RESUMO
The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.
Assuntos
Café/genética , Café/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glicoproteínas/genética , Mariposas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Café/citologia , Café/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
rBbKI and rBbCI, plant recombinant inhibitors from Bauhinia bauhinioides, and BpuTI from Bauhinia purpurea seeds distinctly and specifically block proteolytic enzymes. The secondary structures of those inhibitors were compared and their interactions with phospholipid vesicles were evaluated by the release of calcein and by intrinsic fluorescence of tryptophan residues. The results show that rBbKI, rBbCI and BpuTI are able to interact with phospholipd vesicles and induce membrane permeabilization in a concentration- and pH-dependent manner. The leakage was rapid and extensive at pH 4.5, but at physiological pH, no calcein release was observed. These results may suggest that upon inflammation or microorganism invasion accompanied by lowering of pH, appropriate conditions may occur for the inhibitors to interact with cell membrane and act on specific proteolytic enzyme.
Assuntos
Fosfolipídeos/metabolismo , Inibidores de Proteases/metabolismo , Lipossomas Unilamelares/metabolismo , Animais , Bovinos , Dicroísmo Circular , Fluoresceínas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/química , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Sus scrofa , TemperaturaRESUMO
The kallikrein inhibitor found in Bauhinia bauhinioides seeds (BbKI) differs from classical Kunitz plant inhibitors in the lack of disulfide bridges in its structure [Biochim. Biophys. Acta 1477 (2000) 64-74]. In this study, we examined whether structural properties may be involved in inhibitory specificity and, if so, whether those properties might be useful tools in designing compounds that interfere with enzyme activity. Peptides structurally related to the BbKI (RPGLPVRFESPLRINIIKE-NH(2)) reactive site were synthesized by solid-phase method and assayed for serine proteinase activity. The peptides RPGLPVRFESPLRINIIKE-NH(2), RPGLPVRFESPL-NH(2), and GLPVRFES-NH(2) were efficient tissue kallikrein inhibitors, with I(50) values of 0.54 microM, 0.87 microM, and 0.5mM, respectively. The lasting inhibitory effect was observed in incubation periods of up to 120 min. None of the studied peptides interfere with the activity of thrombin, factor Xa or trypsin, although the native protein BbKI is a potent trypsin inhibitor.