In vitro depalmitoylation of neurospecific peptides: Implication for infantile neuronal ceroid lipofuscinosis

Palmitoyl protein thioesterase 1 (PPT1) removes palmitate from specific cys teine residues in peptides and proteins. We have previously shown that a pa lmitoylated myelin glycoprotein. Po octapeptide (IRYCWLRR) can be specifica lly depalmitoylated by PPT1 in vitro (Cho and Dawson [1998] J. Neurochem. 1 71;323-329). To characterize further the substrate specificity of PPM, we p repared various palmitoylated oligopeptides, based on palmitoylated sequenc es from different proteins. A truncated tetrapeptide from Po (RY[palmitoyl] -CW) was as good a substrate as the octapeptide Po, with optimal activity a t pH 4.0. In contrast, other peptide substrates showed marked differences. Thus, the deacylation of GAP-43 (MLCCMRR), rhodopsin (VTTLCCGKN), and G alp ha subunit (MGCLGNSK) peptides was more efficient at neutral pH (7.4) than at acidic pH (4.0), with the greatest efficiency toward the G alpha peptide (five- to sixfold higher than other substrates). infantile neuronal ceroid lipofuscinosis (INCL) is caused by PPT1 deficiency, and the absence of enz ymatic activity was confirmed with GAP-43 peptide as well as the Po peptide . LA-Nd human neuroblastoma cells overexpressing PPT1. showed increased dep almitoylation of all the peptide substrates, indicating that these peptides are deacylated by PPT1. An amide derivative of a palmitoylated K-Ras pepti de (AcG-palmitoyl diamino propionate-VKIKK) acted as an enzyme pseudosubstr ate and inhibited PPT1 enzyme activity in a dose-dependent manner. The pept ide itself (AcGCVKIKK) did not affect PPT activity. In summary, PPT1 is abl e to hydrolyze a range of cysteinyl peptide sequences found in both neuron- specific and ubiquitous (e.g., G alpha) proteins. The inhibitor of PPT1 act ivity should facilitate the development of a model for INCL and help explai n the neuronal death in this disease. (C) 2000 Wiley-Liss, Inc.