Figure 1.
A Bcl-2-BH4 double-glycine variant (I14G/V15G) has a decreased
α-helical content.
(A) Primary structure of the Bcl-2-BH4 peptide. The key residues involved in the regulation of IP 3Rs are depicted in black/bold. The residues considered for the glycine substitution (I14 and V15) are depicted in red. The α-helical structure underneath represents the best predictive model obtained from I-TASSER web server and drawn using Pymol. The labels for the key peptide residues follow the same color code as in the primary structure. (B) Predicted-secondary structure assignments for the isolated BH4 domain of Bcl-2 (upper panel) and for its IV/GG counterpart (lower panel). Each panel shows the amino acid sequence, the secondary-structure predictions (H = α-helix; C = random coil) and the level of confidence of the predictions (confidence scores from 0 to 9). Residues 14 and 15 of the BH4 domain are highlighted by a semi-transparent red square. (C) CD spectra of synthetic Bcl-2-BH4 (black line) and Bcl-2-BH4 IV/GG peptides (red line). The ellipticity is calculated per mole of amino-acid residue. Bcl-2-BH4-IV/GG peptide lost the native α-helical conformation to adopt a more β-sheet-like structure (210 nm-ellipticity minimum). For the percentages of the other secondary structure features see Table S1.
Figure 2.
IV/GG substitution in BH4-Bcl-2 abolishes the direct interaction with IP 3R1-Domain 3 and the resulting inhibition of IP3R-channel activity.
(A–B) Pull-down assays of BH4 peptides with either purified GST-Domain 3 or GST alone. (A) Specific interactions between Bcl-2-BH4 or Bcl-2-BH4 IV/GG-peptides (30 µg) and the GST proteins (30 µg) detected by total protein staining (GelCode® Blue Stain Reagent) of SDS-PAGE runs. The arrows indicate the bands for GST-domain-3 (upper arrow), GST (middle arrow) and BH4-domain peptides (lower arrow). (B) Bands corresponding to BH4-domain peptides were quantified using ImageJ software. Values were normalized relatively to the binding to GST and corrected for the amount of GST-fusion proteins. The results of at least 4 independent experiments are plotted as means ± SEM. * indicates a statistically significant difference from the GST control. (C) Representative single-channel recordings evoked by low [IP3] (1 µM) at 200 nM Ca2+ and 5 mM ATP, in the presence or absence of the BH4 peptides. (D) Histogram depicting the open probability (Po) ± SD for the IP 3R1 under the previously described conditions. Within every bar is indicated the total number of recordings per each condition. The Po for IP 3R1 was ~5 fold lower when exposed to the Bcl-2-BH4 peptide whereas it was unaffected by the Bcl-2-BH4-IV/GG peptide.
Figure 3.
IV/GG substitution abrogates the inhibitory effect of Bcl-2-BH4 on IICR.
(A) Representative unidirectional 45Ca2+ fluxes in permeabilized MEF cells plotted as fractional loss (% / 2 min) as a function of time. Ca2+ release was activated 10 min after starting the experiment by applying 3 µM IP3 (arrow) in the absence or presence of 100 µM of the different BH4-domain peptides (a gray bar indicates the peptide incubation period). (B) Concentration–response curves ([peptide] = 0,1; 3; 15; 30; 60; 100 µM) are shown for Bcl-2-BH4, Bcl-2-BH4 IV/GG and Bcl-2-BH4 SCR, obtained from 3 independent experiments. IICR was quantified as the difference of the fractional loss after 2 min of incubation with IP3 and the fractional loss before the IP3 addition. The 100% value corresponds to IICR in the presence of the vehicle and all the raw values were normalized to this control. Data points represent means ± SEM. (C) [Ca2+]cyt increases in C6 glioma cells after photoliberation of caged IP3 at 9980 ms of recording (arrow). Traces of individual cells are displayed that were loaded with different BH4-domain peptides (20 μM) together with caged IP3 (50 μM). (D) Quantitative analysis of the area under the curve obtained from 5 or 6 independent experiments (as marked on each bar). Data were normalized to the vehicle condition, which was set as 100%, and are plotted as means ± SEM. These data indicate that Bcl-2-BH4 peptide significantly inhibited IICR (**), whereas Bcl-2-BH4 IV/GG peptide did not. # specifies the statistically significant difference between Bcl-2-BH4 and Bcl-2-BH4-IV/GG results.
Figure 4.
IV/GG substitution abolishes BH4-Bcl-2’s protective function against STS-induced apoptosis.
Simultaneous analysis of caspase activation (FITC-VAD-FMK, green) and nuclear fragmentation (DAPI staining, blue fragments) of STS-treated C6 glioma cells. (A) Representative images of cells electroporated with or without BH4 peptides (20 µM) and successively treated with STS (2 µM for 6 h). The left images are taken outside the electroporation area and are used as negative controls (A, upper right). Electroporation in the absence of peptides (vehicle) (A, middle right). Electroporation of Bcl-2-BH4 peptide (A, lower right). Electroporation of Bcl-2-BH4-IV/GG peptides. Red color is due to the spillover into the FITC channel of the intense DTR signal (the electroporation loading control). (B) Quantitative image based AI (number of apoptotic cells divided by the total cell number). The AI was normalized to the AI outside the electroporated area. All results were obtained from 5 independent experiments and are plotted as means ± SEM. Only Bcl-2-BH4 loading significantly reduced the AI when compared with the control vehicle (**). ### indicates that the results obtained with Bcl-2-BH4 IV/GG were significantly different from Bcl-2-BH4.
Figure 5.
Other glycines substitutions that destabilize BH4-Bcl-2’s
α-helical structure abolish its IP3R-inhibitory properties.
(A, left) Panel showing the values of ΔΔG, in kcal/mol, resulting from the in silico analysis (Eris automated estimator) of the II/GG and VIL/GGG substitutions. Positive ΔΔG values indicate destabilizing mutations (A, right). Predicted-secondary structure assignments for the isolated BH4 domain mutated as described above. Each panel shows (from top to bottom) the primary structure, the secondary-structure predictions (C = random coil, S = strand [black/bold]) and the level of confidence of the predictions (confidence scores from 0 to 9). The key residues involved in the regulation of IP 3Rs are depicted in black/bold while the position of the exchanged residues is indicated by the red G residues in the primary structure. (B) CD spectra of synthetic Bcl-2-BH4 (black line) in comparison with the ones for its G-substituted counterparts [II/GG (purple trace), VIL/GGG (green trace)]. The ellipticity is calculated per mole of amino-acid residue. Both mutant peptides showed a relative decrease in α-helical conformation as assessed by spectra analysis with the CONTIN/LL deconvolution method (see provided change in α-helical percentage for each condition. For the percentages of the other secondary structure features see Table S1 1). (C) Representative unidirectional 45Ca2+ fluxes in permeabilized MEF cells plotted as fractional loss (% / 2 min) as a function of time. Ca2+ release was activated 10 min after starting the experiment by applying 3 µM IP3 (arrow) in the absence or presence of 50 µM of the different BH4-domain peptides (the traces are color coded as in B). The gray bar indicates the peptide-incubation period. Data points represent means ± SD (D) IICR was quantified as the difference of the fractional loss after 2 min of incubation with IP3 and the fractional loss before the IP3 addition in the presence of vehicle (DMSO), Bcl-2-BH4 and the respective mutant peptides. The 100% value corresponds to IICR in the presence of the vehicle. All values were normalized to this control. Data points represent means ± SEM. * indicates a statistically significant difference from vehicle control.