2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

Maria P. Pedro, Aldo Alejandro Vilcaes, Vanesa M. Tomatis, Rafael G. Oliveira, Guillermo A. Gomez, Jose L. Daniotti

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere75232
JournalPLoS One
Volume8
Issue number10
DOIs
StatePublished - Oct 2 2013
Externally publishedYes

Fingerprint

Acylation
acylation
Proteins
proteins
Palmitates
Membranes
acyltransferases
palmitates
Lipoylation
Palmitoyl Coenzyme A
GAP-43 Protein
Kinetics
Enzyme Inhibitors
Substrates
Post Translational Protein Processing
2-bromopalmitate
kinetics
Human Activities
Machinery
Cysteine

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities. / Pedro, Maria P.; Vilcaes, Aldo Alejandro; Tomatis, Vanesa M.; Oliveira, Rafael G.; Gomez, Guillermo A.; Daniotti, Jose L.

In: PLoS One, Vol. 8, No. 10, e75232, 02.10.2013.

Research output: Contribution to journalArticle

Pedro, Maria P. ; Vilcaes, Aldo Alejandro ; Tomatis, Vanesa M. ; Oliveira, Rafael G. ; Gomez, Guillermo A. ; Daniotti, Jose L. / 2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities. In: PLoS One. 2013 ; Vol. 8, No. 10.
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