doi: 10.1016/j.mitoco.2023.08.001.
Epub 2023 Aug 9.
Structure-destabilizing mutations unleash an intrinsic perforation activity of antiapoptotic Bcl-2 in the mitochondrial membrane enabling apoptotic cell death
Affiliations
- PMID: 39239250
- PMCID: PMC11375749
- DOI: 10.1016/j.mitoco.2023.08.001
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Structure-destabilizing mutations unleash an intrinsic perforation activity of antiapoptotic Bcl-2 in the mitochondrial membrane enabling apoptotic cell death
Mitochondrial Commun.
2023.
Abstract
Bcl-2 and Bax share a similar structural fold in solution, yet function oppositely in the mitochondrial outer membrane (MOM) during apoptosis. The proapoptotic Bax forms pores in the MOM to trigger cell death, whereas Bcl-2 inhibits the Bax pore formation to prevent cell death. Intriguingly both proteins can switch to a similar conformation after activation by BH3-only proteins, with multiple regions embedded in the MOM. Here we tested a hypothesis that destabilization of the Bcl-2 structure might convert Bcl-2 to a Bax-like perforator. We discovered that mutations of glutamate 152 which eliminate hydrogen bonds in the protein core and thereby reduce the Bcl-2 structural stability. These Bcl-2 mutants induced apoptosis by releasing cytochrome c from the mitochondria in the cells that lack Bax and Bak, the other proapoptotic perforator. Using liposomal membranes made with typical mitochondrial lipids and reconstituted with purified proteins we revealed this perforation activity was intrinsic to Bcl-2 and could be unleashed by a BH3-only protein, similar to the perforation activity of Bax. Our study thus demonstrated a structural conversion of antiapoptotic Bcl-2 to a proapoptotic perforator through a simple molecular manipulation or interaction that is worthy to explore further for eradicating cancer cells that are resistant to a current Bcl-2-targeting drug.
Keywords: Apoptosis; Bcl-2 protein pore; Cytochrome c release; Mitochondrial membrane.
Conflict of interest statement
Declaration of competing interest The authors declare that there is no conflict of interest.
Figures
(A) Amino acid sequence alignment of Bcl-2 from human (two isoforms), mouse (two isoforms), rat, and Drosophila (denoted by Bcl-2_DROME). The residues that are identical and highly conserved are highlighted in red and yellow, respectively. The α-helical regions (α1, α2, etc.) are indicated above the alignment with cylinders. The C-terminal transmembrane regions are enclosed in a dashed box. The three amino acid residues that form a potential hydrogen bonding network in the Bcl-2 structure (illustrated in Fig. 5A) are indicated below the alignment with triangles. (B) Apoptosis of HeLa cells expressing Bcl-2. Alanine mutagenesis was carried out with human Bcl-2 gene inserted in pSG5 vector. The resulting plasmids were used to transiently transfected HeLa cells for 48 h to express HA-tagged Bcl-2 wide-type (WT) or the mutants that are indicated by the sequence number of the residue that was changed to an alanine. The cells were then stained with anti-HA antibody and FITC-conjugated secondary antibody, Hoechst 33342, and MitoTracker, and subjected to confocal microscopy analysis. Representative confocal images from three independent experiments are shown with green, blue and red colors assigned to the three fluorescent dyes, respectively. The numbers of apoptotic cells were determined by propidium iodide and Annexin V-FITC staining using flow cytometry, and the average percentages of the apoptotic cells from three independent experiments are shown in the lower right corner of the images. Scale bar corresponds to 20 μm.
(A) The plasmid that expresses GFP-tagged wide-type (WT) or the indicated mutant Bcl-2, or Bax, or the control vector that expresses GFP only was used to transfect Bax−/−/Bak−/− MEF cells. After 48 h, the morphology of the cells was examined by light field microscopy. Representative images from one of three independent experiments are shown. Scale bar corresponds to 200 μm. (B) The Bax−/−/Bak−/− MEF cells were transfected with the plasmid that expresses GFP-tagged wide-type or the indicated mutant Bcl-2, or Bax, or the control vector that expresses GFP only. After 48 h, the cells were stained with Hoechst 33342, and fluorescence images were collected by confocal microscopy. Representative images from one of three independent experiments are shown. Scale bar represents 20 μm. (C) Three-hundred cells from each of the indicated groups that displayed GFP fluorescence in an independent experiment described in (B) were examined for nuclear condensation. The percentage of the cells that were apoptotic was calculated and shown as mean ± SD for that group from three independent experiments. (D) The Bax−/−/Bak−/− MEF cells were transfected with the plasmid that expresses HA-tagged wide-type or the indicated mutant Bcl-2, or the control vector. After 48 h, full-length PARP and caspase-cleaved PARP were detected by immunoblotting with an anti-PARP antibody. The total intensity of the PARP band(s) indicates the protein loading from each sample. The levels of different HA-Bcl-2 proteins in these samples were determined by immunoblotting with the anti-HA antibody. The data shown were from one of three independent experiments. Similar data were obtained from the other experiments. (E) The Bax−/−/Bak−/− MEF cells were transfected the plasmid that expresses GFP-tagged wide-type or the indicated mutant Bcl-2, or Bax, or the control vector that expresses GFP only. After 48 h, the cells were sorted by FACS and the histogram is shown on the left side. The cells through P3 gate were collected and seeded on six-well plates. Colonies of cells formed after 5-day culture were visualized by crystal violet staining. Representative images from three independent experiments are shown on the right side. (F) The numbers of cell colonies from the experiments described in (E) were scored. The means ± SD from three independent experiments are shown. Statistical significance of a difference between the means of the data sets that are indicated by a line above them was determined using one-way ANOVA followed by Tukey’s multiple-comparisons test, and *, ** or *** above the line indicates the resulting P value < 0.05, 0.01 or 0.001, respectively. NS above the line indicates a non-significant difference between the means of the indicated data sets with a P value > 0.05.
(A) The Bax−/−/Bak−/− MEF cells were transfected with the plasmid that expresses GFP-tagged wide-type or the indicated mutant Bcl-2, or Bax, or the control vector that expresses GFP only. After 48 h, the cells were stained with an anti-cytochrome c antibody, PE-conjugated secondary antibody and Hoechst 33342. Fluorescence images were collected by confocal microscopy. Representative images from one of three independent experiments are shown. Scale bar represents 20 μm. (B) Two-hundred cells from each of the indicated groups that displayed GFP fluorescence in an independent experiment described in (A) were examined for the cytochrome c that were released from the mitochondria and diffused throughout the cells. The percentage of the cells that released cytochrome c was calculated and shown as mean ± SD for that group from three independent experiments. Statistical significance of a difference between the means of the data sets that are indicated by a line above them was determined using one-way ANOVA followed by Tukey’s multiple-comparisons test, and ** above the line indicates the resulting P value < 0.01. NS above the line indicates a non-significant difference between the means of the indicated data sets with a P value > 0.05.
(A) The Bax−/−/Bak−/− MEF cells were transfected with the plasmid that expresses HA-tagged wide-type or the indicated mutant Bcl-2, or the control vector that expresses HA only. After 48 h, the cells were immunostained with a Bcl-2 BH3 domain specific antibody and a FITC-conjugated secondary antibody, and analyzed by flow cytometry. The cell counts versus the FITC fluorescence intensity histograms obtained from the cells that were side-by-side transfected with the vector or the indicated Bcl-2 plasmid in one of the three independent experiments are shown in black or red, respectively. The mean percentage ± SD of each Bcl-2 plasmid-transfected cells that displayed Bcl-2 immunofluorescence over the background fluorescence from three independent experiments is indicated in the respective histogram. (B) Thermal stability of purified recombinant wild-type and mutant Bcl-2 proteins. The curves are the first derivatives of the fluorescence variations of SYPRO orange dye against temperature at different temperatures, and color-coded for the indicated Bcl-2 proteins and the buffer control. The temperature corresponding to the peak of each curve is the melting temperature of each Bcl-2 protein, and listed beside its color code. (C) One-hundred nM of purified recombinant wide-type or mutant Bcl-2 proteins were incubated with the mitochondria isolated from the Bax−/−/Bak−/− MEF cells at 30 °C for 30 min. For the control, no Bcl-2 protein was incubated with the mitochondria. After centrifugation, the mitochondrial fraction from each incubation was split to two with one treated with the crosslinker DSS and the other with the vehicle DMSO for 30 min. All the samples were immunoprecipitated with an anti-Bcl-2 monoclonal antibody and analyzed by immunoblotting with anti-Bcl-2 polyclonal antibodies. The asterisks on right side of the blot indicate the three crosslinking products with the approximate molecular weights in kDa indicated. The immunoblot for VDAC in each sample serves as the loading control. (D) The indicated purified recombinant Bcl-2 proteins were incubated with the isolated mitochondria at 30 °C for 30 min. The mitochondria were then pelleted and treated with 0.1 M Na2CO3 (pH 11.5) for 20 min. The samples were centrifuged to separate the alkaline extracted (S) and non-extracted (P) fractions. Bcl-2 proteins in these fractions were detected by the immunoblotting and the results are shown in the top panel. The PVDF membrane used in the Bcl-2 immunoblotting was then stripped and re-immunoblotting for VDAC and Cox IV, and the results are shown in the bottom panels, serving as the mitochondrial integral membrane protein and the loading controls.
(A) Potential hydrogen bonds between the sidechain atoms of K22, S105, and E152 in a Bcl-2 structure (PDB entry: 1G5M). In the wild-type structure created in PyMOL, the three residues in stick form are projected from the α-helices 1, 2 and 5 (α1, α2, and α5) in ribbon form. The hydrogen bonding atoms are link by dashed lines with the distance between them indicated in Å. The mutant structures were generated in PyMOL using the Mutagenesis wizard for protein. While the alanine mutation of each of the three residues eliminates the hydrogen donor or acceptor atom, the serine mutation of E152 keeps a potential hydrogen acceptor OG; however, the distance between the S152 OG and the potential hydrogen donor of the K22 NZ or S105 OG is too long to form a hydrogen bond. (B) The Bax−/−/Bak−/− MEF cells were transfected with the plasmid that expresses either GFP-Bcl-2 wide-type or the indicated mutant. After 48 h, the cells were stained with an anti-cytochrome c primary antibody, PE conjugated secondary antibody and Hoechst 33342, and their fluorescent images were collected by confocal microscopy. Representative images from one of three independent experiments are shown. Scale bar represents 20 μm. (C) Three-hundred cells from each of the indicated groups that displayed GFP fluorescence in an independent experiment described in (B) were examined for nuclear condensation. The percentage of the cells that were apoptotic was calculated and shown as mean ± SD for that group from three independent experiments. (D) The Bax−/−/Bak−/− MEF cells were transiently transfected with HA-Bcl-2 wide-type, K22A, S105A, E152 mutants, and control vector. After 48h, full-length PARP and caspase-cleaved PARP were detected by immunoblotting with the specific antibody. The levels of PARP served as the protein loading control for the experiment. The expression levels of Bcl-2 and its mutants were detected by immunoblotting with a Bcl-2 specific antibody. Results shown are representative of three independent experiments with very similar results. Statistical significance of a difference between the means of the data sets that are indicated by a line above them was determined using one-way ANOVA followed by Tukey’s multiple-comparisons test, and ** above the line indicates the resulting P value < 0.01.
(A) Size-exclusion chromatograms of recombinant Bcl-2 proteins. The indicated recombinant Bcl-2 proteins with the C-terminal hydrophobic helix 9 replaced by a His6 tag were purified by their affinity to Ni-NTA agarose resins, and then further purified by size-exclusion chromatography. The eluted proteins were detected by their absorbance at 280 nm (A280). Since the peak fractions eluted between 16 and 19 ml were expected to contain monomeric Bcl-2 proteins based on the elution volumes of the protein markers whose molecular weights in kDa are indicated on the top of the chromatograms, the proteins in these fractions were analyzed by SDS-PAGE and Coomassie Blue staining. The fractions with 95% or more Bcl-2 proteins were pooled. (B) 135 pmole of the indicated recombinant Bcl-2 proteins pooled from the peak fractions described in (A) were analyzed by SDS-PAGE and Coomassie Blue staining alongside with the purified Bax protein of 135 or 270 pmole (indicated by 2x Bax), and the PageRuler prestained protein ladder (ThermoFisher Scientific) with the molecular weights (MW) in kDa indicated. (C–D) The release of the fluorescent dye-conjugated dextran molecules from the liposomes containing 12.5 μM of mitochondrial lipids and 0.125 μM of Ni2+-chelating lipid analog by 50 nM of His6-tagged Bcl-2 wild-type (WT) or E152S mutant protein, or 50 nM of Bax protein in the absence or presence of 15 nM of tBid protein was monitored during a 122-min time course by dye-specific antibodies that quenches the fluorescence of the released molecules. The fraction of the dye release was determined by the fluorescence reduction caused by the protein(s) and normalized to that caused by 0.1% of Triton X-100 that solubilized all the liposomes and released all the fluorescent molecules. The data shown are means and ranges from three replicates with the “Bcl-2 WT + Bid” sample and two replicates with other samples. The data from the “Bcl-2 WT or E152S + tBid” and “Bax + tBid” samples shown in (C) are also shown in (D) to compared with the data from the “Bcl-2 WT or E152S + Bax + tBid” samples. Multiple unpaired t tests were performed with the following data sets to determine whether the difference between the means is significant as indicated by the P values ≤ 0.05. The fractions of the dye release are significantly different between Bcl-2 WT, Bcl-2 E152S or Bax + and − tBid samples after 22, 12 or 22 min, respectively, demonstrating that the pore-forming activity of each is induced by tBid. The fractions of the dye release are not significantly different between Bcl-2 WT and Bcl-2 E152S samples through the entire time course, no matter whether tBid is present, demonstrating that the mutation does not induce or enhance Bcl-2 pore formation in the liposomal membrane.
(A) In cells that have Bax, Bcl-2 functions as an antiapoptotic protein. In these cells, a BH3-only protein such as tBid interacts with Bax changing it from an inactive soluble conformation to an active conformation that inserts into the mitochondrial outer membrane (step i) The active Bax proteins associate with one another, and form an oligomeric pore (step ii) that releases cytochrome c to the cytoplasm (step ii), initiating apoptotic cell death. The antiapoptotic Bcl-2 binds the active Bax (step iv) to prevent it from forming the oligomeric pore with the other Bax (step v), thereby inhibiting the cytochrome c release and apoptosis. (B) In cells lacking Bax (and Bak), a mutation such as E152S can destabilize the antiapoptotic Bcl-2 structure converting it to a proapoptotic molecule (step vi). The proapoptotic Bcl-2 acts like the active Bax forming an oligomeric pore in the mitochondrial membrane (step vii) that releases cytochrome c (step viii) and initiates apoptosis. (C) For a recombinant Bcl-2 protein that lacks the C-terminal transmembrane sequence but is tethered to the liposomal membrane of a typical mitochondrial lipid composition, its pore-forming potential can be unleashed by tBid. Thus, tBid activates the Bcl-2 proteins by inducing conformational changes (step ix) so that they can assemble an oligomeric pore (step x) that releases fluorescent molecules of the size of cytochrome c from the liposome (step xi). BioRender was used to create the schematics of mitochondria.
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