RESUMO
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
A Personal Health Record (PHR) is a health information repository controlled and managed directly by a patient or his/her custodian, or a person interested in his/her own health. PHR System's adoption and compliance with international standards is foremost important because it can help to meet international, national, regional or institutional interoperability and portability policies. In this paper, an interoperable PHR System for supporting the control of type 2 diabetes mellitus is proposed, which meets the mandatory interoperability requirements proposed in the Personal Health Record System Functional Model standard (ISO 16527). After performing a detailed analysis of different applications and platforms for the implementation of electronic Personal Health Records, the adaptation of the Indivo Health open source platform was completed. Interoperability functions were added to this platform by integrating the Mirth Connect platform. The assessment of the platform's interoperability capabilities was carried out by a group of experts, who verified the interoperability requirements proposed in the ISO 16527 standard.
Assuntos
Diabetes Mellitus Tipo 2/terapia , Registros Eletrônicos de Saúde/normas , Interoperabilidade da Informação em Saúde/normas , Autocuidado/normas , HumanosRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteína GAP-43/metabolismo , Palmitatos/farmacologia , Tioléster Hidrolases/antagonistas & inibidores , Acilação/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Cinética , Tioléster Hidrolases/metabolismoRESUMO
The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.
Assuntos
Galactosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Complexos Multienzimáticos/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Rede trans-Golgi/enzimologiaRESUMO
Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L), and a RING finger matrix (Z) protein. The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (J. C. Casabona et al., J. Virol. 83:7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N-coding sequence, and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that N protein displays two functional domains. By using coimmunoprecipitation studies, VLP incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461 to 489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92 to 119) as a newly recognized molecular determinant of N homotypic interactions.
Assuntos
Arenavirus/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Arenavirus/química , Arenavirus/genética , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Nucleoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Proteína GAP-43/metabolismo , Lisofosfolipase/metabolismo , Tioléster Hidrolases/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Western Blotting , Células CHO , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteína GAP-43/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisofosfolipase/genética , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genéticaRESUMO
GAP-43 (growth-associated protein-43) is a dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys3 and Cys4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together, these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via an Arf6-associated endocytic vesicular pathway.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Endossomos/metabolismo , Proteína GAP-43/metabolismo , Vesículas Transportadoras/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Células CHO , Membrana Celular/genética , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Proteína GAP-43/genética , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Vesículas Transportadoras/genética , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.
Assuntos
Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/fisiologia , Glicoproteínas/metabolismo , Nucleocapsídeo/metabolismo , Proteínas Virais/química , Montagem de Vírus , Sequência de Aminoácidos , Animais , Infecções por Arenaviridae/metabolismo , Arenavirus do Novo Mundo/química , Arenavirus do Novo Mundo/genética , Linhagem Celular , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Dados de Sequência Molecular , Nucleocapsídeo/química , Nucleocapsídeo/genética , Domínios RING Finger , Alinhamento de Sequência , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
K-Ras is a small G-protein, localized mainly at the inner leaflet of the plasma membrane. The membrane targeting signal of this protein consists of a polybasic C-terminal sequence of six contiguous lysines and a farnesylated cysteine. Results from biophysical studies in model systems suggest that hydrophobic and electrostatic interactions are responsible for the membrane binding properties of K-Ras. To test this hypothesis in a cellular system, we first evaluated in vitro the effect of electrolytes on K-Ras membrane binding properties. Results demonstrated the electrical and reversible nature of K-Ras binding to anionic lipids in membranes. We next investigated membrane binding and subcellular distribution of K-Ras after disruption of the electrical properties of the outer and inner leaflets of plasma membrane and ionic gradients through it. Removal of sialic acid from the outer plasma membrane caused a redistribution of K-Ras to recycling endosomes. Inhibition of polyphosphoinositide synthesis at the plasma membrane, by depletion of cellular ATP, resulted in a similar subcellular redistribution of K-Ras. Treatment of cells with ionophores that modify transmembrane potential caused a redistribution of K-Ras to cytoplasm and endomembranes. Ca2+ ionophores, compared to K+ ionophores, caused a much broader redistribution of K-Ras to endomembranes. Taken together, these results reveal the dynamic nature of interactions between K-Ras and cellular membranes, and indicate that subcellular distribution of K-Ras is driven by electrostatic interaction of the polybasic region of the protein with negatively charged membranes.
Assuntos
Membrana Celular/química , Membrana Celular/fisiologia , Eletricidade Estática , Proteínas ras/química , Proteínas ras/metabolismo , Animais , Células CHO , Membrana Celular/genética , Cricetinae , Cricetulus , Humanos , Camundongos , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Frações Subcelulares/fisiologia , Transfecção , Proteínas ras/genéticaRESUMO
Gangliosides are sialic acid-containing glycosphingolipids present on mammalian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell membranes and intracellular destinations have not so far been extensively examined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclonal antibody to GD3, R24. By biochemical techniques and detailed confocal microscopic analysis, we demonstrate that the GD3-R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sensitive to brefeldin A and monensin. Taken together, our results indicate that the GD3-R24 complex is endocytosed in GD3-expressing cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.