RESUMO
Reversible S-palmitoylation of cysteine residues critically controls transient membrane tethering of peripheral membrane proteins. Little is known about how the palmitoylation machinery governs their defined localization and function. We monitored the spatially resolved reaction dynamics and substrate specificity of the core mammalian palmitoylation machinery using semisynthetic substrates. Palmitoylation is detectable only on the Golgi, whereas depalmitoylation occurs everywhere in the cell. The reactions are not stereoselective and lack any primary consensus sequence, demonstrating that substrate specificity is not essential for de-/repalmitoylation. Both palmitate attachment and removal require seconds to accomplish. This reaction topography and rapid kinetics allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus. Unidirectional secretion ensures the maintenance of a proper steady-state protein distribution between the Golgi and the plasma membrane, which are continuous with endosomes. This generic spatially organizing system differs from conventional receptor-mediated targeting mechanisms and efficiently counteracts entropy-driven redistribution of palmitoylated peripheral membrane proteins over all membranes.
Assuntos
Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipoilação , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.
Assuntos
Inibidores Enzimáticos/farmacologia , Propiolactona/análogos & derivados , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/química , Proteínas ras/fisiologia , Animais , Linhagem Celular , Cães , Regulação para Baixo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Rim/efeitos dos fármacos , Rim/fisiologia , Ligantes , Lipase/química , Lipase/metabolismo , Lipoilação/efeitos dos fármacos , Modelos Moleculares , Propiolactona/síntese química , Propiolactona/química , Propiolactona/farmacologia , Conformação Proteica , Proto-Oncogene Mas , Transdução de Sinais , Estômago/enzimologia , Tioléster Hidrolases/genética , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
A detailed study on the solid-phase synthesis of lipidated peptides of the Ras family employing the Ellman sulfonamide linker is reported. Using the C-terminal N-Ras sequence, critical issues such as lipidated amino acid resin loading, peptide elongation in the presence of labile groups and optimized conditions for release of the peptides were investigated. A versatile methodology for the synthesis of peptides with diverse lipid motifs and C-terminal methyl esters has accordingly been established.
Assuntos
Peptídeos/química , Peptídeos/síntese química , Sulfonamidas/química , Proteínas ras/química , Sequência de Aminoácidos , Aminoácidos/química , Lipídeos/química , Estrutura Molecular , Peptídeos/genética , Proteínas ras/genéticaRESUMO
Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.