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Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Wilson-Annan J, O'Reilly LA, Crawford SA, Hausmann G, Beaumont JG, Parma LP, Chen L, Lackmann M, Lithgow T, Hinds MG, Day CL, Adams JM, Huang DC - J. Cell Biol. (2003)

Bottom Line: We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis.To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w.These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia.

ABSTRACT
Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

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Fusing wild-type BH3 to Bcl-w does not perturb mitochondrial transmembrane potential (ΔΨm) but inactivates its prosurvival activity. (A) Flow cytometric analysis of untreated (left), IL-3–deprived (middle), or γ-irradiated (right) parental FDC-P1 cells or representative clones stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w stained with the fluorochrome DiOC6(3) and PI. DiOC6(3) staining is shown after appropriate compensation for PI to exclude dead cells. (B) Parental FDC-P1 cells or representative clones of cell lines stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins were deprived of their growth factor IL-3 or γ-irradiated (10 Gy). Cell viability was quantified daily by PI staining revealed by flow cytometric analyses. The data are means ± 1 SD of three or more experiments.
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fig6: Fusing wild-type BH3 to Bcl-w does not perturb mitochondrial transmembrane potential (ΔΨm) but inactivates its prosurvival activity. (A) Flow cytometric analysis of untreated (left), IL-3–deprived (middle), or γ-irradiated (right) parental FDC-P1 cells or representative clones stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w stained with the fluorochrome DiOC6(3) and PI. DiOC6(3) staining is shown after appropriate compensation for PI to exclude dead cells. (B) Parental FDC-P1 cells or representative clones of cell lines stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins were deprived of their growth factor IL-3 or γ-irradiated (10 Gy). Cell viability was quantified daily by PI staining revealed by flow cytometric analyses. The data are means ± 1 SD of three or more experiments.

Mentions: Finally, we tested whether the conformational change mediated by the bound BH3 domain induces a latent killing activity in Bcl-w. That conformer might, for example, upon integration into the membrane, disturb its integrity, as translocated Bax is thought to do (see Discussion). wtBH3/Bcl-w did not, however, affect the viability of untreated FDC-P1 cells, and when they were subjected to cytotoxic stress, it did affect the loss of mitochondrial transmembrane potential (ΔΨm), which sometimes precedes irreversible commitment to apoptosis (Castedo et al., 1996). In contrast, 4EBH3/Bcl-w inhibited the loss of ΔΨm as effectively as wild-type Bcl-w (Fig. 6 A).


Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Wilson-Annan J, O'Reilly LA, Crawford SA, Hausmann G, Beaumont JG, Parma LP, Chen L, Lackmann M, Lithgow T, Hinds MG, Day CL, Adams JM, Huang DC - J. Cell Biol. (2003)

Fusing wild-type BH3 to Bcl-w does not perturb mitochondrial transmembrane potential (ΔΨm) but inactivates its prosurvival activity. (A) Flow cytometric analysis of untreated (left), IL-3–deprived (middle), or γ-irradiated (right) parental FDC-P1 cells or representative clones stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w stained with the fluorochrome DiOC6(3) and PI. DiOC6(3) staining is shown after appropriate compensation for PI to exclude dead cells. (B) Parental FDC-P1 cells or representative clones of cell lines stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins were deprived of their growth factor IL-3 or γ-irradiated (10 Gy). Cell viability was quantified daily by PI staining revealed by flow cytometric analyses. The data are means ± 1 SD of three or more experiments.
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Related In: Results  -  Collection

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fig6: Fusing wild-type BH3 to Bcl-w does not perturb mitochondrial transmembrane potential (ΔΨm) but inactivates its prosurvival activity. (A) Flow cytometric analysis of untreated (left), IL-3–deprived (middle), or γ-irradiated (right) parental FDC-P1 cells or representative clones stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w stained with the fluorochrome DiOC6(3) and PI. DiOC6(3) staining is shown after appropriate compensation for PI to exclude dead cells. (B) Parental FDC-P1 cells or representative clones of cell lines stably expressing Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins were deprived of their growth factor IL-3 or γ-irradiated (10 Gy). Cell viability was quantified daily by PI staining revealed by flow cytometric analyses. The data are means ± 1 SD of three or more experiments.
Mentions: Finally, we tested whether the conformational change mediated by the bound BH3 domain induces a latent killing activity in Bcl-w. That conformer might, for example, upon integration into the membrane, disturb its integrity, as translocated Bax is thought to do (see Discussion). wtBH3/Bcl-w did not, however, affect the viability of untreated FDC-P1 cells, and when they were subjected to cytotoxic stress, it did affect the loss of mitochondrial transmembrane potential (ΔΨm), which sometimes precedes irreversible commitment to apoptosis (Castedo et al., 1996). In contrast, 4EBH3/Bcl-w inhibited the loss of ΔΨm as effectively as wild-type Bcl-w (Fig. 6 A).

Bottom Line: We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis.To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w.These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia.

ABSTRACT
Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Show MeSH
Related in: MedlinePlus