Studies on the Ca2+ - Dependent Regulation of URE3-BP, A Transcription Factor of Entamoeba histolytica: Identification and Characterization of the Plasma Membrane-Binding Partner EhC2A

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, estimated to be the second leading cause of protozoan morbidity and mortality. In order to understand the molecular pathogenesis of the disease, we have studied the transcriptional regulation of important virulence genes. Calcium (Ca 2+ ) is a ubiquitous secondary messenger involved in many cellular signal transduction events. The E. histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds its cognate DNA element (URE3) in a Ca 2+ -inhibitable manner. The protein is located in both the nucleus and the cytoplasm, but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. This led to the identification and characterization of EhC2A, a 22 kDa calcium-dependent binding partner of URE3-BP and a novel member of the C2- domain superfamily. Immunoprecipitations of URE3-BP and EhC2A demonstrated that the two proteins interact, and that the interaction was enhanced in the presence of calcium. Recombinant and native EhC2A bound phospholipid vesicles in a calcium-dependent manner, with half maximal binding occurring at 3.4 µM free Ca 2+ . URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca 2+ concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. In addition, shRNA mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the ii transcriptional activities of URE3-BP via Ca 2+ -dependent anchoring of the transcription factor to the amebic plasma membrane.

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