RESUMO
Protein S-acylation, also known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, is thought to affect over 12% of the human proteome. S-acylation allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane. This review focuses on the consequences of protein S-acylation on intracellular trafficking and membrane association. We summarize relevant information that illustrates how lipid modification of proteins plays an important role in dictating precise intracellular movements within cells by regulating membrane-cytosol exchange, through membrane microdomain segregation, or by modifying the flux of the proteins by means of vesicular or diffusional transport systems. Finally, we highlight some of the key open questions and major challenges in the field.
Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Cisteína/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipoilação , Palmitatos/metabolismo , Transporte ProteicoRESUMO
S-acylation/deacylation cycles and vesicular transport are critical for an adequate subcellular distribution of S-acylated Ras proteins. H-Ras is dually acylated on cysteines 181 and 184, but it is unknown how these residues individually contribute to H-Ras trafficking. In this study, we characterized the acylation and deacylation rates and membrane trafficking of monoacylated H-Ras mutants to analyze their contributions to H-Ras plasma membrane and endomembrane distribution. We demonstrated that dually acylated H-Ras interacts with acyl-protein thioesterases (APTs) 1 and 2 at the plasma membrane. Moreover, single-acylation mutants of H-Ras differed not only in their subcellular distribution, where both proteins localized to different extents at both the Golgi complex and plasma membrane, but also in their deacylation rates, which we showed to be due to different sensitivities to APT1 and APT2. Fluorescence photobleaching and photoactivation experiments also revealed that 1) although S-acylated, single-acylation mutants are incorporated with different efficiencies into Golgi complex to plasma membrane vesicular carriers, and 2) the different deacylation rates of single-acylated H-Ras influence differentially its overall exchange between different compartments by nonvesicular transport. Taken together, our results show that individual S-acylation sites provide singular information about H-Ras subcellular distribution that is required for GTPase signaling.
Assuntos
Membrana Celular/metabolismo , Genes ras/fisiologia , Transporte Proteico/fisiologia , Acilação , Animais , Células CHO , Linhagem Celular , Membrana Celular/fisiologia , Cricetulus , Cisteína/metabolismo , Complexo de Golgi/metabolismo , Mutação , Proteínas/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismoRESUMO
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.