Abstract
S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.
Highlights
S-acylation is catalyzed by protein acyltransferases (PATs) whereas deacylation requires acyl-protein thioesterases (APTs)
This is based on the necessity to modulate the localization and activity of many important intracellular acylated proteins, several of which are involved in pathological processes, with most of the research in this area having been focused on the H- and N-Ras proteins, as they play a causative role in melanoma, leukemia and cancers of the liver and kidney
Having demonstrated that 2-BP inhibited PAT activity in vivo at a range of concentrations between 25 and 150 mM, we investigated whether the a-brominated fatty acid could perturb the deacylation kinetics of monoacylated growthassociated protein-43 (GAP-43) at the same concentrations
Summary
Fatty-acylated peripheral proteins, such as members of the small G-protein Ras family, the neuronal proteins PSD-95 and growthassociated protein-43 (GAP-43) [1,2,3,4,5], are synthesized in the cytosol and post-tranlationally modified by different lipid moieties [6,7,8], with these modifications governing their membrane association and membrane subdomain segregation, as well as their trafficking, function and stability [9,10]. After the discovery and initial characterization of PATs and APTs, it has become of increasing interest to develop pharmacologic inhibitors for these enzymes This is based on the necessity to modulate the localization and activity of many important intracellular acylated proteins, several of which are involved in pathological processes, with most of the research in this area having been focused on the H- and N-Ras proteins, as they play a causative role in melanoma, leukemia and cancers of the liver and kidney. On the basis of these results, we concluded that 2-BP treatment inhibits the APT1 and APT2 activities both in vitro and in vivo This implies that the kinetics analysis of protein acylation using 2-BP should be carefully interpreted because this drug inhibits protein deacylation, and suggests that the 2-BP moiety can be used as a model for the rational design of new drugs that may be able to modify the oncogenic signaling of acylated proteins Ras), which may lead to the development of new therapies for cancer
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
