From the Cover: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

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.

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“…Expression plasmids for N13 GAP-43(C3S)-YFP, N27 GalNAc-T-CFP, APT1-Cherry and APT2-Cherry have been previously described [5], [29], [53].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

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“…Expression plasmids for N13 GAP-43-(YFP), GAP-43 full -(YFP) and its acylation mutants, (YFP)-K-Ras C14 , YFP-H-Ras full , N27 GalNAc-T-CFP and N52 Gal-T2-CFP have been previously described [34], [42], [47], [60]. The expression plasmid encoding YFP-H-Ras G12V was performed by directed mutagenesis of YFP-H-Ras full using PCR.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2010

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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.

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“…Ganglioside synthesis from glc-cer depends upon the action of specific glycosyl transferases which rely upon their appropriate localization in the Golgi, and the sphingosine and fatty acid components comprising the ceramide. Evidence for distinct locations is supported by the observation that different glycosyltransferases are found in different complexes in the Golgi [ 31 ] which can be affected by levels of their expression [ 32 ]. Dependency on the ceramide portion reflects the fact that CNS gangliosides lack the fatty acid diversity found in non-CNS glycolipids, containing predominantly stearic acid [ 33 , 34 ], and either a C18 or C20 sphingosine [ 34 , 35 ].…”

Section: Ganglioside Structure and Biosynthesismentioning

confidence: 99%

Gangliosides and Neuroblastomas

Schengrund

2020

IJMS

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The focus of this review is the ganglio-series of glycosphingolipids found in neuroblastoma (NB) and the myriad of unanswered questions associated with their possible role(s) in this cancer. NB is one of the more common solid malignancies of children. Five-year survival for those diagnosed with low risk NB is 90–95%, while that for children with high-risk NB is around 40–50%. Much of the survival rate reflects age of diagnosis with children under a year having a much better prognosis than those over two. Identification of expression of GD2 on the surface of most NB cells led to studies of the effectiveness and subsequent approval of anti-GD2 antibodies as a treatment modality. Despite much success, a subset of patients, possibly those whose tumors fail to express concentrations of gangliosides such as GD1b and GT1b found in tumors from patients with a good prognosis, have tumors refractory to treatment. These observations support discussion of what is known about control of ganglioside synthesis, and their actual functions in NB, as well as their possible relationship to treatment response.

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From the Cover: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Cited by 36 publications

References 8 publications

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

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

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

Gangliosides and Neuroblastomas

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