers produced a low-intensity signal independent of Bax concentrations (Fig. 1B). The linear range of the ELISA using Bax oligomer standards was between 0.5 and 80 ng/mL, which allowed a linear regression analysis of the data, and thus the calculation of Bax oligomer concentrations in a sample. The coefficient of determination (R2) using Bax oligomer standards was above 0.99 in the experiment reported in Fig. 1B, which is representative of 35 independent ELISAs performed so far. Finally, the limit of detection of Bax oligomers with this ELISA was 5 ng/mL and was defined as the lowest concentration for which the signal is statistically different from the signal generated by the same concentration of Bax monomers (Fig. 1B). Biological samples contain a mix of monomeric and activated/ oligomeric Bax. The ratio between the two conformations of Bax is modified during apoptosis or under pathological conditions. To mimic these conditions, the activated-Bax ELISA was tested with samples containing the same amount of total Bax, but different proportions of Bax monomers vs. oligomers (Fig. 1C). Thus, samples were prepared with mixes of 0/100, 25/75, 50/50, 75/25, and 100/ 0% Bax oligomers/monomers, in which 100% corresponded to 80 ng/mL of total Bax. As expected, the delta ODs were higher as the proportion of oligomeric Bax increased (Fig. 1C). Bax concentration back-calculated from the delta ODs corresponded strictly to the amount of Bax oligomers in each sample tested, demonstrating that the presence of monomeric Bax did not interfere with the ELISA signal (Fig. 1C). Activated Bax ELISA allows the specific quantification of activated/oligomeric Bax in mitochondrial and whole cell proteins extracted from human cells.
Recombinant human Bax
Delta OD (450-540 nm)
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y= 1.0639x R2 = 0.9879
100 75 50 25 0
0/100
25/75
50/50
75/25
100/0
Bax oligomers/monomers ratio (%) Fig. 1. Activated Bax ELISA allows the specific detection and quantification of recombinant human Bax oligomers. (A) Recombinant human Bax oligomers are specifically immunoprecipitated by the 6A7 monoclonal antibody. Equal total amounts of Bax monomers or oligomers (i.e., 10 ng) were submitted to immunoprecipitation using 6A7 anti-Bax monoclonal antibody (Bax Ab) or total mouse IgGs (Control Ab) as described in the Materials and methods section. S: supernatants, P: pellets. (B) A 6A7/N20 antibodies-based sandwich ELISA allows the specific detection and quantification of recombinant human Bax oligomers. Sandwich ELISA was performed on samples containing increasing concentrations of recombinant human Bax monomers and oligomers (0.5e80 ng/mL). During the assay, mouse monoclonal 6A7 and rabbit polyclonal N20 Bax antibodies were used as capture and detection antibodies, respectively. The detection of the immunocomplexes was realized using a biotinylated anti-rabbit tertiary antibody and streptavidin-HRP (see the Materials and methods section for details). Results are representative of 35 independent ELISAs. The detection limit was defined as the lowest concentration at which the signal is statistically different from the signal generated by the same concentration of Bax monomers using a Student's t-test (i.e., p < 0.05). (C) Recombinant human Bax monomers do not interfere with the activated Bax ELISA signals. Sandwich ELISA of sample mixtures of recombinant Bax monomers and oligomers. Samples contained increasing amounts of Bax oligomers (0e100%) and proportional decreasing amounts of Bax monomers (100e0%), to have a final total Bax concentration of 80 ng/mL (Bax oligomers/monomers ratio (%)). The Bax ratio was then recalculated for each sample using the ELISA data, making the assumption that only Bax oligomers contributed to the signal (back-calculated Bax ratio (%)). Results are shown as mean ± s.e.m. (n ¼ 3).
We finally tested the ability to detect and quantify activated Bax in human biological samples comparing the classical immunoprecipitation technique with our new ELISA assay (Fig. 2). Fig. 2A shows the immunoprecipitation of activated Bax from total (top) and mitochondrial (bottom) proteins extracted from HeLa cells treated with the classic apoptotic inducer staurosporine [13]. Immunoprecipitation with the monoclonal anti-Bax 6A7 (Bax
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Fig. 2. Activated Bax ELISA allows the quantification of activated human Bax in biological samples. Apoptosis was induced by incubating HeLa cells in the presence of 0.5 mM staurosporine (STS) for 16 h. Total proteins were extracted from control (untreated) or staurosporine-treated (apoptotic) cells in the presence of 2% CHAPS. Mitochondria were isolated from untreated or apoptotic HeLa cells and mitochondrial proteins extracted in the presence of CHAPS 2% as described in the Materials and Methods section. (A) Endogenous human Bax oligomers are specifically immunoprecipitated by the 6A7 monoclonal antibody in mitochondrial or cell protein extracts. Immunoprecipitation of total protein extracts from untreated and apoptotic whole cells (top) or isolated mitochondria (bottom) using 6A7 anti-Bax antibody (Bax Ab) or total mouse IgGs (Control Ab). S: supernatants, P: pellets. (B) The 6A7/N20 Bax ELISA allows sensitive detection and quantification of Bax activation in protein extracts from whole cells or mitochondrial samples. Cell and mitochondrial protein extracts from HeLa cells were obtained and assayed as described in the Materials and methods section. The amounts of activated oligomeric Bax concentrations (ng Bax/mg total protein) from untreated and apoptotic protein extracts from whole cells (left) or isolated mitochondria (right) were calculated with reference to a standard curve built with increasing concentrations of recombinant human Bax (1e80 ng/mL). Results are shown as mean ± s.e.m. (n ¼ 12e18). Statistical differences were determined by Student's t-test and * corresponds to p < 0.05.
ab) showed a barely visible band in the pellet, corresponding to the activated Bax population, in untreated cells or mitochondria compared with an intense signal in apoptotic samples. Total mouse IgGs (control ab) did not immunoprecipitate any Bax conformation. Similar results were obtained using the polyclonal anti-Bax N20 (Bax antibody) on total and mitochondrial protein extracts from apoptotic HeLa cells, as previously shown in Ref. [13]. These same total and mitochondrial protein extracts were then submitted to the activated-Bax ELISA assay (Fig. 2B). The standard curve was performed with increasing concentrations of recombinant Bax oligomers (1.25e80 ng/mL range, as shown in Fig. 1C). As expected, the activated Bax content was significantly higher in apoptotic whole cells, i.e., 2.7 ± 0.4 vs. 1.0 ± 0.1 ng/mg of total protein (Fig. 2B, left panel), and in apoptotic mitochondria, i.e., 34 ± 6 vs. 13 ± 1 ng/mg of total protein (Fig. 2B, right panel). Interestingly, the high sensitivity of the ELISA allowed a clear and reproducible quantification of activated Bax from untreated HeLa cells, which was, at best, barely detected by the immunoprecipitation assay (compare Fig. 1B with Fig. 1A). The concentration of activated Bax detected by ELISA was also found to be about 10 times higher in mitochondria than in whole cells, in agreement with Bax translocation and activation at the mitochondria during staurosporine-induced apoptosis (Fig. 2B and [25]). However, the relative increase of activated Bax during apoptosis was found to be very similar in both cases (i.e., 2.5-fold increase of activated Bax in
apoptotic whole cells vs. untreated, compared with the 3-fold increase in apoptotic vs. normal in mitochondria), thus allowing clear discrimination between normal and apoptotic situations in both types of samples (Fig. 2B). Discussion Bax activation/oligomerization in mitochondria is a very early event in the apoptotic cascade [1e5,8,11,12]. In this respect, activated Bax content is an early marker for apoptosis, but also a potential biomarker for the clinical diagnosis of diseases associated with defects in apoptosis regulation (e.g., neurodegenerative diseases or cancer). Activated/oligomerized Bax is discriminated from cytosolic Bax by antibodies that recognize the conformational change that leads to the exposure of the Bax N-terminal region. The antibodies used during this study include monoclonal (6A7) and polyclonal (N20) Bax antibodies. Techniques used nowadays to detect activated Bax are based on immunocytochemistry or immunoprecipitation assays using those same antibodies or antibodies directed against similar epitopes [1e5,8,11,12]. However, these time-consuming and multistep methods allow only a few samples to be processed at a time and provide a qualitative, or at best semiquantitative type of detection for activated Bax. Our study describes an ELISA method that allows (i) an absolute quantification of activated Bax, (ii) a higher sensitivity than immunoblots, (iii)
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multiple determinations by performing the experiments on a 96well plate, and (iv) a short processing time. The activated Bax ELISA assay uses monoclonal antibodies (6A7, Sigma-Aldrich), which provide high specificity, and polyclonal antibodies (N20, Santa Cruz Biotechnology, Inc.), which increase the sensitivity of the detection. This method allows the specific detection and quantification of recombinant Bax oligomers, but not monomers, either sampled standalone or in a mix (Fig. 1B and C). The reliability of the ELISA was further tested on human biological samples. In order to specifically determine and quantify activated Bax, total proteins were extracted from HeLa cells or from mitochondria in buffer containing 2% CHAPS, as this detergent does not induce artificial change of the conformation of Bax [21]. The high sensitivity of the ELISA assay allows detection and quantification of activated Bax traces in untreated cells, which are undetectable in the classic Western blot assays. As expected, the amount of activated/oligomeric Bax (ng/mg total protein) was significantly higher in samples from apoptotic cells than from untreated cells (Fig. 2B). Interestingly, the increase of activated Bax in apoptotic extracts (2.5e3 fold) was very similar in whole cells and in isolated mitochondria, confirming that Bax activation and oligomerization occur in the mitochondria during staurosporine-induced apoptosis [25]. Immunoassays such as ELISA are prone to matrix effects when used to quantify membrane proteins [26]. In this study, we observed a decrease of signal linearity vs. Bax oligomer concentrations in protein samples extracted from isolated mitochondria, more specifically when these samples were diluted less than 10 times in PBS 2% CHAPS (not shown). We did not observe this phenomenon in protein samples from whole cells that are generated in the same buffer further used during the ELISA (i.e., PBS 2% CHAPS). Taken together, these results indicate that high concentrations of mitochondrial buffer components such as mannitol, sucrose, or EDTA may generate negative matrix effects on this activated Bax ELISA. We believe that the combination of the results presented in this study validates an ELISA protocol allowing a rapid, specific, and sensitive quantification of activated Bax in human biological samples. Therefore, the application of this protocol to different cell models, including tissue samples, may prove useful in the diagnosis of various pathologies related to defective regulation of apoptosis, such as neurodegenerative diseases or cancer. Acknowledgments We are grateful to Kathleen Kinnally, Pablo Peixoto, and ShinYoung Ryu (NYU CD) for helpful discussions. This study has been supported by NYU Research Challenge Funds and CSU Fresno startup funds to LD. References [1] J.E. Chipuk, D.R. Green, How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol. 18 (2008) 157e164. [2] N.N. Danial, BCL-2 family proteins: critical checkpoints of apoptotic cell death, Clin. Cancer Res. 13 (2007) 7254e7263.
[3] B. Leber, J. Lin, D.W. Andrews, Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes, Apoptosis 12 (2007) 897e911. [4] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99e163. [5] L.M. Dejean, S. Martinez-Caballero, K.W. Kinnally, Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? Cell Death Differ. 13 (2006) 1387e1395. [6] J.P. Meijerink, E.M. Van Lieshout, H.B. Beverloo, E. Van Drunen, E.J. Mensink, M. Macville, R. Pieters, Novel murine B-cell lymphoma/leukemia model to study BCL2-driven oncogenesis, Int. J. Cancer 114 (2005) 917e925. [7] M. Certo, V. Del Gaizo Moore, M. Nishino, G. Wei, S. Korsmeyer, S.A. Armstrong, A. Letai, Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members, Cancer Cell 9 (2006) 351e365. [8] L.M. Dejean, S.Y. Ryu, S. Martinez-Caballero, O. Teijido, P.M. Peixoto, K.W. Kinnally, MAC and Bcl-2 family proteins conspire in a deadly plot, Biochim. Biophys. Acta 1797 (2010) 1231e1238. [9] K.W. Kinnally, P.M. Peixoto, S.Y. Ryu, L.M. Dejean, Is mPTP the gatekeeper for necrosis, apoptosis, or both? Biochim. Biophys. Acta 1813 (2011) 616e622. [10] T.T. Renault, O. Teijido, B. Antonsson, L.M. Dejean, S. Manon, Regulation of Bax mitochondrial localization by Bcl-2 and Bcl-x(L): keep your friends close but your enemies closer, Int. J. Biochem. Cell Biol. 45 (2013) 64e67. [11] K.W. Kinnally, B. Antonsson, A tale of two mitochondrial channels, MAC and PTP, in apoptosis, Apoptosis 12 (2007) 857e868. [12] A. Antignani, R.J. Youle, How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr. Opin. Cell Biol. 18 (2006) 685e689. [13] C. Chinopoulos, A.A. Starkov, S. Grigoriev, L.M. Dejean, K.W. Kinnally, X. Liu, I.S. Ambudkar, G. Fiskum, Diacylglycerols activate mitochondrial cationic channel(s) and release sequestered Ca(2þ), J. Bioenergy Biomembr. 37 (2005) 237e247. [14] L. Guo, D. Pietkiewicz, E.V. Pavlov, S.M. Grigoriev, J.J. Kasianowicz, L.M. Dejean, S.J. Korsmeyer, B. Antonsson, K.W. Kinnally, Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC, Am. J. Physiol. Cell Physiol. 286 (2004) C1109eC1117. [15] S. Martinez-Caballero, L.M. Dejean, M.S. Kinnally, K.J. Oh, C.A. Mannella, K.W. Kinnally, Assembly of the mitochondrial apoptosis-induced channel, MAC, J. Biol. Chem. 284 (2009) 12235e12245. [16] E.V. Pavlov, M. Priault, D. Pietkiewicz, E.H. Cheng, B. Antonsson, S. Manon, S.J. Korsmeyer, C.A. Mannella, K.W. Kinnally, A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast, J. Cell Biol. 155 (2001) 725e731. [17] Y.T. Hsu, R.J. Youle, Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations, J. Biol. Chem. 273 (1998) 10777e10783. [18] Y. Takahashi, M. Karbowski, H. Yamaguchi, A. Kazi, J. Wu, S.M. Sebti, R.J. Youle, H.G. Wang, Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis, Mol. Cell Biol. 25 (2005) 9369e9382. [19] B. Antonsson, S. Montessuit, B. Sanchez, J.C. Martinou, Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells, J. Biol. Chem. 276 (2001) 11615e11623. [20] S. Montessuit, G. Mazzei, E. Magnenat, B. Antonsson, Expression and purification of full-length human Bax alpha, Protein Expr. Purif. 15 (1999) 202e206. [21] Y.T. Hsu, R.J. Youle, Nonionic detergents induce dimerization among members of the Bcl-2 family, J. Biol. Chem. 272 (1997) 13829e13834. [22] O. Teijido, L. Dejean, Upregulation of Bcl2 inhibits apoptosis-driven BAX insertion but favors BAX relocalization in mitochondria, FEBS Lett. 584 (2010) 3305e3310. [23] T.T. Renault, O. Teijido, F. Missire, Y.T. Ganesan, G. Velours, H. Arokium, F. Beaumatin, R. Llanos, A. Athane, N. Camougrand, M. Priault, B. Antonsson, L.M. Dejean, S. Manon, Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737, Int. J. Biochem. Cell Biol. 64 (2015) 136e146. [24] L.M. Dejean, S. Martinez-Caballero, L. Guo, C. Hughes, O. Teijido, T. Ducret, F. Ichas, S.J. Korsmeyer, B. Antonsson, E.A. Jonas, K.W. Kinnally, Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel, Mol. Biol. Cell 16 (2005) 2424e2432. [25] L. Dejean, Y. Camara, B. Sibille, G. Solanes, F. Villarroya, Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis, J. Cell Physiol. 201 (2004) 294e304. [26] A.Y. Kolosova, S. De Saeger, S.A. Eremin, C. Van Petegehem, Investigation of several parameters influencing signal generation in flow-through membranebased enzyme immunoassay, Anal. Bioanal. Chem. 387 (2007) 1095e1104.