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1.
Eur J Pharm Sci ; 24(5): 525-37, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15784342

RESUMEN

Polymethacrylates are vinyl-based polymers that are used for DNA transfection. Cationic polymethacrylates efficiently condense DNA by forming inter-polyelectrolyte complexes. Their use for DNA transfection is, however, limited due to their low ability to interact with membranes. In order to increase their transfection efficiency, we combined polymethacrylates with Penetratin, a 16-residue water-soluble peptide that internalises into cells through membrane translocation. DNA condensation was assessed using physicochemical methods, while transfection efficiency and cellular internalisation were studied using Cos-1 cells. Agarose gel electrophoresis retardation, ethidium bromide exclusion tests and dynamic light scattering measurements showed that the stability of the polymethacrylate-DNA complexes is not affected by addition of Penetratin. Transfection efficiency of polymethacrylate-DNA complexes into Cos-1 cells increased by addition of Penetratin and was higher than that of polyethylenimine (PEI)-DNA complexes and comparable to Lipofectamine. Confocal microscopy and flow cytometry indicated that Penetratin mainly enhances endolysosomal escape polymethacrylate-DNA complexes and increases their cellular uptake. Since the cellular toxicity of polymethacrylate-DNA-Penetratin complexes remains low, especially compared to PEI, this transfection system opens new perspectives for gene therapy.


Asunto(s)
Proteínas Portadoras/farmacología , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Terapia Genética , Ácidos Polimetacrílicos/administración & dosificación , Animales , Células COS , Péptidos de Penetración Celular , Transfección
2.
J Biol Chem ; 279(36): 37779-88, 2004 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-15218032

RESUMEN

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Quinasa de la Caseína II/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Colesterol/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Fosfolípidos/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
3.
Eur J Biochem ; 271(6): 1187-97, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15009197

RESUMEN

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.


Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Dobles de Lípidos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Péptidos de Penetración Celular , Dicroismo Circular , Perros , Yema de Huevo/química , Fluoresceínas/química , Fluoresceínas/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Membrana Dobles de Lípidos/química , Membranas/química , Membranas/metabolismo , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia/métodos , Triptófano/química , Triptófano/metabolismo
4.
Biomacromolecules ; 5(2): 379-88, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15002997

RESUMEN

Recently, we have shown that polymethacrylates containing imidazole side groups (HYMIMMA) or acid functions (MA), which have similar buffering properties as polyethyleneimine, were not able to transfect Cos-1 cells, whereas polymers containing only tertiary amines (DMAEMA) do transfect Cos-1 cells (Dubruel, P. et al. Eur. J. Pharm. Sci. 2003, 18 (3-4), 211-220). In the present work, we investigated to what extent the differences in transfection activity are related to differences in cellular internalization and/or subcellular localization. Therefore, we synthesized a series of polymethacrylates containing primary amine functions, used for the coupling of the fluorescent Oregon Green probe. The polymers containing acid functions were labeled with an amine containing fluorescein derivative (5-aminomethyl)fluorescein hydrochloride. It is demonstrated that the endosomal release of the MA and HYMIMMA-based complexes might be the limiting step in the gene transfer process in Cos-1 cells.


Asunto(s)
Materiales Biocompatibles/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Ácidos Polimetacrílicos/metabolismo , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/toxicidad , Tampones (Química) , Células COS , Cationes , Chlorocebus aethiops , Endosomas/efectos de los fármacos , Endosomas/genética , Endosomas/metabolismo , Técnicas de Transferencia de Gen , Microscopía Confocal , Ácidos Polimetacrílicos/síntesis química , Ácidos Polimetacrílicos/toxicidad , Transfección/métodos
5.
Protein Expr Purif ; 35(1): 102-10, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15039072

RESUMEN

The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Nucleótidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación
6.
Biomacromolecules ; 4(5): 1177-83, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12959581

RESUMEN

Cationic polymers, such as poly-l-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it's not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes.


Asunto(s)
Portadores de Fármacos/farmacología , Terapia Genética/métodos , Ácido Poliglutámico/farmacología , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/normas , Células COS , Bovinos , ADN , Portadores de Fármacos/síntesis química , Portadores de Fármacos/normas , Estabilidad de Medicamentos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemaglutinación/efectos de los fármacos , Hemólisis/efectos de los fármacos , Ensayo de Materiales , Ácido Poliglutámico/síntesis química , Transfección/métodos , Transfección/normas
7.
Eur J Pharm Sci ; 18(3-4): 211-20, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12659932

RESUMEN

We report here the physicochemical and biological evaluation of a series of polymethacrylates with side groups of different pK(a) values, such as tertiary amines, pyridine groups, acid functions and imidazole groups as synthetic vectors for gene delivery. The ability of the different polymers to condense DNA was studied by ethidium bromide exclusion tests and agarose gel electrophoresis. The results show that all polymers are able to condense DNA. Both the molecular weight and the chemical composition of the polymers have an influence on the DNA condensation process. Furthermore, the biological properties of the polymer-DNA complexes were investigated, including their haemolytic activity, cytotoxicity and in vitro transfection efficiency. Complexes based on polymers containing only tertiary amines, have a transfection efficiency similar to that of poly(ethyleneimine) (PEI). Polymers containing pyridine groups have a reduced transfection efficiency compared to polymers containing tertiary amines. Introduction of imidazole groups or acid functions results in a loss of the transfection efficiency of the corresponding complexes with DNA. In general, the viability of cells incubated with complexes based on the polymethacrylates is higher than with PEI. Polymers with high transfection efficiency induce erythrocyte lysis.


Asunto(s)
Vectores Genéticos/síntesis química , Ácidos Polimetacrílicos , Transfección/métodos , Animales , Células COS , Cationes , Bovinos , Fenómenos Químicos , Química Física , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos/métodos , Vectores Genéticos/administración & dosificación , Hemólisis/efectos de los fármacos , Hemólisis/fisiología , Ácidos Polimetacrílicos/administración & dosificación , Ácidos Polimetacrílicos/química , Transfección/tendencias
8.
J Mol Biol ; 325(2): 259-74, 2003 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-12488094

RESUMEN

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/clasificación , Transportadoras de Casetes de Unión a ATP/genética , Células Procariotas/química , Conformación Proteica , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Bases de Datos Factuales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Células Procariotas/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo
9.
Eur J Biochem ; 269(12): 2918-26, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12071955

RESUMEN

Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild-type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F-penetratin variant was larger than for the W48F-penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become alpha helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild-type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to alpha helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.


Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Triptófano/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Péptidos de Penetración Celular , Dicroismo Circular , Fluorescencia , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Estructura Secundaria de Proteína , Triptófano/química
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