Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution

2-bromopalmitate reduces protein deacylation by inhibition of acyl-protein thioesterase enzymatic activities

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

Supporting parents of preschool children in adopting a healthy lifestyle

<p>Abstract</p> <p>Background</p> <p>Childhood obesity is a public health epidemic. In Canada 21.5% of children aged 2–5 are overweight, with psychological and physical consequences for the child and economic consequences for society. Parents often do not view their children as overweight. One way to prevent overweight is to adopt a healthy lifestyle (HL). Nurses with direct access to young families could assess overweight and support parents in adopting HL. But what is the best way to support them if they do not view their child as overweight? A better understanding of parents’ representation of children’s overweight might guide the development of solutions tailored to their needs.</p> <p>Methods/design</p> <p>This study uses an action research design, a participatory approach mobilizing all stakeholders around a problem to be solved. The general objective is to identify, with nurses working with families, ways to promote HL among parents of preschoolers. Specific objectives are to: 1) describe the prevalence of overweight in preschoolers at vaccination time; 2) describe the representation of overweight and HL, as reported by preschoolers’ parents; 3) explore the views of nurses working with young families regarding possible solutions that could become a clinical tool to promote HL; and 4) try to identify a direction concerning the proposed strategies that could be used by nurses working with this population. First, an epidemiological study will be conducted in vaccination clinics: 288 4–5-year-olds will be weighed and measured. Next, semi-structured interviews will be conducted with 20 parents to describe their representation of HL and their child’s weight. Based on the results from these two steps, by means of a focus group nurses will identify possible strategies to the problem. Finally, focus groups of parents, then nurses and finally experts will give their opinions of these strategies in order to find a direction for these strategies. Descriptive and correlational statistical analyses will be done on the quantitative survey data using SPSS. Qualitative data will be analyzed using Huberman and Miles’ (2003) approach. NVivo will be used for the analysis and data management.</p> <p>Discussion</p> <p>The anticipated benefits of this rigorous approach will be to identify and develop potential intervention strategies in partnership with preschoolers’ parents and produce a clinical tool reflecting the views of parents and nurses working with preschoolers’ parents.</p