Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–C) MEFs subjected to SPARKL analysis measuring real-time labeling with fluorescently-tagged Annexin V (100 μg/ml) via imaging with an IncuCyte ZOOM. Left panels: kinetics of cell labeling in response to increasing concentrations of 2t–hexadecenal (2t–16) with DMSO vehicle or co-treated with ABT-737 (ABT); black line reports untreated control. Right panels: endpoint data of replicates at 24 hours. Data shown are the mean of technical triplicates and error bars report SEM. (A) WT MEFs (matched to Bax −/− Bak −/− double knockout MEFs) were treated with 2t–16 (10, 20, 40 μM) and DMSO or ABT–737 (1 μM), imaged every 2 hours, and quantified for number of Annexin V-positive objects. (B) Same as in A with Bim −/− Bid −/− double knockout MEFs. (C) Same as in A with Bax −/− Bak −/− double knockout MEFs. (D–I) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX protein (120 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX (120 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figures S1C . (G–H) LUV permeabilization studies as in D–E with BAX (160 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data as in F with BAX (160 nM) and 16CHO (6.5−50 μM). Data summarized from Figures S1D . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Imaging, Control, Double Knockout, Recombinant, Fluorescence, Liposomes, Spectroscopy, Incubation

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–D) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (A) BAX protein (100 nM) was activated by BIM–BH3 peptide (0.13–2 μM) and added to LUVs. (B) LUVs treated with 2t–16 (16.5–50 μM) in the absence of BAX to confirm no membrane destabilization by 2t–16. (C) Data summarized by . LUVs permeabilized by BAX (120 nM) treated with 2t–16 (6.5– 50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (D) Data summarized by . LUVs permeabilized by BAX (160 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Recombinant, Fluorescence, Liposomes, Generated, Membrane

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–B) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Data shown are the mean of replicate data and error bars report SD. (A) Left: Timetrace thermal shift curves of BAX WT titrated with 2t–16 (0.02–40 μM) and subjected to MST. Right: Thermophoresis and temperature jump value for BAX WT treated with a range of 2t–16 concentrations fitted to determine a K D value. (B) BAX WT was treated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) and MST timetrace thermal shift curves were fitted using a one-step exponential function and compared using the decay (K) constants normalized by the untreated BAX curve. Original data in Figure S2C . (C) LC-MS of recombinant BAX WT alone or incubated with 2t–16. Samples were then alkylated with iodoacetamide to identify unmodified cysteine residues and trypsin digested for analysis. Four cysteine-containing peptide fragments were detected. Values denote peptide abundance, calculated as AUC for each peak. (D–I) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX 2S protein (100 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX 2S and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2B . (G–H) LUV permeabilization studies as in D–E with BAX 2S (100 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2C . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Fluorescence, Liposomes, Spectroscopy

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Timetrace thermal shift curves of BAX WT titrated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) report the mean of replicate data. (B−C) The melting temperature of BAX WT and BAX 2S ± 2t–16 (6.5−50 μM) was measured by thermal shift assay using SYPRO orange and compared. Statistical significance was determined by two-way ANOVA; ns, not significant ( P > 0.05). (D–E) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (D) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (E) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Thermal Shift Assay, Fluorescence, Liposomes, Generated

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Illustration of BAX and BAK–BH3 interactions within FLAMBE. The reporter is a fluorescently-labeled BAK–BH3 peptide that exhibits changes in Polarization measurements as it is bound by BAX. Over time, the BAX population binds BAK–BH3 peptides resulting in increased Polarization, which eventually plateaus if the entire population forms heterodimers (dotted line). In conditions that activate BAX, activation-induced intramolecular rearrangements within BAX result in the dissociation of the BAK–BH3 peptide and a concomitant decrease in Polarization over time as the population of unbound BAK–BH3 peptide increases (solid line). (B) Illustration of FLAMBE data parameterization and analysis. Left: Kinetic Polarization data is collected for a treatment causing BAX activation and exhibiting accelerated kinetics of BAK TAMRA dissociation (blue lines, depicted as a titration exhibiting dose-dependent BAX activation). Kinetic data is parameterized by extracting endpoint Polarization (EP) and time-to-maximum peak (Tmax) for each condition. Right: Parameterized data are normalized to BAK TAMRA and BAX controls (grey and black, respectively) and titrations or separate conditions can be visualized as a two-dimensional plot. Generally, conditions exhibiting minimal or robust activation of the BAX population cluster in the upper-right or lower-left regions, respectively. Conditions plotted above BAX (i.e., EP > 1) form stable non-activating complexes with the BAX:BAK TAMRA heterodimer (red region). (C) BAX WT (60 nM) was treated with BIM–BH3 peptide (0.25–2 μM) and subjected to FLAMBE to visualize dose-dependent activation-induced dissociation of BAK TAMRA . BIM–BH3 at a low concentration (0.25 μM, dark blue data) demonstrated a stable, non-activating interaction with the BAX:BAK TAMRA complex and exhibited increased Polarization. (D) BAX WT (60 nM) was treated with 16CHO (2–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. (E) Same as in D with BAX 2S (60 nM). (F) Left: BAX 2S (60 nM) was treated with three non-activating concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM). Parameterization of this data is included in . Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (G) Left: BAX WT (60 nM) was combined with a non-activating concentrations of BIM–BH3 peptide (0.15 μM) and 2t–16 (4.5 μM), followed by BAK TAMRA (50 nM), and subjected to FLAMBE. Middle: Parameterized FLAMBE data including three concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM) in the absence or presence of BIM–BH3 (circle and square datapoints, respectively). Annotations report the magnitude of shift between data with and without BIM–BH3. Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (H) Fluorescence polarization competition assay with recombinant BCL-xL ΔC protein treated with 2t–16 (2–50 μM) and combined with BAK TAMRA .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Activation Assay, Titration, Concentration Assay, Fluorescence, Competitive Binding Assay, Recombinant

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated with the indicated 2t–alkenals (0.16–5 μM), and subjected to MST. Timetrace data are shown as the mean of replicates. Thermophoresis metrics for each 2t–alkenal are summarized in . (B) BAX 2S (60 nM) was treated with the indicated 2t–alkenal (3–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. Data are shown as the mean of replicates. Parameterized data reporting EP and Tmax for each experiment are provided in Figures 5D –E . (C) LUVs permeabilized by BAX 2S (100 nM) treated with the indicated 2t–alkenal (6.5–50 μM). Data are shown as the mean of replicates. Normalized endpoint permeabilization data summarized in .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation