RESUMO
Curculin from Curculigo latifolia is a unique sweet protein that exhibits both sweet-tasting and taste-modifying activities. We isolated a gene that encodes a novel protein highly homologous to curculin. Using cDNAs of the previously known curculin (designated as curculin1) and the novel curculin isoform (curculin2), we produced a panel of homodimeric and heterodimeric recombinant curculins by Escherichia coli expression systems. It was revealed that sweet-tasting and taste-modifying activities were exhibited solely by the heterodimer of curculin1 and curculin2.
Assuntos
Curculigo/química , Curculigo/genética , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Edulcorantes/química , Edulcorantes/farmacologia , Paladar/efeitos dos fármacos , Sequência de Aminoácidos , Dicroísmo Circular , Clonagem Molecular , Dimerização , Dissulfetos/metabolismo , Humanos , Dados de Sequência Molecular , Oxirredução , Proteínas de Plantas/genética , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Paladar/fisiologiaRESUMO
beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.
Assuntos
Apoptose , Proteínas de Choque Térmico HSP90/fisiologia , Naftoquinonas/farmacologia , Acetilcisteína/química , Acetilcisteína/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Northern Blotting , Western Blotting , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Corantes/farmacologia , Citocromos c/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Regulação da Expressão Gênica , Vetores Genéticos , Células HL-60 , Humanos , Células K562 , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio , Frações Subcelulares/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermum radix, induces apoptosis in various lines of human tumor cells. To identify genes involved in beta-HIVS-induced apoptotic process, we performed cDNA array analysis and found that beta-HIVS suppresses the expression of the gene for a polo-like kinase 1 (PLK1) that is involved in control of the cell cycle. When U937 and HL60 cells were treated with 10(-6) M beta-HIVS for 0.5 h, both the amount of PLK1 itself and the kinase activity of this enzyme were decreased. By contrast, Bcr-Abl-positive K562 cells were resistant to the induction of apoptosis by beta-HIVS and this compound did not suppress the kinase activity of PLK1 in these cells. However, simultaneous treatment of K562 cells with both beta-HIVS and STI571, which selectively inhibits the protein tyrosine kinase (PTK) activity of Bcr-Abl, strongly induced apoptosis. Moreover, beta-HIVS increased the inhibitory effect of STI571 on PTK activity. Treatment of K562 cells with antisense oligodeoxynucleotides (ODNs) specific for PLK1 sensitized these cells to the beta-HIVS-induced fragmentation of DNA. These results suggest that suppression of the activity of PLK1 via inhibition of tyrosine kinase activity by beta-HIVS might play a critical role in the induction of apoptosis.
