RESUMEN
Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-ß1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-ß1.
Asunto(s)
Echinococcus granulosus/enzimología , Echinococcus granulosus/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencias de Aminoácidos , Animales , Clonación Molecular , Echinococcus granulosus/fisiología , Femenino , Perfilación de la Expresión Génica , Modelos Moleculares , Conformación Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Conejos , Análisis de Supervivencia , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
OBJECTIVE: To investigate whether Echinococcus granulosus cyst fluid-infected host liver cells had differential expression of mitogen-activated protein kinases (MAPKs) or differential cell cycle activity. METHODS: Human liver cells cultured with different concentrations of hydatid cyst fluid (HCF) were tested by the MTT method to determine effects on proliferation. The cell cycle was assessed by flow cytometry. Western blotting was used to detect changes in protein expressions of p-ERK, PCNA, cyclin-A, cyclin-B1, cyclin-D1, and cyclin-E. RESULTS: Forty-eight, 72 and 96 h of HCF at 15%, 30% and 60% concentrations in the cell media significantly promoted cell proliferation (F=67.845, P less than 0.01) and compared to controls (P less than 0.05). Cells exposed to 15% HCF for 48 h showed significantly induced expression of p-ERK (F=1.916, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 24 h showed significantly induced expression of cyclin-Dl (F=3.901, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h showed significantly induced expression of PCNA (F=91.140, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h shed significantly induced expression of cyclin-A (F=18.587, P=0.002), higher than controls (P less than 0.01). Cells exposed to 15% HCF for either 48 h or 72 h showed significantly induced expression of cyclin-B1 (F=2.064, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 30% HCF for 96 h showed significantly induced expression of cyclin-E (F=1.068, P less than 0.01), higher than controls (P less than 0.01). CONCLUSION: Hydatid cyst fluid exerts no inhibitory effect on primary cultured host liver cells, but may promote cellular proliferation.