RESUMO
Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renais Coletores/citologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.
Assuntos
Domínios Proteicos Ricos em Prolina , Mapeamento de Interação de Proteínas , Animais , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Drosophila melanogaster/metabolismo , Esterificação , Imunofluorescência , Humanos , Cinética , Ligantes , Proteínas dos Microfilamentos/química , Modelos Moleculares , Peso Molecular , Peptídeos/química , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Pseudópodes , Fibras de Estresse/metabolismo , Zixina/químicaRESUMO
Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Glicosídeos/química , Lipídeos/química , Peptídeos/química , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Nanopartículas/química , Saponaria/química , TransfecçãoRESUMO
The human lutropin/choriogonadotropin receptor (hLHR) for the gonadotropic hormones human luteinizing hormone (hLH; lutropin) and human choriogonadotropin (hCG) is crucial for normal sexual development and fertility. We aimed to unravel differences between the two hLHR hormones in molecular activation mechanisms at hLHR. We utilized a specific hLHR variant that lacks exon 10 (hLHR-delExon10), which maintains full cAMP signaling by hCG, but decreases hLH-induced receptor signaling, resulting in a pathogenic phenotype. Exon 10 encodes 27 amino acids within the hinge region, which is an extracellular segment that is important for signaling and hormone interaction. Initially, we assumed that the lack of exon 10 might disturb intermolecular trans-activation of hLH, a mechanism that has been reported for hCG at hLHR. Coexpression of signaling-deficient hLHR and binding-deficient hLHR can be used to examine the mechanisms of receptor signaling, in particular intermolecular cooperation and intramolecular cis-activation. Therefore, hLHR-delExon10 was combined with the hLHR Lys605âGlu mutant, in which signaling is abolished, and the hLHR mutant Cys131âArg, in which binding is deficient. We found that hCG signaling was partially rescued, indicating trans-activation. However, the hLH signal could not be restored via forced trans-activation with any construct. Fluorescence cross-correlation spectroscopy detected oligomerization in all combinations, indicating that these functional differences cannot be explained by monomerization of hLHR-delExon10. Thus, our data demonstrate not only that the different behavior of hLH at hLHR-delExon10 is unlikely to be related to modified intermolecular receptor activation, but also that hLH may exclusively stimulate the targeted hLHR by cis-activation, whereas hCG is also capable of inducing trans-activation.
Assuntos
Gonadotropina Coriônica/metabolismo , Hormônio Luteinizante/metabolismo , Receptores do LH/metabolismo , Substituição de Aminoácidos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Éxons , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estrutura Quaternária de Proteína , Receptores do LH/química , Receptores do LH/genética , Deleção de Sequência , Transdução de Sinais , Ativação TranscricionalRESUMO
Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Humanos , Imunotoxinas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponinas/química , Saporinas , Trastuzumab , Triterpenos/químicaRESUMO
Macromolecular interaction of protein toxins with certain plant triterpenoids holds potential for application in tumor therapy. The ability of only certain saponins to enhance the endosomal escape of toxins specifically in tumor cells was evaluated and set into correlation with the electrophoretic mobility. Saponins from Saponaria officinalis Linn, were selected as a lead to understand this evolutionarily conserved principle in detail. Agarose gel electrophoresis was utilized to procure pure saponin fractions with different electrophoretic mobility, which were tested for their ability to enhance the toxicity by live cell monitoring. Five fractions (SOG1-SOG5) were isolated with a relative electrophoretic mobility of (-0.05, 0.41, 0.59, 0.75 and 1.00) and evaluated using thin layer chromatography, HPLC, and mass spectroscopic analysis. Cytotoxicity experiments revealed highest effectiveness with SOG3. Live cell imaging experiments with SOG3 revealed that this saponin with a specific REM of 0.59 could assist in the lyso/endosomal release of the toxic payload without affecting the integrity of plasma membrane and could lead to the induction of apoptosis. This charge dependent enhancement was also found to be highly specific to type I ribosome inactivating proteins compared to bacterial toxins. Charge interaction of plant toxins and saponins with tumor cells, plays a major role in toxin specific modulation of response. The finding opens up newer ways of finding protein saponin interaction conserved evolutionarily and to test their role in endosomal escape of therapeutic molecules.
Assuntos
Saponinas/química , Toxinas Biológicas/química , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Camundongos , Extratos Vegetais/química , Saponaria/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.
Assuntos
Aquaporina 2/metabolismo , Benzodioxóis/farmacologia , Benzofuranos/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/fisiologia , Fluconazol/farmacologia , Complexo de Golgi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Microscopia de Fluorescência , Fosforilação , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.
Assuntos
Ativação Enzimática/fisiologia , GTP Fosfo-Hidrolases/química , Proteínas de Ligação ao GTP/química , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica , Linfócitos T/metabolismo , Calorimetria , Linhagem Celular , Cristalização , Dimerização , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Hidrólise , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , UltracentrifugaçãoRESUMO
Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Dextranos/química , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dextranos/síntese química , Dextranos/farmacologia , Relação Dose-Resposta a Droga , Eletroporação , Células HeLa , Humanos , Células Jurkat , Maleimidas/química , Microscopia Confocal , Oligopeptídeos/química , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/farmacologia , Espectrometria de FluorescênciaAssuntos
Luz , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/química , Peptídeos/farmacologia , Animais , Ligantes , Camundongos , Conformação Molecular , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K(+) and one Cl(-), as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K(+) ions in the presence of a transepithelial K(+) gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P(f) by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K(+)-H(+) exchanger nigericin, indicating that KCC1 affects water transport solely by K(+) extrusion. Intracellular K(+) retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.
Assuntos
Aquaporina 2/metabolismo , Células Epiteliais/metabolismo , Reologia , Simportadores/metabolismo , Água/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Ácidos Carboxílicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Indenos/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Túbulos Renais Coletores/citologia , Modelos Biológicos , Nigericina/farmacologia , Osmose/efeitos dos fármacos , Ratos , Ratos Wistar , Reologia/efeitos dos fármacos , Solventes , Simportadores/antagonistas & inibidores , Cotransportadores de K e Cl-RESUMO
Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.
Assuntos
Aquaporina 2/metabolismo , Arginina Vasopressina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Colforsina , AMP Cíclico/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Ratos , Transcrição GênicaRESUMO
The aim of this study was to investigate the effect of liposomal membrane properties on cellular uptake and transcytosis across a tight Madin-Darby canine kidney (MDCK) cell barrier in vitro. More than 25 small vesicles were prepared by lipid film hydration/extrusion to generate small unilamellar vesicles. The fluorescence marker calcein was encapsulated to mimic hydrophilic drug transport. Marker uptake by MDCK cells seems to be mediated by different mechanisms for the liposomes used. It was mainly depending on membrane fluidity and vesicle charge. Liposomes L2 with a positive charge (325 +/- 3 pmol/well) and vesicles L3 containing the helper lipid dioleylphosphatidylethanolamine (DOPE) in their membrane (216 +/- 42 pmol/well) were taken up to the most. Selected liposomes were tested for their transcytotic transport across a MDCK monolayer. Liposomes L4 containing equimolar DOPE and octadecyl-1,1-dimethylpiperidin-1-ium-4-yl phosphate (OPP) were the most efficient vesicles for transcellular transport resulting in 808 +/- 30 pmol calcein/cm(2) in the basal medium (28.1% of total liposomal marker added). Transcytosis was positively correlated with membrane fluidity in the outer part of the bilayer, as electron paramagnetic resonance measurements revealed. We expect that an increase in membrane fluidity of vesicles should also improve the restricted transport of hydrophilic drugs across the blood-brain barrier.
Assuntos
Portadores de Fármacos/química , Células Epiteliais/metabolismo , Fluidez de Membrana , Nanopartículas/química , Lipossomas Unilamelares/química , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cães , Portadores de Fármacos/farmacocinética , Composição de Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Células Epiteliais/efeitos dos fármacos , Fluoresceínas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Microscopia Confocal , Microscopia de Fluorescência , Lipossomas Unilamelares/farmacocinéticaRESUMO
A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Motivos de Aminoácidos/fisiologia , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
A 12-mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell-penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell-penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA-peptide conjugates to be not primarily related to the cell-penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA-peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors.
Assuntos
Permeabilidade da Membrana Celular , Ácidos Nucleicos Peptídicos/farmacologia , Ácidos Nucleicos Peptídicos/farmacocinética , Peptídeos/química , Animais , Linhagem Celular , Eletroforese Capilar , Humanos , Microscopia Confocal , Ácidos Nucleicos Peptídicos/química , Espectrometria de FluorescênciaRESUMO
Ketalization of the biomolecule progesterone with (6-bromo-7-hydroxycoumarin-4-yl)ethane-1,2-diol gives the photolabile progesterone derivatives 3 and 4. These compounds display dramatically reduced bioactivity and release progesterone upon irradiation with UV/vis or IR light. In particular, 4 can be used to perform concentration-jump experiments with high temporal and spatial resolution that allows one to study elegantly the mechanisms of rapid nongenomic cellular events evoked by progesterone. The usefulness of 4 was demonstrated by measurement of changes in swimming behavior of single human sperm caused by progesterone-induced Ca(2+) influx in the sperm flagellum.
Assuntos
Processos Fotoquímicos , Progesterona/análogos & derivados , Progesterona/farmacologia , Álcoois , Cálcio/metabolismo , Flagelos/efeitos dos fármacos , Humanos , Luz , Masculino , Progesterona/química , Progesterona/efeitos da radiação , Projetos de Pesquisa , Espermatozoides/efeitos dos fármacosRESUMO
Tissue morphogenesis and cell sorting are major forces during organ development. Here, we characterize the process of tissue morphogenesis within the zebrafish lateral line primordium, a migratory sheet of cells that gives rise to the neuromasts of the posterior lateral line organ. We find that cells within this epithelial tissue constrict actin-rich membranes and enrich apical junction proteins at apical focal points. The coordinated apical membrane constriction in single Delta D-positive hair cell progenitors and in their neighbouring prospective support cells generates cellular rosettes. Live imaging reveals that cellular rosettes subsequently separate from each other and give rise to individual neuromasts. Genetic analysis uncovers an involvement of Lethal giant larvae proteins in the maturation of apical junction belts during cellular rosette formation. Our findings suggest that apical constriction of cell membranes spatially confines regions of strong cell-cell adhesion and restricts the number of tightly interconnected cells into cellular rosettes, which ensures the correct deposition of neuromasts during morphogenesis of the posterior lateral line organ.
Assuntos
Adesão Celular/fisiologia , Sistema da Linha Lateral , Morfogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Polaridade Celular , Forma Celular , Peptídeos e Proteínas de Sinalização Intracelular , Sistema da Linha Lateral/citologia , Sistema da Linha Lateral/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The tight junctions-associated MAGUK protein nagie oko is closely related to Drosophila Stardust, mouse protein associated with lin-seven 1 (Pals1), and human MAGUK p55 subfamily member 5 (Mpp5). As a component of the evolutionarily conserved Crumbs protein complex, nagie oko is essential for the maintenance of epithelial cell polarity. Here, we show that nagie oko contains a predicted nuclear export and two conserved nuclear localization signals. We find that loss of the predicted nuclear export signal results in nuclear protein accumulation. We show that nagie oko nuclear import is redundantly controlled by the two nuclear localization signals and the evolutionarily conserved region 1 (ECR1), which links nagie oko with Par6-aPKC. Finally, deletion forms of nagie oko that lack nuclear import and export signals complement several nagie oko mutant defects in cell polarity and epithelial integrity. This finding provides an entry point to potentially novel and unknown roles of this important cell polarity regulator.
Assuntos
Núcleo Celular/metabolismo , Guanilato Ciclase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Western Blotting , Polaridade Celular/genética , Biologia Computacional , Células Epiteliais/metabolismo , Guanilato Ciclase/genética , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Transporte Proteico , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Adenilil Ciclases/análise , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Arginina Vasopressina/farmacologia , Linhagem Celular , Cloroquina/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Lisossomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos , Estrutura Terciária de Proteína , Receptores de Vasopressinas/genética , Frações Subcelulares/metabolismo , Transfecção , Ubiquitinação , Proteína com ValosinaRESUMO
The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18delta and PKA. We show that AKAP18delta acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca(2+) re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca(2+) re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.