RESUMO
Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.
Assuntos
Núcleosídeo-Difosfato Quinase/química , Trypanosoma cruzi/química , Difração de Raios X/métodos , Sequência de Aminoácidos , Animais , Domínio Catalítico , Clonagem Molecular , Modelos Moleculares , Estrutura Molecular , Mutagênese , Núcleosídeo-Difosfato Quinase/genética , Proteínas de Protozoários , Trypanosoma cruzi/genéticaRESUMO
The activity of dehydroleucodine, a sesquiterpene lactone obtained from Artemisia douglasiana, was studied in mice small intestinal transit. Its mechanism was evaluated in the presence of several adrenergic and cholinergic antagonist drugs and one opioid antagonist. Docking of dehydroleucodine into the homology model of the α2-adrenergic receptor allowed us to analyze the structural basis of their interactions. The experiments showed that dehydroleucodine delayed intestinal transit. The docking of dehydroleucodine showed a unique binding site, equivalent to the binding site of carozolol in the ß-adrenergic receptor. The results suggested that dehydroleucodine produced an inhibitory effect on intestinal transit. Its action could be mediated, at least in part, through the α2-adrenergic receptor.
Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Lactonas/química , Lactonas/farmacologia , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 2/química , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Animais , Intestino Delgado/efeitos dos fármacos , Camundongos , Fentolamina/antagonistas & inibidores , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/metabolismo , Ioimbina/antagonistas & inibidoresRESUMO
The anti-inflammatory activity of quercetin was evaluated through serotonin-induced rat-paw edema. The experiments showed that quercetin had an important effect on acute inflammatory processes. Docking of serotonin and quercetin into the homology model of the 5-Hydroxytryptamine Type 2 Receptor allowed to analyze the structural basis of the anti-inflammatory activity. Results showed that serotonin and quercetin bind in the same region of the active site with a similar binding energy but quercetin has a much bigger inhibition constant. Therefore, it seems possible that quercetin may act as a natural inhibitor of the receptor blocking the acute inflammation generated by serotonin.