Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters.

Non-enzymatic thioesterification of specific cysteinyl peptides with fatty acyl-CoA has been previously demonstrated in both liposomes and aqueous medium. To identify the molecular basis for the differential reactivity of polypeptides in aqueous solutions, 26 synthetic cysteinyl peptides encompassing the palmitoylation sites of well known proteins (protein zero, proteolipid protein, beta-adrenergic receptor, p21(K-ras), transferrin receptor, CD-4 and SNAP-25) and six small thiol compounds were incubated separately with [3H]palmitoyl-CoA, [14C]acetyl-CoA and p-nitrophenyl thioacetate (NPTA). For each peptide, both the observed reaction rate constant at pH 7.5 and the pH-independent rate constant (k(2)) were calculated, and reactivity of the attacking sulfhydryl group was characterized using the Brønsted equation (log k(2)=beta(nuc) pK(a)+C). In general, peptides bearing basic and aromatic amino acid residues showed the lowest thiol pK(a)s, and consequently displayed the highest acylation rates. Reaction with palmitoyl-CoA was complicated to analyze because of the variable partition of peptides in the acyl chain donor/detergent micelles. In contrast, a linear Brønsted relationship was found for the reaction of the peptides with the water-soluble acetyl-CoA (beta(nuc)=0.59). A similar beta(nuc) value was obtained with the neutral NPTA, indicating that electronic effects other than those responsible for the acid-base properties of the thiol are less important. Thus, the concentration of the thiolate anion appears to be the major factor influencing the rate of the nucleophilic substitution reaction. These findings and the fact that the acylation sites in most proteins are surrounded by basic amino acids may partially explain the specificity of non-enzymatic palmitoylation regarding the acceptor sequences.

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath.

Myelin proteolipid protein (PLP) contains thioester-bound, long-chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post-translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mM hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein-bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mM dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP-less myelin-deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side-effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle-clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.