Serine Palmitoyltransferase Regulates de NovoCeramide Generation during Etoposide-induced Apoptosis

Abstract

The de novo pathway of sphingolipid synthesis has been identified recently as a novel means of generating ceramide during apoptosis. Furthermore, it has been suggested that the activation of dihydroceramide synthase is responsible for increased ceramide production through this pathway. In this study, accumulation of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted in an accumulation of label in ceramide upon etoposide treatment. This result suggests that the activation of serine palmitoyltransferase is the event responsible for increased ceramide generation during de novo synthesis initiated by etoposide. Importantly, the ceramide generated from de novo synthesis appears to have a distinct function from that induced by sphingomyelinase action in that it is not involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system. These results implicate serine palmitoyltransferase as the enzyme controlling de novo ceramide synthesis during apoptosis and begin to define a unique function of ceramide generated from this pathway.It is increasingly apparent that sphingolipids, and in particular ceramide, are important mediators in regulating the response to stress of a cell. The agents that induce ceramide generation include physiological factors, such as tumor necrosis factor and the Fas ligand, as well as therapeutic agents, such as chemotherapy drugs and radiation. Many of these agents induce ceramide generation via the hydrolysis of sphingomyelin by the activation of one or more sphingomyelinases. Additional studies, however, have begun to implicate ceramide generated from the de novo pathway of sphingolipid synthesis as having a signaling function (1-8).Studies of de novo sphingolipid biosynthesis have been advanced by the realization that a class of fungal metabolites known as fumonisins share structural similarities with the sphingoid backbone. During investigation of the effects of fumonisin on sphingolipid metabolism in hepatocytes, it was observed that the synthesis of complex sphingolipids was significantly inhibited. It was also determined that the primary site of action of fumonisin was dihydroceramide synthase (9), an enzyme in the de novo pathway that catalyzes the N-acylation of sphinganine to produce dihydroceramide.

Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities

“…This screen allowed us to identify 2 short membrane proteins that, by a variety of criteria, appear to be the functional orthologs of yeast Tsc3p. Although SPT activity is apparently not highly regulated, a variety of stimuli have been reported to modulate activity a few fold without altering the levels of the LCB1 or LCB2 mRNAs or proteins (20)(21)(22). While it is possible that the ssSPTs contribute to regulation of enzyme activity, given the observation that all 3 proteins copurify it seems more likely that they are integral subunits of the SPT holoenzymes, a conclusion entirely consistent with the extremely small increase in SPT activity observed when the hLCB1 and hLCB2 subunits are overexpressed by themselves.…”

Detection of drug-induced apoptosis and necrosis in human cervical carcinoma cells using 1H NMR spectroscopy

“…10 An increase in ceramide, a sphingolipid that is a major membrane component, has also been observed in a recent study during etoposideinduced apoptosis of Molt-4 human leukemia cells. 27 This study provided evidence that ceramide, generated de novo, functions as a regulatory molecule in mediating membrane related apoptotic events. The increase in ceramide, with its long -CH 2 -chain, could also explain the increase in the 1.3 p.p.m.…”

Vitamin E prevents the age-dependent and palmitate-induced disturbances of sphingolipid turnover in liver cells

“…This might reflect impact of de novo pathway of sphingolipid synthesis in ceramide accumulation in old cells. There is growing evidence implicating SPT as the enzyme controlling de novo ceramide synthesis (Perry et al 2000;Watson et al 2009). Taking into account that α-tocopherol reduced activity of a key enzyme in the de novo synthesis of ceramide from palmitoyl-CoA and serine and that this effect was imitated by myriocin, it could be suggested that SPT is an important target for antioxidant action in the old cells.…”