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Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity
Julie Wilson-Annan1, Lorraine A O'Reilly, Simon A Crawford
1Walter 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.
Insights
Prosurvival Bcl-w protein membrane association increases during apoptosis. BH3-only protein binding alters Bcl-w conformation, enhancing mitochondrial membrane affinity but neutralizing its survival function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Prosurvival Bcl-2-like proteins, such as Bcl-w, are typically localized to organelles like mitochondria via their hydrophobic COOH-terminal domain.
- Their role in regulating cell death pathways is crucial for maintaining cellular homeostasis.
Purpose of the Study:
- To investigate the unexpected observation that Bcl-w membrane association is enhanced during apoptosis.
- To elucidate the mechanism by which BH3-only proteins influence Bcl-w's interaction with membranes.
Main Methods:
- Studying Bcl-w's membrane association in healthy versus apoptotic cells.
- Utilizing cytotoxic signals and BH3 peptides to induce changes in Bcl-w localization.
- Creating a chimeric protein by tethering the Bim BH3 domain to Bcl-w to mimic ligand binding.
Main Results:
- Bcl-w transitions from a loosely attached to an integral membrane protein during apoptosis, triggered by BH3-only proteins like Bim.
- BH3 ligand binding to Bcl-w displaces its COOH-terminal domain, facilitating hydrophobic residue insertion into the membrane.
- A Bcl-w/Bim BH3 chimera demonstrated avid membrane binding, dependent on the COOH-terminal domain, without affecting apoptosis.
Conclusions:
- BH3-only protein ligation induces a conformational change in Bcl-w, leading to enhanced membrane association.
- This enhanced membrane association appears to neutralize the survival function of Bcl-w.
- The findings provide new insights into the dynamic regulation of Bcl-w during apoptosis.