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BAX activation is initiated at a novel interaction site.

Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu HC, Kim H, Cheng EH, Tjandra N, Walensky LD - Nature (2008)

Bottom Line: Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins.The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location.Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

ABSTRACT
BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

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Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosisa, Impact of BAX K21E mutagenesis in oligomerization and b, cytochrome c release assays. c, Apoptotic response of Bax-/-Bak-/- MEFs reconstituted with BAX or BAXK21E to treatment with BIM SAHBA or BIM SAHBA(R153D). d, Impact of BAX K21E mutagenesis on staurosporine (STS)-induced apoptosis of BAX-reconstituted DKO MEFs. Error bars, mean ± s.d.
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Figure 5: Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosisa, Impact of BAX K21E mutagenesis in oligomerization and b, cytochrome c release assays. c, Apoptotic response of Bax-/-Bak-/- MEFs reconstituted with BAX or BAXK21E to treatment with BIM SAHBA or BIM SAHBA(R153D). d, Impact of BAX K21E mutagenesis on staurosporine (STS)-induced apoptosis of BAX-reconstituted DKO MEFs. Error bars, mean ± s.d.

Mentions: To examine the impact of BAX mutagenesis at the α1-α6 interaction site, we mutated residue K21, which exhibited the most pronounced chemical shift upon BIM SAHB binding (Fig. 1a). We generated recombinant BAX bearing a K21E point mutation (Supplementary Fig. 9) and observed that BIM SAHB-induced BAXK21E activation was significantly reduced compared to wild type BAX as measured by the oligomerization assay (Fig. 5a). BAXK21E-mediated cytochrome c release in response to BIM SAHB was likewise blunted (Fig. 5b). To determine if this single point mutation within the novel BAX binding site impacted the capacity to activate BAX in a cellular context, we retrovirally reconstituted Bax-/-Bak-/- mouse embryonic fibroblasts (DKO MEFs) with either wild type or BAXK21E and monitored apoptosis induction in response to BIM SAHB. Whereas BIM SAHB induced time-dependent apoptosis of BAX-reconstituted MEFs, as quantitated by annexin-V binding, single K21E point mutation within the new BAX binding site reduced BIM SAHB-triggered apoptosis (Fig. 5c). This decrease in BAXK21E-mediated apoptosis correlates with impaired activation of BAXK21E by BIM SAHB in both oligomerization and cytochrome c release assays (Fig. 5a, 5b). As a further measure of specificity, we found that R153D mutagenesis of BIM SAHB, which eliminated its BAX activating capacity in the oligomerization and cytochrome c release assays (Fig. 4e, 4f), likewise abolished BIM SAHB-induced activation of BAX and BAXK21E in a cellular context (Fig. 5c).


BAX activation is initiated at a novel interaction site.

Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu HC, Kim H, Cheng EH, Tjandra N, Walensky LD - Nature (2008)

Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosisa, Impact of BAX K21E mutagenesis in oligomerization and b, cytochrome c release assays. c, Apoptotic response of Bax-/-Bak-/- MEFs reconstituted with BAX or BAXK21E to treatment with BIM SAHBA or BIM SAHBA(R153D). d, Impact of BAX K21E mutagenesis on staurosporine (STS)-induced apoptosis of BAX-reconstituted DKO MEFs. Error bars, mean ± s.d.
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Related In: Results  -  Collection

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Figure 5: Mutagenesis of the BAX interaction site impairs activation and BAX-mediated apoptosisa, Impact of BAX K21E mutagenesis in oligomerization and b, cytochrome c release assays. c, Apoptotic response of Bax-/-Bak-/- MEFs reconstituted with BAX or BAXK21E to treatment with BIM SAHBA or BIM SAHBA(R153D). d, Impact of BAX K21E mutagenesis on staurosporine (STS)-induced apoptosis of BAX-reconstituted DKO MEFs. Error bars, mean ± s.d.
Mentions: To examine the impact of BAX mutagenesis at the α1-α6 interaction site, we mutated residue K21, which exhibited the most pronounced chemical shift upon BIM SAHB binding (Fig. 1a). We generated recombinant BAX bearing a K21E point mutation (Supplementary Fig. 9) and observed that BIM SAHB-induced BAXK21E activation was significantly reduced compared to wild type BAX as measured by the oligomerization assay (Fig. 5a). BAXK21E-mediated cytochrome c release in response to BIM SAHB was likewise blunted (Fig. 5b). To determine if this single point mutation within the novel BAX binding site impacted the capacity to activate BAX in a cellular context, we retrovirally reconstituted Bax-/-Bak-/- mouse embryonic fibroblasts (DKO MEFs) with either wild type or BAXK21E and monitored apoptosis induction in response to BIM SAHB. Whereas BIM SAHB induced time-dependent apoptosis of BAX-reconstituted MEFs, as quantitated by annexin-V binding, single K21E point mutation within the new BAX binding site reduced BIM SAHB-triggered apoptosis (Fig. 5c). This decrease in BAXK21E-mediated apoptosis correlates with impaired activation of BAXK21E by BIM SAHB in both oligomerization and cytochrome c release assays (Fig. 5a, 5b). As a further measure of specificity, we found that R153D mutagenesis of BIM SAHB, which eliminated its BAX activating capacity in the oligomerization and cytochrome c release assays (Fig. 4e, 4f), likewise abolished BIM SAHB-induced activation of BAX and BAXK21E in a cellular context (Fig. 5c).

Bottom Line: Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins.The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location.Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

ABSTRACT
BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

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