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1.
Neuroscience ; 155(2): 423-38, 2008 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-18619525

RESUMEN

Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.


Asunto(s)
Barrera Hematoencefálica/fisiología , Circulación Cerebrovascular/fisiología , Plexo Coroideo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Biotinilación , Barrera Hematoencefálica/ultraestructura , Western Blotting , Línea Celular , Plexo Coroideo/irrigación sanguínea , Plexo Coroideo/ultraestructura , Epéndimo/irrigación sanguínea , Epéndimo/metabolismo , Epéndimo/ultraestructura , Perfilación de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Riñón/citología , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Microcirculación/fisiología , Microcirculación/ultraestructura , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ratas , Ratas Sprague-Dawley , Simportadores/genética , Simportadores/metabolismo , Transfección
3.
Stem Cells ; 16 Suppl 2: 21-9, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-11012174

RESUMEN

We have screened a variety of L-amino acid peptide libraries against the extracellular domain of the human thrombopoietin (HuTPO) receptor, c-Mpl. A large number of peptide ligands were recovered and categorized into two families. Peptides from each family compete with the binding of HuTPO and with the binding of peptides from the other familiy. Representative peptides were synthesized and found to activate the full-length HuTPO receptor expressed in Ba/F3 cells to promote proliferation. These peptide families show no apparent homology to the primary sequence of TPO. We have focused our optimization efforts on one of the peptides, a linear 14-mer (IEGPTLRQWLAARA) with an IC50 of 2 nM in a competition binding assay and an EC50 of 400 nM in the proliferation assay. In order to enhance the potency of the compound, we constructed dimeric peptides by linking the carboxy-termini of the 14-mers to a lysine branch. These molecules exhibited slightly higher affinity (0.5 nM) and greatly increased potency (0.1 nM). The EC50 of the dimeric peptide was equivalent to that of the 332 aa form of baculovirus-expressed recombinant HuTPO. As previously shown for the erythropoietin-mimetic peptides, the TPO-mimetic peptides probably activate the TPO receptor by binding and inducing receptor dimerization. This supposition is supported by the observation that covalent dimerization of the peptide enhances its potency by 4,000-fold over that of the monomer. The peptide dimer is also active in stimulating in vitro proliferation of progenitors and maturation of megakaryocytes from human bone marrow, and in promoting an increase in platelet count when administered to normal mice.


Asunto(s)
Proteínas de Neoplasias , Péptidos/química , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/química , Receptores de Citocinas , Trombopoyetina/análogos & derivados , Trombopoyetina/química , Secuencia de Aminoácidos , Animales , Unión Competitiva/fisiología , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Péptidos/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Receptores de Trombopoyetina , Trombopoyetina/farmacología
4.
Science ; 276(5319): 1696-9, 1997 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-9180079

RESUMEN

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.


Asunto(s)
Proteínas de Neoplasias , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas/agonistas , Receptores de Citocinas , Trombopoyetina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Plaquetas/citología , División Celular , Línea Celular , Células Cultivadas , Secuencia de Consenso , Dimerización , Eritropoyetina/farmacología , Hematopoyesis/efectos de los fármacos , Humanos , Megacariocitos/citología , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/metabolismo , Péptidos/farmacología , Recuento de Plaquetas , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Trombopoyetina , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Trombopoyetina/farmacología , Transfección
5.
Proc Natl Acad Sci U S A ; 93(14): 7381-6, 1996 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-8693002

RESUMEN

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.


Asunto(s)
Interleucina-1/metabolismo , Péptidos/química , Péptidos/farmacología , Receptores de Interleucina-1/antagonistas & inhibidores , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Células Cultivadas , Cartilla de ADN , Bases de Datos Factuales , Dinoprostona/metabolismo , Receptores ErbB/biosíntesis , Escherichia coli , Haplorrinos , Humanos , Interleucina-1/farmacología , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Péptidos/síntesis química , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptores de Interleucina-1/biosíntesis , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Piel/efectos de los fármacos , Piel/inmunología , Piel/metabolismo , Bazo/inmunología
6.
J Biol Chem ; 270(36): 21129-36, 1995 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-7545665

RESUMEN

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adhesión Celular/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Selectina E , Femenino , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis , Neutrófilos/citología , Péptidos/síntesis química , Péptidos/farmacología , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología
7.
Cell ; 75(4): 717-28, 1993 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-7902213

RESUMEN

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.


Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Oligopéptidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Chaperoninas , Secuencia de Consenso , Retículo Endoplásmico/metabolismo , Activación Enzimática , Biblioteca de Genes , Técnicas In Vitro , Inovirus/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Unión Proteica , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad
8.
Anal Biochem ; 204(2): 357-64, 1992 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-1443536

RESUMEN

Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kd's greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.


Asunto(s)
Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Cápside/genética , Colifagos/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Vectores Genéticos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ligandos , Datos de Secuencia Molecular , Péptidos/inmunología , betaendorfina/inmunología
9.
Proc Natl Acad Sci U S A ; 87(16): 6378-82, 1990 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-2201029

RESUMEN

We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.


Asunto(s)
Colifagos/genética , Escherichia coli/genética , Biblioteca de Genes , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/genética , Ligandos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Péptidos
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