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

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.

BCL-2 in the crosshairs: tipping the balance of life and death

“…Indeed, binding of BH3 peptides to the hydrophobic groove of BCL-w is impaired by a-9, as reflected by decreased BH3 peptide affinity for full-length versus C-terminally truncated BCL-w. 82,83 The distinctive C-terminal structure likely accounts for the cytosolic disposition of BAX and BCL-w by optimizing solubility until triggered to undergo a conformational change, which releases a9 for membrane insertion. 52,82,83,99 By obstructing the protein interaction site, a9 may also contribute to maintaining BAX and BCL-w in the monomeric form. Of note, a9 of BCL-w is less hydrophobic and more mobile than that of BAX by NMR, suggesting that the activation criteria for BAX a9 disengagement and resultant BAX translocation are stringent by design.…”

Mitochondrial factors with dual roles in death and survival

“…In this scenario, the drugs would have an effect similar to proteolytic cleavage of Bcl-2 and Bcl-xL, whereby Bcl-2 and Bcl-xL gain the ability to potently kill cells and to release cytochrome c from intracellular and isolated mitochondria and from pure lipid vesicles (Cheng et al, 1997;Clem et al, 1998;Basan˜ez et al, 2001). An important outcome of the work on various BH3-only proteins (including Bid, Bad, Hrk, Bim, Puma, Noxa and several others) is that we are now armed with the understanding that the binding capacities of Bcl-2 family proteins differ in healthy and dying cells (Suzuki et al, 2000;Wilson-Annan et al, 2003;Jeong et al, 2004). This was facilitated by the generation of antibodies that distinguish 'dormant' from death-activated Bax and Bak (Griffiths et al, 1999;Nechushtan et al, 1999).…”

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

“…Following SDS-PAGE, proteins were transferred onto nitrocellulose membranes and immunoblotted with either rat anti-HA 3F10 (Roche) or FLAG 9H1 antibodies. 37 …”