RESUMO
PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.
Assuntos
Proteínas de Transporte , Domínios PDZ , Ligação Proteica , Receptor Nicotínico de Acetilcolina alfa7 , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteínas de Transporte/metabolismo , Ligação Proteica/fisiologia , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Sítios de Ligação/fisiologiaRESUMO
Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.
Assuntos
Carboidratos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Acetilglucosamina/farmacologia , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Fungos/ultraestrutura , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Fusarium/ultraestrutura , Glucosamina/farmacologia , Glucose/farmacologia , Ligação Competitiva/fisiologia , Microscopia Eletrônica , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Sacarose/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologiaRESUMO
Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine. (AU)
Assuntos
RESEARCH SUPPORT, NON-U.S. GOVT , Ligação Competitiva/efeitos dos fármacos , Carboidratos/farmacologia , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Acetilglucosamina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Fungos/ultraestrutura , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Fusarium/ultraestrutura , Glucosamina/farmacologia , Glucose/farmacologia , Microscopia Eletrônica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Sacarose/farmacologiaRESUMO
Both hydropathy plot and in vitro translation results predict the topology of SR-BI; the receptor is an integral membrane protein of 509 amino acids, consisting of a short cytoplasmic N-terminus of 9 amino acids followed by a first transmembrane domain of 22 amino acids, the extracellular domain of 408 amino acids, the second transmembrane domain of 22 amino acids, and the cytoplasmic C-terminus of 47 amino acids. The immunoblot of rBBMV in the presence or absence of pAb589 peptide antigen (the C-terminal 22 amino acid residues of SR-BI) confirmed that the bands at apparent molecular weight of 140 and 210 kDa are SR-BI related protein which might be multimeric forms of SR-BI. 125I apo A-I overlay analysis showed that SR-BI can bind to its ligand, apo A-I, only when it is thoroughly matured - glycosylated and dimerized. The antibody which was generated against extracellular domain of SR-BI (pAb230) not only prevented 125I-labeled apo A-I from binding to 140 kDa band but also inhibited the esterified cholesterol uptake of rabbit BBMV with its IC50 value of 40 microgram/ml of IgG. In contrast, the antibody generated against the C-terminal domain of SR-BI (pAb589) did not show any effect either on cholesterol uptake of rabbit BBMV or 125I-labeled apo A-I binding to 140 kDa band. Overall results show that the ligand binding site of SR-BI in rabbit BBMV is located in extracellular domain, and SR-BI is only functional when it is part of dimeric forms which rationalize the previously found cooperative nature of the binding interaction and maybe a fundamental finding towards the so far poorly understood mechanism of SR-BI function.
Assuntos
Animais , Humanos , Coelhos , Sequência de Aminoácidos , Antígenos CD36/metabolismo , Apolipoproteína A-I/metabolismo , Sítios de Ligação/fisiologia , Western Blotting , Células CACO-2 , Ésteres do Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Radioisótopos do Iodo , Proteínas de Membrana/metabolismo , Microvilosidades/metabolismo , Dados de Sequência Molecular , Receptores Imunológicos , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Propriedades de SuperfícieRESUMO
A troponina C é o componente do complexo heterotrimérico troponina ao qual o Ca²+ se associa. Pearlstone et al. (Biochemistry 31, 6545, (1992)) utilizaram o cDNA da troponina C de músculo esquelético de galinha (sTnC) para construir um mutante onde a fenilalanina da posição 29 foi substituída por triptofano (mutante F29W). Esse mutante permitiu que a ligação de Ca²+ aos sítios regulatórios amino terminais fosse acompanhada através de técnicas fluorescentes. Entretanto, algumas propriedades da sTnC foram alteradas pela mutação (Li et al. (1995) Biochemistry 34, 8330). No presente estudo, ensaios de ligação direta de Ca²+ bem como titulações de Ca²+ seguidas por fluorescência foram usadas para melhor se entenderem...
Assuntos
Animais , Biofísica , Proteínas de Ligação ao Cálcio , Contração Muscular/fisiologia , Sequência de Aminoácidos/fisiologia , Troponina C , Diálise/métodos , Diálise , Microscopia Eletrônica/métodos , Microscopia Eletrônica , Sítios de Ligação/fisiologia , Espectrometria de FluorescênciaRESUMO
Polylysine is a polycation used in histology to glue tissue sections to glass slides. It was successfully used to eliminate positively charged proteins from heparin-agarose beds applied for affinity chromatography facilitating the preparative steps in the isolation and purification of heparin binding lectins from the ovary of the frog Leptodactylus occellatus. The addition pretreatment of agarose-heparin beds with polylysine increased more than 3-fold the hemagglutinating capacity of eluted lectin
Assuntos
Animais , Anuros , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Cromatografia de Afinidade , Heparina/química , Heparina/diagnóstico , Lectinas/química , Lectinas/isolamento & purificação , Polilisina/química , Polilisina/diagnósticoRESUMO
Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha(5)beta(1) integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation an a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.
Assuntos
Fibronectinas/química , Receptores de Fibronectina/química , Sítios de Ligação/fisiologia , Colágeno/química , Matriz Extracelular/química , Fibrina/química , Testes de PrecipitinaRESUMO
Fucosylated glycoconjugates play an important role in fertilization as the recognition signal of the zona pellucida. In this work, using "critical" concentrations of either, FITC Lotus tetragonolobus lectin or FITC alpha-L-fucosyl-BSA neoglycoprotein as molecular probes, population densities of fucosylated glycoconjugates and of their "complementary" molecules (carrying fucosyl receptors), were found all over the sperm surface with higher population densities in post acrosomal sheath, neck and midpiece. These results were compared with previously reported data on the population densities of lactosaminic compounds and their "complementary" molecules, obtained on same samples of spermatozoa. Statistical data demonstrate that fucosylated glycoconjugates share the same domains with biantennary N-acetyllactosaminic oligosaccharides carrying outer galactose and bisected N-acetylglucosamine residues. These domains highly differ with those of the lactosaminic glycoproteins carrying tri and tetraantennary N-acetyllactosaminic oligosaccharides. These studies also show that the domains of fucosylated glycoconjugates and their "complementary" molecules (carrying fucosyl receptors) locate in different zones of the spermatozoon than those of the compounds carrying beta-galactosyl receptors. Besides, the results suggest structural differences between fucosylated glycoconjugates of the acrosome, equatorial zone and post acrosomal sheath. This may be relevant to the different biological behavior of these compounds and zones, in fertilization.
Assuntos
Humanos , Masculino , Amino Açúcares/química , Fucose , Galactose , Glicoconjugados , Espermatozoides , Amino Açúcares/metabolismo , Hormônio Foliculoestimulante , Fucose , Galactose , Glicoconjugados , Hormônio Luteinizante/química , Hormônio Luteinizante/metabolismo , Lectinas , Estrutura Terciária de Proteína , Sítios de Ligação/fisiologia , EspermatozoidesRESUMO
Fucosylated glycoconjugates play an important role in fertilization as the recognition signal of the zona pellucida. In this work, using "critical" concentrations of either, FITC Lotus tetragonolobus lectin or FITC alpha-L-fucosyl-BSA neoglycoprotein as molecular probes, population densities of fucosylated glycoconjugates and of their "complementary" molecules (carrying fucosyl receptors), were found all over the sperm surface with higher population densities in post acrosomal sheath, neck and midpiece. These results were compared with previously reported data on the population densities of lactosaminic compounds and their "complementary" molecules, obtained on same samples of spermatozoa. Statistical data demonstrate that fucosylated glycoconjugates share the same domains with biantennary N-acetyllactosaminic oligosaccharides carrying outer galactose and bisected N-acetylglucosamine residues. These domains highly differ with those of the lactosaminic glycoproteins carrying tri and tetraantennary N-acetyllactosaminic oligosaccharides. These studies also show that the domains of fucosylated glycoconjugates and their "complementary" molecules (carrying fucosyl receptors) locate in different zones of the spermatozoon than those of the compounds carrying beta-galactosyl receptors. Besides, the results suggest structural differences between fucosylated glycoconjugates of the acrosome, equatorial zone and post acrosomal sheath. This may be relevant to the different biological behavior of these compounds and zones, in fertilization.(AU)
Assuntos
Estudo Comparativo , Humanos , Masculino , RESEARCH SUPPORT, NON-U.S. GOVT , Amino Açúcares/química , Fucose/química , Galactose/química , Glicoconjugados/química , Espermatozoides/química , Amino Açúcares/metabolismo , Sítios de Ligação/fisiologia , Fluoresceína-5-Isotiocianato , Hormônio Foliculoestimulante/química , Hormônio Foliculoestimulante/metabolismo , Fucose/metabolismo , Galactose/metabolismo , Glicoconjugados/metabolismo , Lectinas , Hormônio Luteinizante/química , Hormônio Luteinizante/metabolismo , Estrutura Terciária de Proteína , Espermatozoides/ultraestruturaRESUMO
Membranes derived from bovine pituitary glands free of the neural lobe were used to investigate the presence of binding sites for inhibin, a glycoprotein produced by the ovarian granulosa cells capable of selectively suppressing FSH secretion from the pituitary gland. Optimal concentration of membranes (400 micrograms prot) and 125I-bovine inhibin (2 nM) were incubated in a medium containing 50 mM Tris-HCl pH 7.4, 0.01 M MgCl2 and BSA 0.01 percent in a final assay volume of 200 microliters at 37 degrees C for different time intervals. Non-specific binding was estimated using unlabelled inhibin in excess. The time course of specific 125I-bovine inhibin (2 nM) binding to bovine pituitary membranes is slow with 50 percent binding at approximately 20 min of incubation and reaching equilibrium at 90 min of incubation. The kinetic analysis shows an apparent pseudo first order association rate constant (Kob) equivalent to 4 x 10(-2) min-1. Following equilibrium with the tracer, a large excess of unlabelled inhibin (1.27 microM) was able to displace 84 percent of the specific binding within 120 min of incubation and 50 percent of the binding at approximately 40 min. The analysis under displacing conditions showed an apparent dissociation rate constant (K2) equals to 1.5 x 10(-2) min-1 and an apparent association rate constant (K1) equals to 1.3 x 10(9) M min-1. Thus, the estimation of the apparent kinetic equilibrium dissociation constant (Kd = K2/K2) of the binding of inhibin to bovine pituitary membranes was 1.2 nM. These results show for the first time the existence of bovine inhibin specific binding sites in bovine pituitary, and also that such a binding can take place in the absence of either gonadal and/or hypothalamic influences. They also contribute to the better understanding of the role of non-steroidal hormones such as inhibin, in the regulation of gonadotrophin secretion
Assuntos
Animais , Bovinos , Feminino , Membrana Basal/fisiologia , Sítios de Ligação/fisiologia , Inibinas/biossíntese , Inibinas/farmacologia , Hipófise/fisiologia , Hormônio Foliculoestimulante/metabolismo , Líquido Folicular/metabolismoRESUMO
Affinity labeling has proved to be a very useful tool for searching important amino acid residues located in active or allosteric sites of enzymes. In this article, the general principles and specific examples of the use of affinity labeling are discussed