Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Bioorg Med Chem ; 24(23): 6215-6224, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27756508

RESUMO

An aqueous acetone extract from the fruit of Alpinia galanga (Zingiberaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50=7.3µg/mL). Through bioassay-guided separation of the extract, a new 7-O-9'-linked neolignan, named galanganol D diacetate (1), was isolated along with 16 known compounds including 14 phenylpropanoids (2-15). The structure of 1, including its absolute stereochemistry in the C-7 position, was elucidated by means of extensive NMR analysis and total synthesis. Among the isolates, 1 (IC50=2.5µM), 1'S-1'-acetoxychavicol acetate (2, 5.0µM), and 1'S-1'-acetoxyeugenol acetate (3, 5.6µM) exhibited a relatively potent inhibitory effect without notable cytotoxicity at effective concentrations. The following structural requirements were suggested to enhance the inhibitory activity of phenylpropanoids on melanogenesis: (i) compounds with 4-acetoxy group exhibit higher activity than those with 4-hydroxy group; (ii) 3-methoxy group dose not affect the activity; (iii) acetylation of the 1'-hydroxy moiety enhances the activity; and (iv) phenylpropanoid dimers with the 7-O-9'-linked neolignan skeleton exhibited higher activity than those with the corresponding monomer. Their respective enantiomers [1' (IC50=1.9µM) and 2' (4.5µM)] and racemic mixtures [(±)-1 (2.2µM) and (±)-2 (4.4µM)] were found to exhibit melanogenesis inhibitory activities equivalent to those of the naturally occurring optical active compounds (1 and 2). Furthermore, the active compounds 1-3 inhibited tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA expressions, which could be the mechanism of melanogenesis inhibitory activity.


Assuntos
Alpinia/química , Lignanas/farmacologia , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Frutas/química , Expressão Gênica , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lignanas/química , Lignanas/isolamento & purificação , Melanócitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Mensageiro/metabolismo , Estereoisomerismo , Teofilina/farmacologia
2.
J Biol Chem ; 286(51): 44078-44085, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22052907

RESUMO

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.


Assuntos
Flavinas/química , Oxirredutases/metabolismo , Thermus thermophilus/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/química , Ferro/química , Cinética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
3.
Biochim Biophys Acta ; 1803(5): 527-33, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20206212

RESUMO

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.


Assuntos
Apoptose , Inibidores de Caspase , NAD/metabolismo , Pentosiltransferases/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Sobrevivência Celular , Células Cultivadas , Citoplasma/metabolismo , Dactinomicina/farmacologia , Ativação Enzimática , Células HeLa/enzimologia , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Fígado/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Biochem Biophys Res Commun ; 367(3): 535-41, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18177738

RESUMO

The YdjC-family protein is widely distributed, from human to bacteria, but so far no three-dimensional structure and functional analysis of this family of proteins has been reported. We determined the three-dimensional structure of the YdjC homolog TTHB029 at a resolution of 2.9A. The overall structure of the monomer consists of (betaalpha)-barrel fold forming a homodimer. Asp21, His60, and His127 residues coordinate to Mg(2+) as a possible active site. TTHB029 shows structural similarity to the peptidoglycan N-acetylglucosamine deacetylase from Streptococcus pneumoniae (SpPgdA). The active site groove of SpPgdA includes the Zn(2+) coordinated to Asp276, His326, and His330. Despite the low sequence identity, metal-binding residues of Asp-His-His were conserved among the two enzymes. There were definitive differences, however, in that one of the histidines of the metal-binding site was substituted for the other histidine located on the other loop. Moreover, these important metal-binding residues and the residues of the presumed active site are fully conserved in YdjC-family protein.


Assuntos
Amidoidrolases/química , Proteínas de Bactérias/química , Thermus thermophilus/química , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Enterococcus faecalis/enzimologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/enzimologia
5.
J Mol Biol ; 365(2): 362-78, 2007 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-17069851

RESUMO

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.


Assuntos
Proteínas Arqueais/química , Endonucleases/química , Thermoproteus/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Arqueais/genética , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Endonucleases/genética , Estabilidade Enzimática , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
6.
FEBS Lett ; 581(30): 5743-50, 2007 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-18036345

RESUMO

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts.


Assuntos
Proteínas do Olho/metabolismo , Fibroblastos/enzimologia , Metaloproteinase 3 da Matriz/biossíntese , Glicoproteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Indução Enzimática/efeitos dos fármacos , Proteínas do Olho/química , Proteínas do Olho/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Masculino , Metaloproteinase 3 da Matriz/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Células NIH 3T3 , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
J Mol Biol ; 345(2): 325-37, 2005 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-15571725

RESUMO

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.


Assuntos
Glutamato Desidrogenase/química , NAD/química , Pyrobaculum/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Ligação de Hidrogênio , Íons , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pyrobaculum/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , Difração de Raios X
8.
J Cancer Res Clin Oncol ; 136(6): 911-21, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20049476

RESUMO

PURPOSE: Tumor necrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS: Tipalpha binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS: Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha to recombinant His-tagged nucleolin fragment (284-710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipalpha-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipalpha and expression of TNF-alpha gene. CONCLUSIONS: All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.


Assuntos
Proteínas de Bactérias/metabolismo , Carcinógenos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Imunoprecipitação , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Nucleolina
9.
J Physiol Sci ; 58(2): 105-11, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18307834

RESUMO

The oligomeric structure and the residual propeptide are distinct characteristics of cathepsin C from other members in the papain superfamily. In this study, we examined the physiological role of the cathepsin C propeptide. The stable overexpression of cathepsin C propeptide significantly decreased the activities of intestinal alkaline phosphatase (IAP) and sucrase in human Caco-2 intestinal epithelial cells, whereas it did not change the proliferation and cathepsin C activity. The overexpression of cathepsin C propeptide significantly decreased the amounts of IAP protein in differentiated Caco-2 cells, compared with the transfection of mock vector, whereas the amounts of IAP transcripts were not changed. Pulse-chase analysis confirmed that the reduction in IAP activity was due to an increase in IAP degradation, but not a decrease in IAP expression. For the mechanism of the enhanced IAP degradation, we identified proteins interacting with cathepsin C propeptide in Caco-2 cells by immunoprecipitation and mass spectrometry. Cathepsin C propeptide interacted with proteins with a molecular mass of approximately 70 kDa, including IAP and heat shock cognate protein 70. Our present results suggest that the propeptide of cathepsin C may stimulate the sorting to the lysosome, at least in part, contributing to the degradation of IAP in Caco-2 cells.


Assuntos
Adenocarcinoma/metabolismo , Antígenos de Neoplasias/metabolismo , Catepsinas/metabolismo , Neoplasias do Colo/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Adenocarcinoma/patologia , Fosfatase Alcalina , Células CACO-2 , Catepsina C , Diferenciação Celular , Neoplasias do Colo/patologia , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Humanos , Lisossomos/metabolismo , Sacarase/metabolismo
10.
Biochem Biophys Res Commun ; 355(2): 385-91, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17303072

RESUMO

D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO.


Assuntos
Aminoácido Oxirredutases/metabolismo , Di-Hidroxifenilalanina/metabolismo , Dopamina/biossíntese , Cristalização , Di-Hidroxifenilalanina/química , Modelos Moleculares , Oxirredução , Conformação Proteica , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
11.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 11): 2006-8, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15502310

RESUMO

Two crystal forms, rhombohedral and hexagonal, of a homing endonuclease from Thermoproteus sp. IC-061 (I-Tsp0611) were obtained by the hanging-drop and sitting-drop method, respectively. The hexagonal crystals belong to space group P6(3)22, with unit-cell parameters a = b = 111.4, c = 97.6 A, and diffract to 3.2 A resolution on beamline BL44 at SPring-8 (Harima, Japan). The rhombohedral crystals belong to space group R32, with unit-cell parameters a = b = 95.4, c = 192.9 A, and diffract to 2.7 A resolution using a Cu Kalpha rotating-anode generator with an R-AXIS VII detector. The crystal asymmetric unit contained one protein molecule and the solvent contents of the two crystal forms were estimated to be 68.3 and 67.6% by volume, respectively.


Assuntos
Desoxirribonuclease I/química , Thermoproteus/enzimologia , Cristalização , Difração de Raios X
12.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 4): 715-7, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039563

RESUMO

NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum was crystallized in the apo- and holoenzyme forms. Crystals were obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3(1)21 or its enantiomorph P3(2)21. The asymmetric unit contains three subunits; the values of the Matthews coefficient (VM) and the solvent content are 2.9 A3 Da-1 and 57%, respectively. A native data set was collected to a highest resolution limit of 4.0 A on an in-house X-ray source using a rotating-anode generator (overall Rsym of 12.3% and completeness of 97%). The holoenzyme crystals belong to the orthorhombic space group P2(1)2(1)2(1); the asymmetric unit contains one hexamer, giving a VM of 2.79 A3 Da-1 and a solvent content of 55%. Native and derivative data sets were collected. The crystals diffract to a maximum resolution of 2.8 A on the KEK-NW12 beamline at the Photon Factory and gave a data set with an overall Rsym of 7.9% and a completeness of 91%. Attempts are being made to solve the structure by the SIRAS method.


Assuntos
Proteínas Arqueais/química , Cristalização , Desidrogenase de Glutamato (NADP+)/química , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa