Bax and Bcl-xL exert their regulation on different sites of the ceramide channel

Abstract

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

Metabolic control of cell death

“…In response to ionizing radiation, acidic spingomyelinase is activated, catalyzing the generation of ceramide on the plasma membrane of endothelial intestinal cells, where it coalesces to form microdomains that transmit apoptotic signals (129). Ceramide that accumulates in mitochondria can trigger MOMP either by stimulating the insertion of BAX into the OMM (130) or by forming lipid pores that are under the control of proteins from the Bcl-2 family (131). …”

Ceramide channels: destabilization by Bcl-xL and role in apoptosis

“…Mitochondria were isolated from the liver of male Sprague Dawley rats as described originally (33) and as modified (29). The animal use protocols were approved by the Institutional Animal Care and Use Committee.…”

“…Whereas, it is generally agreed that Bcl-xL inhibits Bax channel formation by forming heterodimers with activated Bax monomers (27, 28), the mechanism by which Bcl-xL inhibits ceramide channels is not well understood. Since ceramide channels are large assemblies of ceramide monomers, there is no precedent to indicate possible mechanisms by which such a structure could be prevented from forming or be destabilized by a protein although a mechanism has been proposed (29). Experiments with ceramide analogs demonstrated that the hydrophobic tails of ceramide are important for the inhibition by Bcl-xL (29).…”

“…Since ceramide channels are large assemblies of ceramide monomers, there is no precedent to indicate possible mechanisms by which such a structure could be prevented from forming or be destabilized by a protein although a mechanism has been proposed (29). Experiments with ceramide analogs demonstrated that the hydrophobic tails of ceramide are important for the inhibition by Bcl-xL (29). In addition, small molecule inhibitors (2-methoxyantimycin A3, ABT-737 and ABT-263), known as BH3 peptide mimetics because they bind to a hydrophobic grove on Bcl-xL and thus block its ability to bind to Bax, were also found to inhibit the ability of Bcl-xL to inhibit the formation of ceramide channels in the MOM (29).…”

“…Experiments with ceramide analogs demonstrated that the hydrophobic tails of ceramide are important for the inhibition by Bcl-xL (29). In addition, small molecule inhibitors (2-methoxyantimycin A3, ABT-737 and ABT-263), known as BH3 peptide mimetics because they bind to a hydrophobic grove on Bcl-xL and thus block its ability to bind to Bax, were also found to inhibit the ability of Bcl-xL to inhibit the formation of ceramide channels in the MOM (29). These inhibitors interfere with the anti-apoptotic activity of Bcl-xL (30–32) suggesting that the ability of Bcl-xL to interfere with ceramide channel formation may somehow be linked to its anti-apoptotic function.…”