Mitochondrial targeting of N-myristoylated dihydroceramide D4-desaturase: potential regulation of ceramide synthesis and apoptosis induction by myristic acid

Abstract : Background: Dihydroceramide Δ4-desaturase 1 (DES1), the enzyme which catalyses the desaturation of dihydroceramide into ceramide, has recently been shown to be N-myristoylated1. Compared to a recombinant unmyristoylable mutant form of DES1 (N-terminal Gly replaced by an Ala), the desaturase activity of the myristoylable wild-type DES1 is two times higher, in the presence of myristic acid incubated with COS-7 cells. Objectives: This study was performed to understand the effect of myristic acid and DES1 myristoylation on ceramide synthesis and metabolism, on the potential mitochondrial targeting of DES1, and on ceramide induced apoptosis. Procedure: DES activity in COS-7 cells expressing rat DES1 and in rat hepatocytes was measured in different organelles after subcellular fractionation by ultracentrifugation and iodixanol gradient separation. Subcellular localization of myristoylated or unmyristoylated DES1 was also investigated by immunofluorescence assay. Caspase 3 and cytochrome C release analysis were performed using western blot protocol. Caspase activity was measured using DEVD-AMC fluorometric assay. Results: Iodixanol gradient separation showed that DES activity was found in two different fractions from rat hepatocytes. Both mitochondrial and microsomal fractions from COS-7 cells expressing DES1-Gly both exhibited a higher activity than fractions from cells expressing DES1-Ala. Moreover, western blot analyses suggested that DES1-Gly is in higher proportion in the mitochondrial fraction. Only the myristoylable form of DES1 is targeted to the mitochondria considering the immunofluorescence assay. Finally, these preliminary results suggested that the association of DES1 myristoylation and its localization in the mitochondria may have an effect on the regulation of apoptosis process. Conclusion: These results show the importance of myristic acid in the regulation of sphingolipid metabolism and its potential effect on the induction of apoptosis through the mitochondrial targeting of DES1. 1Beauchamp et al. (2007) Biochimie 89, 1553-1561.