RESUMEN
The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and beta-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrP(Sc)) form of the protein and its amyloidic transformation.
Asunto(s)
Membrana Celular/metabolismo , Priones/química , Priones/metabolismo , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Péptidos/química , Proteínas PrPSc/metabolismo , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
A diversity of cell-penetrating peptides (CPPs), is known, but so far the only common denominator for these peptides is the ability to gain cell entry in an energy-independent manner. The mechanism used by CPPs for cell entry is largely unknown, and data comparing the different peptides are lacking. In order to gain more information about the cell-penetrating process, as well as to quantitatively compare the uptake efficiency of different CPPs, we have studied the cellular uptake and cargo delivery kinetics of penetratin, transportan, Tat (48-60) and MAP (KLAL). The respective CPPs (labelled with the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pentapeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a disulfide bond. The cellular uptake of the cargo is registered as an increase in fluorescence intensity when the disulfide bond of the CPP-S-S-cargo construct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48-60) and, last, penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48-60) and, last, penetratin. Since some CPPs have been found to be toxic at high concentration, we characterized the influence of CPPs on cellular 2-[(3)H]deoxyglucose-6-phosphate leakage. Measurements on this system show that the membrane-disturbing potential appears to be correlated with the hydrophobic moment of the peptides. In summary, the yield and kinetics of cellular cargo delivery for four different CPPs has been quantitatively characterized.
Asunto(s)
Permeabilidad de la Membrana Celular , Portadores de Fármacos , Péptidos/química , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Proteínas Portadoras/química , Péptidos de Penetración Celular , Cistina/química , Fluorescencia , Galanina , Humanos , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Recombinantes de Fusión/química , Células Tumorales Cultivadas , Tirosina/química , Venenos de AvispasAsunto(s)
Proteínas Portadoras/metabolismo , Portadores de Fármacos/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Células COS , Galanina , Proteínas Fluorescentes Verdes , Transporte de Proteínas , Estreptavidina/metabolismo , Venenos de Avispas , Xantenos/metabolismoRESUMEN
It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Proteínas de Unión al GTP Heterotriméricas/química , Receptores de Glucagón/química , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/química , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Toxina del Cólera , AMP Cíclico/biosíntesis , Activación Enzimática/efectos de los fármacos , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP , Receptor del Péptido 1 Similar al Glucagón , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Toxina del Pertussis , Unión Proteica , Receptores de Glucagón/genética , Transducción de Señal , Factores de Virulencia de BordetellaRESUMEN
Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.
Asunto(s)
Proteínas Portadoras/síntesis química , Portadores de Fármacos , Péptidos/síntesis química , Péptidos/farmacocinética , Proteínas Recombinantes de Fusión/síntesis química , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Péptidos de Penetración Celular , Galanina , Humanos , Melanoma , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Proteínas Recombinantes de Fusión/farmacocinética , Células Tumorales Cultivadas , Venenos de AvispasRESUMEN
Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.
Asunto(s)
Membrana Celular/química , Fosfolípidos/química , Proteínas Recombinantes de Fusión/química , Alcoholes , Secuencia de Aminoácidos , Permeabilidad de la Membrana Celular , Diseño de Fármacos , GTP Fosfohidrolasas/química , Galanina , Humanos , Radioisótopos de Yodo , Membrana Dobles de Lípidos/química , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Células Tumorales Cultivadas , Venenos de Avispas , AguaRESUMEN
The established view in cellular biology dictates that the cellular internalization of hydrophilic macromolecules can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromolecules with molecular weights several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.
Asunto(s)
Proteínas Portadoras/farmacocinética , Permeabilidad de la Membrana Celular , Péptidos/farmacocinética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismoRESUMEN
PNA is a nucleic acid analog with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units (figure 1). The purine or pyrimidine bases are linked to the each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid (4). PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules (5-7). The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks (8). These properties of PNA oligomers suggest that they could potentially serve as efficient antisense or antigene reagents. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional (9) and translational level (10,11), and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells (12). However, contrary to the "normal" nucleic acid analogs, PNA oligomers are not efficiently delivered into the cytoplasm of the cell, and until recently this has hindered the application of PNA oligomers as antisense reagents. In this work we summarize some recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.
Asunto(s)
ADN sin Sentido/uso terapéutico , Terapia Genética/métodos , Ácidos Nucleicos de Péptidos/farmacocinética , Ácidos Nucleicos de Péptidos/uso terapéutico , Animales , Proteínas Portadoras/farmacocinética , ADN sin Sentido/farmacocinética , Liposomas/farmacocinéticaRESUMEN
Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Proteínas de Unión al GTP/metabolismo , Insulina/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Venenos de Avispas/farmacología , Animales , Membrana Celular/metabolismo , GTP Fosfohidrolasas/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/efectos de los fármacos , Glucosa/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Péptidos/síntesis química , Péptidos/metabolismo , Péptidos/farmacología , Toxina del Pertussis , Unión Proteica , Ratas , Ratas Wistar , Receptores de Vasopresinas/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Vasopresinas/metabolismo , Vasopresinas/farmacología , Factores de Virulencia de Bordetella/farmacología , Venenos de Avispas/metabolismoRESUMEN
Ca2+ efflux, Ca(2+)-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca2+ release over a range of concentrations from 0.3-10 microM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca(2+)-ATPases and directly activate the ryanodine receptor (RyR) to release Ca2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP3 receptor (InsP3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca(2+)-mobilising action of MP and chimeric constructs.
Asunto(s)
Calcio/metabolismo , Venenos de Avispas/farmacología , Secuencia de Aminoácidos , Permeabilidad de la Membrana Celular , Cerebelo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Datos de Secuencia Molecular , Péptidos , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.
Asunto(s)
Proteínas Nucleares , Dolor/fisiopatología , Ácidos Nucleicos de Péptidos/metabolismo , Receptores de Neuropéptido/metabolismo , Transducción de Señal , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Secuencia de Bases , Regulación hacia Abajo , Femenino , Galanina , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patología , Melanoma/fisiopatología , Datos de Secuencia Molecular , Dolor/metabolismo , Fragmentos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptor de Galanina Tipo 1 , Receptores de Galanina , Receptores de Neuropéptido/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Médula Espinal/metabolismo , Células Tumorales Cultivadas , Venenos de AvispasRESUMEN
Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.