Investigation of the binding of ceramide and palmitoyl-CoA to murine t-ACBP using heteronuclear NMR spectroscopy
Murine t-ACBP is a member of the family of acyl-CoA binding proteins expressed specifically in testis. Acyl-CoA binding proteins (ACBPs, also know as 'diazepam binding inhibitors' or DBIs') bind long-chain acyl-CoA esters with high affinity and act as intracellular transporters and pool formers for acyl-CoA. They are also endogenous ligands for the peripheral benzodiazepine receptor (PBR), which is localized in the mitochondrial membrane, and is thought to regulate the opening of the permeability transition pore complex (PTPC), a central event in apoptosis. We have shown previously that knock-out of t-ACBP leads to resistance to ceramide-induced apoptosis in Chinese hamster ovary (CHO) cells. We have also shown that transduction of recombinantly-produced t-ACBP into CHO cells leads to apoptosis by activation of caspase 3. One possible explanation for this behaviour is that ceramide up-regulates the interaction of t-ACBP with the PBR, leading to opening of the PTPC. A second explanation is that knock-out of t-ACBP leads to reduction in the availability of palmitoyl-CoA, and consequently to a lowering of the level of endogenous ceramide, rendering the cells less sensitive to exogenously introduced ceramide. We have recombinantly expressed 13C- and 15N-enriched samples of murine t-ACBP and used heteronuclear NMR spectroscopy to show that it binds strongly to palmitoyl-CoA. No significant binding was observed to either C2-ceramide or C16-ceramide. We conclude from this that if t-ACBP does play a role in the opening of the PTPC in response to ceramide, it does not do so by direct interaction with ceramide. We therefore conclude that it is the interaction of t-ACBP with palmitoyl-CoA that holds the key to its role in ceramide-induced apoptosis. This preliminary study serves to illustrate the power of NMR spectroscopy as a tool for probing protein-ligand and protein-protein interactions, which has not previously been exploited in South Africa.