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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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BH3 peptides induce tight membrane association of Bcl-w. (A) Damage signal (10 Gy γ irradiation) to FD/FLAG–Bcl-w cells provokes BimEL and BimL isoforms to associate with FLAG–Bcl-w. (B) Wild-type BimL protein and its BH3 peptide bind tightly to Bcl-w. The affinities of recombinant BimLΔC27 or 26-mer Bim BH3 peptides to recombinant Bcl-w ΔC10 determined in optical biosensor. (C) Bim BH3 peptide (wtBH3) binds Bcl-w as well as BimL ΔC27. The ability of free BimLΔC27 or wtBH3 peptide to compete with immobilized BimLΔC27 for Bcl-w binding was assessed in biosensor experiments. (D) Addition of wild-type, but not mutant, BH3 peptide induces tight membrane association of FLAG–Bcl-w. Lysates of healthy cells were incubated with increasing amounts (0–100 μg/ml) of wtBH3 (top) or L94ABH3 (bottom) peptides before fractionation. The blots were probed with rat anti–FLAG 9H1 to detect FLAG–Bcl-w. (E) Bim BH3 peptide induces tighter association of endogenous Bcl-w from FDC-P1 cells. Lysates of healthy cells were incubated with increasing amounts of BH3 peptides and fractionated. In D and E, the blots were probed for endogenous Bcl-w. (F) BH3 peptide induces membrane integration of endogenous Bcl-w. Lysates prepared from healthy FDC-P1 cells and incubated with wtBH3 peptide were fractionated without or with alkali treatment.
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fig3: BH3 peptides induce tight membrane association of Bcl-w. (A) Damage signal (10 Gy γ irradiation) to FD/FLAG–Bcl-w cells provokes BimEL and BimL isoforms to associate with FLAG–Bcl-w. (B) Wild-type BimL protein and its BH3 peptide bind tightly to Bcl-w. The affinities of recombinant BimLΔC27 or 26-mer Bim BH3 peptides to recombinant Bcl-w ΔC10 determined in optical biosensor. (C) Bim BH3 peptide (wtBH3) binds Bcl-w as well as BimL ΔC27. The ability of free BimLΔC27 or wtBH3 peptide to compete with immobilized BimLΔC27 for Bcl-w binding was assessed in biosensor experiments. (D) Addition of wild-type, but not mutant, BH3 peptide induces tight membrane association of FLAG–Bcl-w. Lysates of healthy cells were incubated with increasing amounts (0–100 μg/ml) of wtBH3 (top) or L94ABH3 (bottom) peptides before fractionation. The blots were probed with rat anti–FLAG 9H1 to detect FLAG–Bcl-w. (E) Bim BH3 peptide induces tighter association of endogenous Bcl-w from FDC-P1 cells. Lysates of healthy cells were incubated with increasing amounts of BH3 peptides and fractionated. In D and E, the blots were probed for endogenous Bcl-w. (F) BH3 peptide induces membrane integration of endogenous Bcl-w. Lysates prepared from healthy FDC-P1 cells and incubated with wtBH3 peptide were fractionated without or with alkali treatment.

Mentions: Apoptosis seems to be initiated when BH3-only proteins, unleashed by damage signals, bind to prosurvival relatives (Huang and Strasser, 2000; Cory and Adams, 2002). As the enhanced membrane association of Bcl-w appeared to involve a step before caspase activation, we hypothesized that it was triggered by engagement of Bcl-w by a BH3-only protein such as Bim. When FLAG–Bcl-w was immunoprecipitated from lysates of FD/FLAG–Bcl-w cells that had been exposed to a damaging agent, the complex included both the major endogenous Bim isoforms (BimL and BimEL), but neither was detectable in parallel immunoprecipitates from untreated cells (Fig. 3 A), confirming that apoptosis induces their association.


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)

BH3 peptides induce tight membrane association of Bcl-w. (A) Damage signal (10 Gy γ irradiation) to FD/FLAG–Bcl-w cells provokes BimEL and BimL isoforms to associate with FLAG–Bcl-w. (B) Wild-type BimL protein and its BH3 peptide bind tightly to Bcl-w. The affinities of recombinant BimLΔC27 or 26-mer Bim BH3 peptides to recombinant Bcl-w ΔC10 determined in optical biosensor. (C) Bim BH3 peptide (wtBH3) binds Bcl-w as well as BimL ΔC27. The ability of free BimLΔC27 or wtBH3 peptide to compete with immobilized BimLΔC27 for Bcl-w binding was assessed in biosensor experiments. (D) Addition of wild-type, but not mutant, BH3 peptide induces tight membrane association of FLAG–Bcl-w. Lysates of healthy cells were incubated with increasing amounts (0–100 μg/ml) of wtBH3 (top) or L94ABH3 (bottom) peptides before fractionation. The blots were probed with rat anti–FLAG 9H1 to detect FLAG–Bcl-w. (E) Bim BH3 peptide induces tighter association of endogenous Bcl-w from FDC-P1 cells. Lysates of healthy cells were incubated with increasing amounts of BH3 peptides and fractionated. In D and E, the blots were probed for endogenous Bcl-w. (F) BH3 peptide induces membrane integration of endogenous Bcl-w. Lysates prepared from healthy FDC-P1 cells and incubated with wtBH3 peptide were fractionated without or with alkali treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172834&req=5

fig3: BH3 peptides induce tight membrane association of Bcl-w. (A) Damage signal (10 Gy γ irradiation) to FD/FLAG–Bcl-w cells provokes BimEL and BimL isoforms to associate with FLAG–Bcl-w. (B) Wild-type BimL protein and its BH3 peptide bind tightly to Bcl-w. The affinities of recombinant BimLΔC27 or 26-mer Bim BH3 peptides to recombinant Bcl-w ΔC10 determined in optical biosensor. (C) Bim BH3 peptide (wtBH3) binds Bcl-w as well as BimL ΔC27. The ability of free BimLΔC27 or wtBH3 peptide to compete with immobilized BimLΔC27 for Bcl-w binding was assessed in biosensor experiments. (D) Addition of wild-type, but not mutant, BH3 peptide induces tight membrane association of FLAG–Bcl-w. Lysates of healthy cells were incubated with increasing amounts (0–100 μg/ml) of wtBH3 (top) or L94ABH3 (bottom) peptides before fractionation. The blots were probed with rat anti–FLAG 9H1 to detect FLAG–Bcl-w. (E) Bim BH3 peptide induces tighter association of endogenous Bcl-w from FDC-P1 cells. Lysates of healthy cells were incubated with increasing amounts of BH3 peptides and fractionated. In D and E, the blots were probed for endogenous Bcl-w. (F) BH3 peptide induces membrane integration of endogenous Bcl-w. Lysates prepared from healthy FDC-P1 cells and incubated with wtBH3 peptide were fractionated without or with alkali treatment.
Mentions: Apoptosis seems to be initiated when BH3-only proteins, unleashed by damage signals, bind to prosurvival relatives (Huang and Strasser, 2000; Cory and Adams, 2002). As the enhanced membrane association of Bcl-w appeared to involve a step before caspase activation, we hypothesized that it was triggered by engagement of Bcl-w by a BH3-only protein such as Bim. When FLAG–Bcl-w was immunoprecipitated from lysates of FD/FLAG–Bcl-w cells that had been exposed to a damaging agent, the complex included both the major endogenous Bim isoforms (BimL and BimEL), but neither was detectable in parallel immunoprecipitates from untreated cells (Fig. 3 A), confirming that apoptosis induces their association.

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