RESUMO
Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Micelas , Peptídeos/química , Animais , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Lipossomas Unilamelares/químicaRESUMO
Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide These enzymes, which are found in fungi, bacteria, and plants, show very similar structures. Despite an abundance of urease in vegetal tissues, the physiological role of this enzyme in plants is still poorly understood. It has been previously described that ureases from the legumes jackbean ( Canavalia ensiformis) and soybean ( Glycine max) have insecticidal activity and antifungal properties. This work presents the physicochemical purification and characterization of a urease from cotton ( Gossypium hirsutum) seeds, the first description of this enzyme in Malvaceae. The urease content varied among different cotton cultivars. Cotton seed urease (98.3 kDa) displayed low ureolytic activity but exhibited potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. As described for other ureases, the antifungal effect of cotton urease persisted after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of these proteins in plant defense.