Multimodal interaction with BCL-2 family proteins underlies the proapoptotic activity of PUMA BH3

Abstract

PUMA is a proapoptotic BCL-2 family member that drives the apoptotic response to a diversity of cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent proapoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally stabilized PUMA BH3 helices that, in addition to broadly targeting antiapoptotic proteins, directly bind to proapoptotic BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage an α1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1, and BAX. Thus, we find that PUMA BH3 is a dual antiapoptotic inhibitor and proapoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.

Figure 1. Sequence Composition and Anti-apoptotic Binding Activity of Hydrocarbon-stapled PUMA BH3 Peptides

(A, B) A series of FITC-PUMA SAHB_A peptides of differential BH3 domain length (A) and FITC-PUMA BH3 (131–153) constructs with varying staple positions (as reflected by the “staple walk” along the pictured helical wheel) (B) were synthesized and screened for BCL-X_LΔC binding activity by fluorescence polarization assay. See also Figure S1B for separated views of the PUMA SAHB A1-L1 plots. (C) Circular dichroism of PUMA SAHB_A1 and its A139L/L141A mutant demonstrated the marked α-helical structure of both stapled peptides in aqueous solution. *, exceeds the calculated ideal α-helicity of an undecapeptide standard. (D) FITC-PUMA SAHB_A1 (solid lines) exhibits high affinity binding to the diversity of anti-apoptotic BCL-2 family proteins, whereas A139L/L141A point mutagenesis (dashed lines) markedly impairs binding activity, highlighting the BH3 sequence-dependence of PUMA SAHB_A1 engagement. Data are mean ± S.D. for experiments performed in triplicate. X, stapling amino acid; B, norleucine. See also Figure S1.

Figure 2. NMR Analysis of ¹⁵N-BAX upon PUMA SAHB_A1 Titration

(A, B) At a 1:1 ratio of ¹⁵N-PUMA SAHB_A1/BAX, a series of chemical shift changes localize to surface residues of α1 and α6, corresponding to the previously defined trigger site (purple) for BIM BH3-induced direct BAX activation (A). Allosteric changes involving residues of the α1-α2 loop (yellow), α2 (teal), and α9 (pink), as transmitted through the hydrophobic core (α4, α5), are also evident (A), and become more prominent upon titration up to a PUMA SAHB_A1/BAX ratio of 4:1 (B). Cα atoms of BAX residues that undergo significant chemical shift change upon exposure to PUMA SAHB_A1 are represented as orange bars on the plot (top) and orange spheres on the ribbon diagrams of monomeric BAX protein (calculated significance threshold >0.007 p.p.m. for 1:1 titration and >0.011 for 4:1 titration).

Figure 3. BAX Interaction Site Analysis by PUMA SAHB Photoaffinity Labeling and Mass Spectrometry

(A) PUMA pSAHB-1 was incubated with full-length BAX and the mixture subjected to UV irradiation, streptavidin pulldown, electrophoresis, excision of the crosslinked protein, trypsin proteolysis, and LC-MS/MS analysis. A single arginine substitution (A150R) was made in PUMA pSAHB-1 to facilitate tryptic digestion into shorter and more identifiable fragments by MS. The plot (left) depicts the frequency of crosslinked sites identified across the BAX polypeptide sequence, with crosslinked residues mapped onto the solution structure of monomeric BAX (PDB ID 2K7W) (right) and colored according to the frequency of occurrence. The C-terminal α9 helix of BAX has been removed from the structure to better visualize the crosslinked residues of the canonical BH3-binding pocket. (B–D) The corresponding analysis was performed for PUMA pSAHB-2 with full length BAX (B), PUMA pSAHB-1 with C-terminal helix-deleted BAX (BAXΔC) (C), and PUMA pSAHB-2 with BAXΔC (D). X, stapling amino acid; B, norleucine; U, 4-benzoyl-phenylalanine (Bpa). See also Figure S2.

Figure 4. PUMA SAHBs Bind to Two Geographically Distinct Sites on BAX

(A) Covalent tethering of the BAX C-terminal helix to its binding groove by an installed A112C/V177C disulfide bridge (GSSG, 0.5 mM) occludes the canonical BH3-binding surface of full-length BAX. In this context, only the α1/α6 trigger site is available for BH3 interaction. (B) Both structural and MS analyses indicate that activator BH3 helices bind to the exposed canonical pocket of BAXΔC, a truncated BAX construct in which the C-terminal helix is removed. In this context, the canonical BH3-binding site on BAX is preferred. (C) FITC-PUMA SAHB_F1 binds to BAX A112C/V177C and BAXΔC with K_Ds of 90 and 70 nM, respectively, as measured by FP assay. (D) FITC-PUMA SAHB_A1 binds to BAX A112C/V177C and BAXΔC with K_Ds of 717and 221 nM, respectively. (E) A139A/L141A mutagenesis of FITC-PUMA SAHB_A1 abrogates BAX-binding activity, highlighting the sequence specificity of the measured FITC-PUMA SAHB_A1 interactions with BAX A112C/V177C and BAXΔC. (F) FITC-PUMA BH3 (aa 131–153) exhibited no binding activity toward BAX A112C/V177C, but engaged BAXΔC with a K_D of 306 nM, suggesting that the exposed canonical pocket is more amenable to induced folding of an unstructured PUMA BH3 peptide.Data are mean ± S.D. for experiments performed in triplicate.

Figure 5. PUMA SAHB_A1 Triggers BAX-mediated Liposomal and Cytochrome c Release

(A) PUMA SAHB_A1 triggered dose-responsive BAX-mediated liposomal release of entrapped fluorophore (left), whereas BAX or PUMA SAHBs alone, or the combination of PUMA SAHB_A1mut with BAX (right), had little to no effect. Data are mean ± S.D. for experiments performed in triplicate and repeated twice. (B) BAX/BAK-deficient mitochondria dose-responsively released cytochrome c upon incubation with recombinant BAX and increasing amounts of PUMA SAHBA1, whereas BAX or PUMA SAHBs alone, or the combination of PUMA SAHB_A1mut with BAX, had little to no effect. Data are mean ± S.D. for experiments performed in sextuplicate and repeated three times.

Figure 6. Development and Evaluation of a Cell-Permeable PUMA SAHB

(A, B) SH SY5Y cells were treated with the indicated FITC-PUMA BH3 peptides (5 μM) in serum-free media for 6 hours, followed by washing, trypsinization, cellular lysis, electrophoresis of the isolated supernatant, and fluorescence detection. Whereas little to no unmodified PUMA BH3 peptide or PUMA SAHB_A1 fluorescence was detected in the corresponding cellular lysates, PUMA SAHB_A2 demonstrated robust fluorescence. (C) The cellular uptake of FITC-PUMA SAHB_A2 was time-dependent, as reflected by a progressive increase in intracellular peptide that co-migrated with FITC-PUMA SAHB_A2 marker peptide. (D) Live cell confocal microscopy of SH SY5Y cells treated with FITC- PUMA SAHB_A2 demonstrated its intracellular distribution. Prior to imaging, the cells were treated with Hoechst dye (blue) to label nuclei and CellMask Orange (orange-red) to delineate the plasma membrane. Original magnification, 100×. (E) Cellular uptake of FITC-PUMA SAHB_A2 was dose-responsively reduced by the addition of fetal bovine serum (FBS; 0%, 2.5%, 5%, and 10% [v/v]) to the tissue culture medium. (F) Addition of the indicated amounts of serum to PUMA SAHB_A2-treated SH SY5Y cells during the initial 6 hour incubation period, dose-responsively reduced cytotoxicity, as measured at 24 hours (18 hours after serum replacement to 10% [v/v]) by CellTiter-Glo assay. Incubating SH SY5Y cells with vehicle and media containing 0%, 2.5%, 5%, or 10% serum for 6 hours, followed by serum replacement to 10% (v/v), had no independent effect on cell viability, as measured by CellTiter-Glo at 24 hours. Data are mean ± S.D. for experiments performed in triplicate. See also Figure S3.

Figure 7. Pro-apoptotic Activity and Multimodal Interactions of PUMA BH3

(A–C) PUMA SAHB_A2 dose-responsively impaired the viability of a series of neuroblastoma cell lines including, SH SY5Y, Kelly, and IMR5, as measured by CellTiter-Glo at 24 hours after treatment (left column). The observed decrease in cell viability corresponded to dose-responsive caspase 3/7 activation, as measured by Caspase-Glo at eight 8 hours after treatment (middle column). Data are mean ± S.D. for experiments performed in triplicate. Incubation of cellular lystates with biotinylated PUMA SAHB_A3, followed by streptavidin pull-down, washing, electrophoresis of eluates, and BCL-2 family western analysis, documented engagement of native MCL-1, BCL-2, and BAX in all three neuroblastoma lines (right column). (D, E) PUMA SAHB_A2 dose-responsively impaired the viability of wild-type mouse embryonic fibroblasts (MEFs), but had little to no effect on Bax^−/−Bak^−/− MEFs, as measured by CellTiter-Glo at 24 hours after treatment (left column). Correspondingly, dose-responsive caspase 3/7 was observed in wild-type MEFs but not in Bax^−/−Bak^−/− MEFs, as measured by Caspase-Glo at 8 hours after treatment (right column). Data are mean ± S.D. for experiments performed in triplicate and repeated four times. (F, G) Flag-Pumaα, its R142E or D147K mutants, or vector control was transiently transfected into human embryonic kidney 293T (HEK 293T) cells and lysates subjected to immunoprecipitation using Flag affinity beads, followed by washing, electrophoresis of eluates, and anti-MCL-1, BCL-2, BAX, PUMA, and GAPDH western analysis. See also Figure S4.