Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of Entamoeba histolytica

Abstract

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA 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. The parasite Entamoeba histolytica is the causative agent of amebiasis, estimated to be the second leading protozoan cause of morbidity and mortality in humans worldwide (2). The molecular mechanisms that regulate the outcome of infection have yet to be elucidated; however, we hypothesize that transcriptional regulation may play an essential role in determining such outcomes.The calcium ion (Ca 2ϩ ) is a universal regulator of many cellular processes in eukaryotic cells, including cell signaling events, gene expression, and cell death. In Entamoeba, Ca 2ϩ has been implicated in many aspects of the parasite's pathophysiology, including cell motility (35), adhesion to fibronectin (6), cytolytic activity (36), transcriptional regulation (16-18), and growth and developmental stage transition (25, 26). In eukaryotes, these processes are generally mediated by proteins possessing Ca 2ϩ -binding motifs, such as EF-hands, endonexin folds, and C2 domains, which, in response to changes in intracellular Ca 2ϩ levels, are responsible for the transduction of signaling events, several of which have been identified in Entamoeba (for a review, see reference 4).The upstream regulatory element 3-binding protein (URE3-BP) is an E. histolytica Ca 2ϩ -regulated transcription factor first identified for its roles in the control of the expression of ferredoxin (fdx1) and the heavy subunit of the galactose/N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin (hgl5). The protein was isolated by a yeast one-hybrid screen using the cognate URE3 DNA motif (17), though the amino acid sequence of URE3-BP revealed no DNA-binding motifs. The protein did, however, possess two canonical and one noncanonical EF-hand motifs. URE3-BP localized to both the nucleus and the cytoplasm of trophozoites and was also found to be enriched in the plasma membrane. Ca 2ϩ induced the release of binding of URE3-BP from the URE3 motif in vitro and in vivo (18). Mutation of the second EF-hand motif at residues critical for Ca 2ϩ coordination abrogated this effect and allowed for the genome-wide identification of targets of URE3-BP in parasites overexpressing the mutant protein (16).In this study, we explored the reason for the unusual plasma membrane localization of the URE3-BP transcription factor. We discovered a novel URE3-BP binding partner, EhC2A, containing an N-terminal Ca 2ϩ -binding C2 domain and a proline-rich C-terminal region. C2 domains are ϳ120-residue motifs that confer phospholipid-binding activity on the majority of proteins which possess them (reviewed in references 9, 22, and 37). EhC2A coimmunoprecipitated with URE3-BP in a Ca 2ϩ -dependent manner and ...

Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica

“…The localization and distribution of EHI_108720 was confirmed using an immunofluorescence assay (data not shown). Cytoplasmic localization is not uncommon for transcription factors; for example, URE3-BP is present in both the nucleus and cytoplasm (46). Changes in the cellular localization of URE3-BP have been linked to a regulatory mechanism for this transcription factor (46).…”

“…Cytoplasmic localization is not uncommon for transcription factors; for example, URE3-BP is present in both the nucleus and cytoplasm (46). Changes in the cellular localization of URE3-BP have been linked to a regulatory mechanism for this transcription factor (46). The transfected cell line overexpressing EHI_108720 was exposed to H 2 O 2 stress, and a Western blot was performed to determine if there was any change in abundance, size, or localization of EHI_108720.…”

Structural genomics of infectious disease drug targets: the SSGCID

“…SSGCID works closely with members of the scientific community to publish protein structural data produced by the consortium and this has resulted in a number of collaborative publications (Yamada et al, 2010;Edwards et al, 2010;Jaffe et al, 2011;Zhang et al, 2011;Buchko et al, 2010Buchko et al, , 2011Li et al, 2010a,b;Moreno et al, 2010).…”

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase

“…Keck Biomedical Mass Spectrometry Laboratory as described previously [39] but with the following changes: the liquid chromatography (LC)-MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm × 75 μm id Phenomenex Jupiter 10 μm C18 reversed-phase capillary column. 7.5 μl of the extract was injected, and the peptides were eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 μl/min over 30 min.…”