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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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Fusion of Bim BH3 to Bcl-w blocks engagement of BH3-only proteins. (A) Representation of the constructs fusing Bim BH3 to the NH2 terminus of Bcl-w. The shaded box below represents the core BH3 domain, and asterisks mark the hydrophobic BH3 residues critical for Bcl-w binding and altered to alanine (A) or glutamate (E) in the mutant peptides. (B) Bcl-w interacts avidly with Bim. 35S metabolically labeled lysates prepared from 293T cells overexpressing FLAG–Bcl-w and EE BimEL were immunoprecipitated with the anti–FLAG M2 (αF), anti-EE (αE), or anti-HA (αH) antibodies, and the immunoprecipitations were size fractionated on SDS-PAGE gels. (C) Fusion of wild-type Bim BH3 peptide to Bcl-w blocks its binding to Bim. wtBH3 (filled triangle), linked to Bcl-w via a flexible linker (in gray), is likely to bind onto the groove of Bcl-w, thereby displacing the COOH terminus (left). As expected, FLAG–wtBH3/Bcl-w could not be coimmunoprecipitated with BimEL. (D) Fusing a BH3 with the key BH3 hydrophobic residues mutated (filled circle) did not affect binding to BH3-only proteins. In the 4EBH3/Bcl-w fusion, the groove should be unoccupied (left), and that chimera readily associated with BimEL.
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fig4: Fusion of Bim BH3 to Bcl-w blocks engagement of BH3-only proteins. (A) Representation of the constructs fusing Bim BH3 to the NH2 terminus of Bcl-w. The shaded box below represents the core BH3 domain, and asterisks mark the hydrophobic BH3 residues critical for Bcl-w binding and altered to alanine (A) or glutamate (E) in the mutant peptides. (B) Bcl-w interacts avidly with Bim. 35S metabolically labeled lysates prepared from 293T cells overexpressing FLAG–Bcl-w and EE BimEL were immunoprecipitated with the anti–FLAG M2 (αF), anti-EE (αE), or anti-HA (αH) antibodies, and the immunoprecipitations were size fractionated on SDS-PAGE gels. (C) Fusion of wild-type Bim BH3 peptide to Bcl-w blocks its binding to Bim. wtBH3 (filled triangle), linked to Bcl-w via a flexible linker (in gray), is likely to bind onto the groove of Bcl-w, thereby displacing the COOH terminus (left). As expected, FLAG–wtBH3/Bcl-w could not be coimmunoprecipitated with BimEL. (D) Fusing a BH3 with the key BH3 hydrophobic residues mutated (filled circle) did not affect binding to BH3-only proteins. In the 4EBH3/Bcl-w fusion, the groove should be unoccupied (left), and that chimera readily associated with BimEL.

Mentions: As the tighter membrane association of Bcl-w correlated with the recruitment of Bim to Bcl-w, we wished to test whether the Bim BH3 domain could induce the transition in lysates from healthy cells. To validate the use of BH3 peptides, we first measured the affinity for Bcl-w of a BimL polypeptide, or a BH3 peptide from it, using an optical biosensor (Fig. 3, B and C). Purified recombinant BimL lacking its COOH-terminal hydrophobic 27 amino acids (BimLΔC27) binds purified Bcl-w with high (nM) affinity (Fig. 3 B; Hinds et al., 2003). Notably, a 26-mer peptide spanning the BH3 domain of Bim (denoted wtBH3) bound Bcl-w as avidly as BimLΔC27, and it competed effectively for the binding of BimLΔC27 (Fig. 3, B and C). Consequently, we focused on this peptide and two derivatives that have reduced affinity for Bcl-w, due to replacement of one or more of the four hydrophobic BH3 residues that mediate interaction with the hydrophobic groove of the prosurvival proteins (Fig. 4 A) (Sattler et al., 1997; Petros et al., 2000; Hinds et al., 2003). Replacement of the invariant BH3 leucine (L94 of the mouse BimL) with alanine (L94ABH3) reduced the binding over 50-fold, whereas a glutamate replacement of all four key residues (4EBH3) abolished binding altogether (Fig. 3 B).


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)

Fusion of Bim BH3 to Bcl-w blocks engagement of BH3-only proteins. (A) Representation of the constructs fusing Bim BH3 to the NH2 terminus of Bcl-w. The shaded box below represents the core BH3 domain, and asterisks mark the hydrophobic BH3 residues critical for Bcl-w binding and altered to alanine (A) or glutamate (E) in the mutant peptides. (B) Bcl-w interacts avidly with Bim. 35S metabolically labeled lysates prepared from 293T cells overexpressing FLAG–Bcl-w and EE BimEL were immunoprecipitated with the anti–FLAG M2 (αF), anti-EE (αE), or anti-HA (αH) antibodies, and the immunoprecipitations were size fractionated on SDS-PAGE gels. (C) Fusion of wild-type Bim BH3 peptide to Bcl-w blocks its binding to Bim. wtBH3 (filled triangle), linked to Bcl-w via a flexible linker (in gray), is likely to bind onto the groove of Bcl-w, thereby displacing the COOH terminus (left). As expected, FLAG–wtBH3/Bcl-w could not be coimmunoprecipitated with BimEL. (D) Fusing a BH3 with the key BH3 hydrophobic residues mutated (filled circle) did not affect binding to BH3-only proteins. In the 4EBH3/Bcl-w fusion, the groove should be unoccupied (left), and that chimera readily associated with BimEL.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Fusion of Bim BH3 to Bcl-w blocks engagement of BH3-only proteins. (A) Representation of the constructs fusing Bim BH3 to the NH2 terminus of Bcl-w. The shaded box below represents the core BH3 domain, and asterisks mark the hydrophobic BH3 residues critical for Bcl-w binding and altered to alanine (A) or glutamate (E) in the mutant peptides. (B) Bcl-w interacts avidly with Bim. 35S metabolically labeled lysates prepared from 293T cells overexpressing FLAG–Bcl-w and EE BimEL were immunoprecipitated with the anti–FLAG M2 (αF), anti-EE (αE), or anti-HA (αH) antibodies, and the immunoprecipitations were size fractionated on SDS-PAGE gels. (C) Fusion of wild-type Bim BH3 peptide to Bcl-w blocks its binding to Bim. wtBH3 (filled triangle), linked to Bcl-w via a flexible linker (in gray), is likely to bind onto the groove of Bcl-w, thereby displacing the COOH terminus (left). As expected, FLAG–wtBH3/Bcl-w could not be coimmunoprecipitated with BimEL. (D) Fusing a BH3 with the key BH3 hydrophobic residues mutated (filled circle) did not affect binding to BH3-only proteins. In the 4EBH3/Bcl-w fusion, the groove should be unoccupied (left), and that chimera readily associated with BimEL.
Mentions: As the tighter membrane association of Bcl-w correlated with the recruitment of Bim to Bcl-w, we wished to test whether the Bim BH3 domain could induce the transition in lysates from healthy cells. To validate the use of BH3 peptides, we first measured the affinity for Bcl-w of a BimL polypeptide, or a BH3 peptide from it, using an optical biosensor (Fig. 3, B and C). Purified recombinant BimL lacking its COOH-terminal hydrophobic 27 amino acids (BimLΔC27) binds purified Bcl-w with high (nM) affinity (Fig. 3 B; Hinds et al., 2003). Notably, a 26-mer peptide spanning the BH3 domain of Bim (denoted wtBH3) bound Bcl-w as avidly as BimLΔC27, and it competed effectively for the binding of BimLΔC27 (Fig. 3, B and C). Consequently, we focused on this peptide and two derivatives that have reduced affinity for Bcl-w, due to replacement of one or more of the four hydrophobic BH3 residues that mediate interaction with the hydrophobic groove of the prosurvival proteins (Fig. 4 A) (Sattler et al., 1997; Petros et al., 2000; Hinds et al., 2003). Replacement of the invariant BH3 leucine (L94 of the mouse BimL) with alanine (L94ABH3) reduced the binding over 50-fold, whereas a glutamate replacement of all four key residues (4EBH3) abolished binding altogether (Fig. 3 B).

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