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Abstract

We have investigated the effect of documented protein palmitoylation inhibitors on the fatty acylation and intracellular transport of myelin proteolipid protein (PLP). To this end, brain slices from 20-day-old rats were incubated with either [3H]palmitate or [3H]leucine in the presence or absence of various concentrations of 2-fluoropalmitate (FP), cerulenin (CER), or tunicamycin (TM). FP (> or = 10 microM) decreased the cellular uptake of [3H]palmitate and consequently reduced the labeling of palmitoyl-CoA, glycerolipids and PLP. CER (> or = 1 mM) reduced the palmitoylation of PLP with a concomitant decline in protein thiols. Consistent with being a fatty acyl-CoA analogue, TM (> or = 200 microM) diminished the palmitoylation of PLP and lipids while increasing the amount of [3H]palmitoyl-CoA. Although both CER and TM decreased protein palmitoylation, only the latter affected the appearance of newly synthesized PLP into myelin. Because TM, but not CER, also reduced the formation of lipids, it is concluded that palmitoylation is not required for intracellular transport. Finally, comparison of the effect of TM in brain slices and in a cell-free system suggests that palmitoylation of PLP in whole cells may be an enzymatic process.

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

“…These findings suggest that cerulenin has a minimal effect on the incorporation of exogenously added long chain fatty acid into cellular lipids. However, treatment with higher (> 1mM) concentrations of cerulenin result in reduced palmitate incorporation into phosphatidylcholine, but not other phospholipids [30]. No changes in uptake of labeled palmitate or its conversion to palmitoyl CoA were observed with cerulenin.…”

“…A recent study with a related halogen substituted palmitate analog, 2-fluoropalmitate, concluded that this compound inhibited uptake of [3H] palmitate into brain cells, perhaps by competing with palmitate for cellular entry. As a result, 2-fluoropalmitate treatment leads to decreases in [ 3 H]palmitoyl CoA levels and decreased fatty acylation of phospholipids and myelin proteolipid protein [30].…”

“…Cerulenin is a natural product antibiotic that inhibits fatty acid synthesis by alkylating β-ketoacyl acyl-carrier protein synthase. Cerulenin treatment inhibits palmitoylation of myelin proteolipid protein [30] and CD36 [32]. A palmitoyl analog of cerulenin has been synthesized and has been shown to block palmitoylation of p21Ras without inhibiting fatty acid synthesis.…”

“…Two groups have suggested that the compound chemically modifies sulfhydryl groups [30,33]. Thus, the ability of cerulenin to inhibit protein palmitoylation may be due to the formation of chemical adducts with critical thiols on either the palmitoyl acyltransferase or the protein acceptor substrate.…”

“…The structural similarity between tunicamycin and palmitoyl CoA led to the finding that tunicamycin can also inhibit protein palmitoylation [36]. Inhibitory effects of tunicamycin on palmitoylation of GAP-43, N-type Ca++ channels, estrogen receptor α variant, and myelin proteolipid protein have been documented [30,[36][37][38].…”