Bax, Bcl-xl Exert their Regulation on Different Sites on the Ceramide Channel

Abstract

Ceramide is a sphingolipid that has been shown to play a vital role in the commitment of a cell to apoptosis. There is increasing evidence that ceramide channels may be the pathway through which cytochrome c is released from mitochondria, a critical step in the apoptotic process. Ceramide content increases in mitochondria upon an apoptotic signal and can form stable channels in mitochondrial membranes which are large enough for the passage of proteins. The Bcl-2 family of proteins regulate apoptosis and have pro-apoptotic members, like Bax, and anti-apoptotic members, like Bcl-xL. These proteins have been shown to directly interact with ceramide channels; Bax directly enlarges ceramide channels and Bcl-xL directly disassembles ceramide channels. The molecular site of interaction of Bax and Bcl-xL with ceramide was probed using structural analogs of ceramide which retain channel forming ability. Mitochondrial outer membrane permeability, and thus channel formation, was assessed using the dynamic cytochrome c accessibility assay. The results indicate that Bax was most sensitive to changes in the head group of ceramide while Bcl-xL was most sensitive to changes in tail length. These changes were not simply an effect of kinetics as longer incubations did not yield different results. Furthermore, when we tested known inhibitors of Bcl-xL, 2-methoxyantimycin A3 and ABT-737, the inhibitory effects of Bcl-xL on ceramide could be reversed. The results obtained in this study paint a very novel picture of how proteins may indeed be able to identify and regulate ceramide channels during apoptosis. Supported by a grant from NSF (MCB-0641208).