Perera, Meenu NadishaA critical event in apoptosis is the release of intermembrane space proteins from mitochondria following mitochondrial outer membrane (MOM) permeabilization (MOMP). The Bcl-2 family of proteins regulates MOM integrity and includes pro- and anti-apoptotic members, like Bax and Bcl-xL respectively. Preceding MOMP, the MOM becomes enriched with the sphingolipid, ceramide, which can self-assemble to form ceramide channels, contributing to MOMP. Bax and ceramide channels were found to act synergistically in the generation of MOMP and a direct interaction between these was observed in phospholipid membranes. The apparent affinity of activated Bax for ceramide channels increases with ceramide channel size, consistent with an induced fit mechanism; Bax drives the enlargement of ceramide channels to an optimum fit for the Bax binding site. A ceramide channel specific inhibitor prevented the enhanced MOMP in the presence of Bax and ceramide indicating ceramide channels were the primary permeabilizing entity. Analogs with changes to all the major structural features of ceramide were used to assess the molecular basis of stability of ceramide channels. Methylation of the C1- hydroxy group abrogated channel formation in mitochondria. Methylation of the amide nitrogen or a change in chirality at C2, which influences the C1-hydroxy group orientation, greatly reduced channel-forming ability whereas other changes were well tolerated. Competition experiments between ceramide and analogs resulted in synergism or antagonism, depending on compatibility of the analog structure with the ceramide channel model. The results provide evidence for ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions. Analogs that retained channel-forming ability were used to assess the structural features of ceramide channels required for regulation by Bcl-2 family proteins. The stereochemistry of the ceramide head group and access to the amide nitrogen is indispensible for regulation by Bax, implicating the polar portion of the channel as the Bax binding site. Bcl-xL's ability to disassemble ceramide channels depends on the length of the hydrophobic chains of ceramide. Specific Bcl-xL inhibitors reveal that BclxL binds ceramide channels through its hydrophobic groove and this is supported by simulated docking. The opposite effects of pro-and anti-apoptotic proteins are achieved at different sites on the ceramide channel.DissertationBiophysicsBiologyMolecular biologyApoptosisBaxBcl-xLBcl-2 Family ProteinsCeramide AnalogsCeramide ChannelsMitochondrial Outer Membrane Permeabilization