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1.
Methods Mol Biol ; 524: 77-86, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19377938

RESUMO

The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple "library" approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin alpha -subunit with four different proteases and detected antibody binding to fragments by Western blot. Using this approach, the epitope for this antibody was localized within the amino-terminal 17 residues of transducin alpha -subunit.


Assuntos
Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos/métodos , Epitopos/análise , Fragmentos de Peptídeos/imunologia , Peptídeo Hidrolases/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Epitopos/imunologia , Humanos , Hidrólise , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Transducina/química , Transducina/imunologia
2.
Med Chem ; 4(4): 298-308, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18673140

RESUMO

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, exerts its physiological effects by binding and activating specific G protein-coupled receptors, named ET(A) and ET(B). An unique property of ET-1 is its ability to bind almost irreversibly to its receptors. Aspirin and salicylic acid (SA) are allosteric inhibitors of ET-1 binding to ET(A) receptors. Dihalogenated derivatives of SA have been identified as more potent allosteric inhibitors than aspirin. In this study, disubstituted benzohydroxamic acid, benzaldoximes and dihalosalicylic acid dimers were synthesized and tested as inhibitors of [(125)I]ET-1 binding to ET(A) receptors in rat embryonic cardiomyocyte (H9c2 cell) membranes. Some dihalosalicylic acid dimers 2h showed good inhibitory activity, the most active compounds are the hydroxamic acids derived from anthranilic acid. Among these compounds, the 3, 5-diiodo-2-aminobenzohydroxamic acid e compound 2a is three-folds more potent as inhibitor of [(125I)] ET-1 binding to ET(A) receptors than the 3; 5-diiodosalicylic acid reported in literature. Most aryl aldoximes in this study were biologically inactive as inhibitors of [(125I)] ET-1 binding to ET(A) receptors.


Assuntos
Antagonistas do Receptor de Endotelina A , Endotelina-1/metabolismo , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Oximas/química , Oximas/farmacologia , Receptor de Endotelina A/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Ácidos Hidroxâmicos/síntese química , Radioisótopos do Iodo , Estrutura Molecular , Oximas/síntese química , Ratos , Relação Estrutura-Atividade
3.
J Recept Signal Transduct Res ; 28(3): 265-83, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18569527

RESUMO

In cardiomyocytes, certain extracellular stimuli that activate heterotrimeric G protein-coupled receptors (GPCRs) can induce hypertrophy by regulating gene expression and increasing protein synthesis. We investigated if rat embryonic cardiomyocytes (H9c2) underwent variations in the expression levels and subcellular distribution of key components of GPCR-activated signaling pathways during endothelin-1 (ET-1)-induced hypertrophic response. A significant increase of p115RhoGEF protein level was evident in ET-1-treated cells. Real-time quantitative PCR showed RhoGEF mRNA levels were significantly increased. Inhibition of the Rho-associated kinase (ROCK) caused a significant decrease of p115RhoGEF protein in the nuclear fraction, whereas an inhibitor of PKC induced a redistribution of the protein between membrane/organelle and nuclear fractions. The ROCK inhibitor also decreased H9c2 cell hypertrophic response. These results indicate that ROCK and its downstream target molecules, which are involved in inducing the hypertrophic response, are also implicated in signaling the up-regulation of the p115RhoGEF protein.


Assuntos
Embrião de Mamíferos/citologia , Endotelina-1/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipertrofia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Subunidades Proteicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
4.
J Med Chem ; 50(7): 1458-64, 2007 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-17348636

RESUMO

A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.


Assuntos
Proteínas de Transporte/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Membranas Artificiais , Fragmentos de Peptídeos/química , Proteínas/química , Peptídeos Penetradores de Células , Dicroísmo Circular , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Micelas , Microscopia de Fluorescência , Modelos Moleculares , Peptídeos , Permeabilidade , Fosforilcolina/análogos & derivados , Dodecilsulfato de Sódio , Eletricidade Estática , Tensoativos
5.
Mol Pharmacol ; 69(3): 727-36, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16332984

RESUMO

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.


Assuntos
Adenosina-5'-(N-etilcarboxamida)/antagonistas & inibidores , Membrana Celular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Membrana/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células , AMP Cíclico/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Isoproterenol/farmacologia , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Células PC12 , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos , Permeabilidade , Conformação Proteica , Proteínas , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
6.
Haematologica ; 90(7): 988-90, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15996942

RESUMO

The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases , Estaurosporina/farmacologia
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